Eur. J. Biochem. 61, 271 -286 (1976)

Stereochemistry of a Methyl-Group Rearrangement during the Biosynthesis of Lanosterol Gareth T. PHILLlPS and Kenneth H. CLIFFORD Shell Research Limited, Milstead Laboratory, Sittingbourne Research Centre, Sittingbourne (Receivcd August 19/October 8, 1975)

1. (3RS,6R)-[6-2Hl,6-3H1,6-14C], (3RS,6S)-[6-2H1,6-3HH,, 6-I4C] and (3RS)-[6-3HH,,6-14C]mevalonolactones were synthesised from R-[2Hl?H,,2-'4C], S-[2H1,3Hl,2-14C]and [3H1,2-14C]acetic acids respectively. 2. Each mevalonate was converted into cholesterol by a rat liver preparation. 3. Each cholesterol specimen was converted into androsta-l,4-diene-3,17-dioneby incubation with Mycobacteriumphlei in the presencd of 2,2'-dipyridyl. Each specimen of androsta-l,4-diene-3,17dione was converted into androsta-l,4-dien-3-one-l7-ethylene ketal. 4. The samples of androsta- 1,4-dien-3-one-17-ethyleneketal were each converted chemically into oestrones in which the methyl group at C-18 is the only carbon atom that originated from C-6 in mevalonolactone. 5. The oestrone from (3RS)-[6-3H1,6-'4C]mevalonolactone was oxidised chemically to acetic acid which was converted into p-bromophenacyl acetate and the 3H/14C ratio was measured. 6. There was no overall loss of tritium from the methyl group of acetic acid, as measured by determining the 3H/14Cratios of the p-bromophenacyl esters, when the synthetic and degradative procedures 1- 5 were tested with [3H,,2-'4C]acetic acid. 7. The oestrones derived from the 6R and 6S-mevalonolactones were oxidised. The chiralities of the resulting acetates were determined by an established procedure whereby the acetates were converted into 2s-malates which were examined for loss of tritium on equilibration with fumarate hydratase. 8. The oestrone from (3RS,6R)-[6-2Hl,6-3Hl,6-'4C]mevalonate gave acetic acid which was converted into 2s-malate that retained 68.6 % of its tritium after treatment with fumarate hydratase ; the configuration of this acetic acid was R. 9. The oestrone from (3RS,6S)-[6-2Hl,6-3Hl,6-'4C]mevalonate was oxidised to acetic acid which was converted into 2s-malate that retained 31.9 % of its tritium after treatment with fumarate hydratase; the configuration of this acetic acid was S. 30. There was no overall change in the configuration of a chiral methyl group between C-6 of mevalonate and C-18 of oestrone. It is concluded that the intramolecular migration of a chiral methyl group from C-15 in 2,3-oxidosqualene to C-13 in lanosterol is stereospecific and occurs with overall retention of configuration.

The stereochemical synthesis and configurational assay of the chiral methyl group [1,2] have led to the elucidation of the substrate stereochemistry of many enzymic reactions. The chiral methyl has been used to study the stereochemistry of two types of biological Dedicated to Professor J. W . Cornforth and Dr R. H. Cornforth. Enzymes. Malate dehydrogenase or L-mdkdte :"3 oxidoreductase (EC 1.1.1.37); phosphotransacetylase or acetyl-CoA :orthophosphate acetyltransacetylase (EC 2.3.13 ) ;si-citrate synthase or citrate oxaloacetate lyase [CoA-acetylating] (EC 4.1.3.7); acetate kinase or ATP: acetate phosphotransferase (EC 2.7.2.1); malate synthase or L-malate glyoxalate-lyase (EC 4.1.3.2); lactate dehydrogenase or L-1actate:NAD oxidoreductase (EC 1.1.1.27); fumarate hydratase or L-malate hydrolase (EC 4.2.1.2).

reactions. The first type is the addition of an isotope of hydrogen to a double bond where the chiral centre is stereospecifically labelled with the two other isotopes of hydrogen ;such a reaction is catalysed by isopentenyl pyrophosphate isomerase [3] and pyruvate kinase [4]. The second type involves the enzymic interconversion of a chiral methyl group and chiral methylene; representative examples of the elucdation of the stereochemistry of this process are concerned with enzymic citrate synthesis and cleavage [5 - 91, 3s-3-hydroxy3-methylglutaryl coenzyme A synthase [lo], pyruvate carboxylase [4] and malic enzyme [4,11]. The work described in this paper represents a novel use of the chiral methyl group to elucidate the stereochemistry

212

of the intramolecular rearrangement of a methyl group during the biosynthesis of lanosterol by enzymes from rat liver. The final stage in the biosynthesis of lanosterol in liver and yeast is the oxidative cyclisation of squalene in a two-step process for which 2,3-oxidosqualene is the intermediate [12- 151. The enzymes that catalyse these reactions are squalene epoxidase [16,17] which converts squalene into (3S)-2,3-oxidosqualene and 2,3-oxidosqualene-lanosterolcyclase [I8- 211 which converts (3S)-2,3-oxidosqualene into lanosterol. N o intermediate has been detected between (3s)2,3-oxidosqualene and lanosterol, and the cyclisation [22,23] is thought to proceed to the protolanosterol carbonium ion [23] or its stabilised equivalent [24] followed by a concerted series of 1 :2-hydrogen and 1 :2-methyl group migrations, which are terminated by the loss of the 9P-hydrogen. The sequence of these migrations is in accordance with the mode of cyclisation of (3S)-2,3-oxidosqualene shown in Scheme 1. The sequence of 1 : 2 migrations is supported by the fact that squalene containing six p r o 4 R protons from mevalonate is converted into lanosterol with the loss of one of the pro-4R protons [25] and that in cholesterol two of the p r o 4 R protons of mevalonate are located at C-17a and C-20D [25,26]. The process has been shown to involve two 1 :2 hydrogen migrations as evidenced by the fact that the hydrogen attached to C-14 in squalene becomes the 17cr hydrogen in lanosterol in both rat liver [27] and yeast [28].Maudgal etal. [29] showed that the methyl group that is attached to C-14 in lanosterol has arrived there by either an intermolecular or intramolecular migration from its attachment to C-10 in squalene. Of particular importance to the subject matter of this paper is the work of Cornforth et al. [30] who showed that the C-18 methyl group in cholesterol, and consequently the C-18 methyl group in lanosterol, had arrived there by a 1 : 2 intramolecular rearrangement which occurred within a structure that was derived from a single molecule of mevalonic acid. The stereochemistry of this migrating methyl group, when it is chirally labelled with the isotopes of hydrogen, is the subject of this paper. Bloch rt af. [31] have shown that no deuterium is present in lanosterol biosynthesised from squalene in a rat liver preparation containing deuterium oxide. Therefore it is unlikely that the migration of the methyl groups occurs via the formation and opening of cyclopropane intermediates, for which there is an analogy in the biosynthesis of cycloartenol and its conversion into plant sterols [32]. A chiral methyl group can prima facie migrate from C-15 in (3s)2,3-oxidosqualene to C-13 in lanosterol (Scheme 1) with either retention or inversion of configuration. In order to distinguish between the two stereochemical possibilities it is necessary to know the configuration

Stereochemistry of a Biological Methyl-Group Rearrangement

of a chiral methyl group that is attached to C-15 in (3S)-2,3-oxidosqualene prior to its cyclisation into lanosterol, and to determine the configuration of the methyl group that becomes attached to C-13 in this lanosterol. The evidence on the mode of formation of squalene from [2-'4C]mevalonate [33], together with the information on the detailed stereochemical aspects of this process [34], is consistent with the assumption that the configuration of a chiral methyl group at C-6 in mevalonate does not change during the conversion of this mevalonate into squalene. Therefore it is sufficient to synthesise mevalonolactones containing chiral C-6 methyl groups whose configurations are known by their method of synthesis from potassium R and S-[2H,, 3Hl]acetate (Scheme 2). The biosynthetic pathway from mevalonate to cholesterol [35] uses (3S)-2,3-oxidosqualeneand lanosterol as intermediates and the final stages of the conversion of lanosterol into cholesterol do not involve any modification of the C-18 methyl group in lanosterol. The solution to the stereochemical problem is therefore reduced to defining the configuration of the chiral C-18 methyl group in cholesterol which is biosynthesised from a mevalonate of known chirality at C-6. The possible configuration of the methyl groups in cholesterol which is biosynthesised from (3R, 6R)[6-2H,,6-3Hl]mevalonateis shown in Scheme 3. The chiral mevalonates were therefore converted directly into cholesterol (Scheme 3) which was converted into androsta-l,4-diene-3,17-dioneusing a previously described [lo] microbiological procedure. The chiral C-19 methyl group was removed by a chemical reductive aromatisation [36] that converted androstd-l,4-dien-3-one-l7-ethylene ketal into oestrone. The chiral C-18 methyl was isolated as acetic acid, and its configuration was determined by an established method [l]. This work has been the subject of a preliminary communication [37]. MATERIALS AND METHODS Enzymes, Substrates and Intermediates

Malate synthase was a gift from Professor H. Eggerer (Biochemie I, Fachbereich Biologie, Regensburg). NAD, NADH, coenzyme A, sodium fumarate, sodium glyoxylate and all enzymes were purchased from Boehringer Co. (London) Ltd. ATP, NADPH, nicotinamide, 2,2'-dipyridyl, oestrone, Tris base, cholesterol, androsta-l,4-diene-3,17-dione and 3-hydroxy3-methylglutaric acid were from Sigma (London) Chemical Company. Cholesterol, m.p. 148.5- 149 "C (sealed tube) was purified via the dibromide [38]and androsta-1,4-dien-3-one-17-ethylene ketal, m.p. 171.5173 "C, was prepared as described by Gentles et al. 1391. Oestrone was recrystallised from ethanol :

G. T. Phillips and K . H. Clifford

M + 270 (mass spectrometry), i : : "282 nm ( E 2118 M-' . cm-'). Benzyl chloride, benzyl acetate, ally1 bromide and p-bromophenacyl bromide were from British Drug House (Poole, Dorset). p-Bromophenacyl bromide was crystallised from ethanol prior to use. Tetraethylammonium hydroxide was purchased from the Aldrich Chemical Company Ltd. (3RS)-Mevalonolactone and (3RS)-mevalonyl benzhydrylamide were a gift from Dr R. H. Cornforth. Sodium [2-14C]acetate (specific radioactivity 60 pCi . pmol-I), sodium ['H1]acetate (specific radioactivity 250 pCi . pmol-') and 2S-[U-14C]malicacid (specific radioactivity 58.5 pCi . pmol-') were purchased from the Radiochemical Centre, Amersham. Potassium R and S-[2Hl,3Hl]acetates [8] (specific radioactivity 0.82 and 0.85 pCi . pmol-' respectively) were a gift from Professor J. W. Cornforth and Dr R. Mallaby.

Chronzatographic Materials and Systems Analytical grade silicic acid (100 mesh) was from Mallinckrodt Chemical Company ; Celite 545 was from Johns-Manville Corporation and neutral alumina was purchased from M. Woelm (Eschwege, Germany). Thin-layer chromatogram plates were purchased from Merck (Darmstadt, Germany). Thin-layer chromatograms were run in four systems: system I, kieselgel F254 developed in ethyl acetate/chloroform/acetic acid (15/5/1, v/v/v); system 11, kieselgel F254 developed in chloroform/methanol (17/3, v/v) ; system 111, kieselgel F254 developed in hexane/ethyl acetate (131, v/v); system IV aluminiumoxid F254 (type E) developed in hexane/ethyl acetate ( l / l , v/v). Compounds were detected by ultraviolet absorption and exposure to iodine vapour except for 3-hydroxy-3-methylgutaric acid and 3-methylpentane-l,3,5-triolwhich were detected with 0.2% KMnO,. Dowex 50WX2 (100-200 mesh, H') was purchased from Sigma Chemical Company and AG 50WX4 (200-400 mesh, H + ) and AG 1x8 (200-400mesh, formate) were purchased from Bio Rad Laboratories Limited. Gas-liquid chromatography was carried out with nitrogen as carrier gas in the following systems. System A, 91.4 cm x 0.64 cm Carbowax 20M 10% on Chromosorb W 100/120 mesh; conditions: flow rate 20 ml . min-', temperature 70- 120 "C at 4 "C . min-l; (the retention times obtained with this column were : 4-hydroxy-4-methyl-hepta-1,6-diene, 1.8 min; benzyl chloride, 4.4 min ; benzyl acetate, 9.1 min; benzyl alcohol, 12.6min). System B, same column and conditions as in system A except that the column temperature was 180 "C (retention time of mevalonolactone 4.8 min). System C, 182.8 cm x 0.64 cm QF-1 3 %, on Chromosorb W-AW-DMS; conditions: flow rate 10 ml . min-', temperature 220 "C (retention time of oestrone, 6.2 min).

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Measurement of Radioactivity The measurement of 3H and 14C in compounds associated with the synthesis of mevalonolactones and for the series of compounds in the conversion of the mevalonolactones to acetic acid was carried out with the same equipment and scintillator solutions as previously described [lo]. The measurement of 'H and 14C in compounds associated with the assay of c h i d acetic acids was carried out with the same equipment [lo] in the scintillator solution, Permafluor 11, purchased from Packard Instruments. Synthesis of (3RS)-[6-3 H , ,6-14C],

(3RS,6R)-[6-2H1,6-3H1,6-14C] and (3RS,6S)-[6-2Hl,6-3H, ,6-'4C]Mevalonoluctones The synthesis of (3RS)-mevalonolactone (VI) containing a chiral methyl group at C-6 was based partly on previously reported procedures. The synthetic route (Scheme 2) from potassium acetate (I) was tested with unlabelled compounds in which the intermediates, benzyl acetate (11) 4-methylhepta-l,6-dien4-01 (111) [40], 3-hydroxy-3-methylglutaric acid (IV) [40] and 3-methylpentane-l,3,5-triol(V) [41] were isolated and characterised by comparison of their physical and spectral data with authentic compounds or against literature values. These compounds were used as standards for gas-liquid chromatography and thin-layer chromatography of the intermediates during the small-scale synthesis of the radioactive mevalonolactones. (3RS ,6S) -(6-=H I ,6-3Hl ,6-14C]Mevalonolactone (Mevalonolactone A ) Benzyl 2S-[2-'H1 ,2-3H,,2-'4C]Acetate. The preparation of this compound was based on a previously reported procedure [42]. Sodium [2-14C]acetate (0.1 mmol, x 25 pCi) was converted to the free acid on passage through 5.0 ml of Dowex-50 (H+) which was then washed with water (50 ml). Similarly potassium S-['H,,'HH,]acetate (0.3 mmol, 246 pCi 'H) was mixed with unlabelled potassium acetate (2.0 mmol) and converted into the free acid by passage through 7.0ml of Dowex-50 (H') which was then washed with water (50 ml). The combined eluates (25.3pCi 14C, 251 pCi 3H) were titrated with 0.43 M tetraethylammonium hydroxide to pH 7.2; the titre was 4.6 ml (1.97 mmol). The solution of tetraethylammonium acetate was evaporated to an oil and stirred with a solution of benzyl chloride (0.25 ml, 2.2 mmol) in dimethylformamide (2.0 ml) for 18 h. The reaction mixture was poured into water (20 ml) which was extracted with ether (3 x 20 ml). The ethereal solution was dried (Na2S0,) and evaporated at

274

Stereochemistry of a Biological Methyl-Group Rearrangement

-'

20 "C to give an oil (315 mg) which was analysed by ( 3 RS ,6S) - [6 H1,6 - H , ,6-I4C]3 - Me thylpentane 1,3,5-triol. The reduction of 3-hydroxy-3-methylglugas-liquid chromatography (system A) and contained benzyl 2S-[2-2H,,2-3H1,2-'4C]acetate (98%, taric acid to 3-methylpentane-1,3,5-triolwas carried out by the method of Brown et al. [43]. A solution of 1.93 mmol, 80.4% recovery based on acetate) and (3RS,6S)- [6-'H1,6-,H1,6 -14C]3- hydroxy- 3 -methylbenzyl chloride (2%). The sample contained 21.86 pCi glutaric acid (183.1 mg, 1.13 mmol, 12.82 pCi I4C, I4C (86.4% based on [2-'4C]acetic acid) with ,H/I4C 3H/14C = 10.89) in dry tetrahydrofuran (3.3 ml) was = 10.63. (4RS,8 S)-[8-2Hl,8-3H1,8-14C]4-Methylhepta-1,6added to a stirred solution of BH, (5.44mmol) in tetrahydrofuran (12.04 ml) at 0 "C, under a stream of dien-4-01. Benzyl 2S-[2-'Hl ,2-3Hl,2-14C]acetate nitrogen over 2 min. The mixture was stirred at 0 "C (309 mg, 1.93 mmol, 21.86 pCi I4C with 3H/14C for 1 h and warmed to 20 "C over 4 h, when water = 10.63) and ally1 bromide (0.70 ml, 8.1 mmol) in (10 ml) was added and the mixture stirred for 1 h. dry ether (1.33 ml) and tetrahydrofuran (2.66 ml) The solvent was removed by evaporation under rewere added to a suspension of magnesium turnings duced pressure and the residue dried by azeotropic (244 mg, 10 mmol) in ether (0.38 ml) and tetrahydrodistillation with benzene (2 x 10 ml). Dry methanol furan (0.38 ml) over a period of 15 min, and the reac(3 x 25 ml) was added and the solution was evaporated tion allowed to proceed for another 1.75 h. Water to give (3RS,6S)-[6-2H,,6-3H,,6-'4C]3-methylpentans (10 ml) and 6N H2S04 were added and the reaction 1,3,5-triol that contained 10.99 pCi 14C with ,H/I4C mixture was extracted with ether (3 x 20 ml) which = 11.07. The material was analysed on thin-layer was washed with 5 % NaHCO, and dried over molecular sieve No. 4A. The ether was partly removed by chromatography in system 11. The plate was scanned evaporation under reduced pressure at 20 "C and the for radioactivity and contained 93% of (3RS,6S)product distilled at atmospheric pressure. The frac[6-2H1,6-3H1,6-14C]3-methylpentane-l,3,5-triol R, tion boiling between 75 "C and 140 "C was analysed 0.22 and 7 % of an impurity at R, 0.02. It was estimated by gas-liquid chromatography (system A) and conthat the product contained 0.9 mmol of (3RS,6S)tained (4RS,8S)-[8-2H,,8-3H1,8-14C]4-methylhepta[6-2Hl,6-3H,,6-14C]3-methylpentane-1,3,5-triol with 1,6-dien-4-01 (78 %) and benzyl alcohol (22 %). The 10.22 pCi I4C (79.7% based on 14C content of 3radioactive content was 15.27 pCi I4C (69.9% based hydroxy-3-methylglutaric acid). on 14C content of the benzyl acetate) with ,H/14C (3RS,6S)-[6-2H,,6-3H,,6-'4C]Mevalonolactone. = 10.77 from which it is calculated that the sample The oxidation described is based on the method of contained (4RS,8S)-[8-2Hl,8-3H,,8-'4C]4-methylhepFetizon et al. [41]. A stirred suspension of silver ta-1,6-dien-4-01 (170.1 mg, 1.35 mmol) and benzyl carbonate on Celite [44] (12.2 g) in dry benzene alcohol (46.9 mg, 0.43 mmol). (60ml) was heated under a reflux condenser and (3RS,6S)-[6-2Hl,6-3Hl,6-14C]3-Hydr~~y-3-methDean-Stark trap until 20 ml of benzene was removed. ylglutaric Acid. A mixture of (4RS,8S)-[8-'H1,8A solution of (3RS,6S)-[6-2H,,6-3H1,6-'4C]3-methyl3H,,8-14C]4-methylhepta-l,6-dien-4-ol(170.1 mg, pentane-1,3,5-triol (% 0.9 mmol, 10.22 pCi I4C) in 1.35 mmol, 15.27 pCi I4C, 3H/'4C = 10.77)and benzyl hot benzene (40 ml) was added and part of the benzene alcohol (46.9 mg, 0.43 mmol) in methylene chloride (20 ml) removed by distillation into the Dean-Stark trap for 1 h. The flask containing the trio1 was washed (9 ml) and glacial acetic acid (0.5 ml) was treated with an excess of ozone at -70 "C. The solution was with benzene (80 ml) which was added to the reaction mixture and the benzene (80 ml) removed by distillawarmed to room temperature, acetic acid (3.6 ml) was added and the methylene chloride removed by tion. After a total time of 3 h the reaction mixture was evaporation. The solution was mixed with hydrogen cooled, filtered and the residue was washed with peroxide (1.5 ml, 30%) in acetic acid (5.1 ml) and methylene chloride (120 ml). The solvent was removed heated under reflux for 4.5 h, after which the reaction and the product was chromatographed on a column of silicic acid (1 cm x 17 cm) packed in chloroform. mixture was cooled, diluted with ether (10 ml) and powdered ferrous sulphate added. The solution The sample was applied in chloroform (0.5 ml) was filtered, dried (Na'SO,), filtered and the solvent which was the initial eluting solvent (28 ml). The removed. The residue was dried by azeotropic distillafractions which were eluted with methanol/chlorotion with benzene, and washed with cold hexane form (119) were assayed by thin-layer chromatography in system 11.The fractions containing pure mevalonol(2 x 2 ml) to remove traces of benzyl acetate, leaving (3RS,6S)- [6-'H1 ,6-3H,,6-'4C]3 - hydroxy- 3 -methylactone RF 0.43 (fractions 50- 54, 2 ml each) were glutaric acid, 12.82 pCi I4C (84.0% based on 14Cconcombined and the solvent removed. The product was with 3H/ tent of 4-hydroxy-4-methylhepta-1,6-diene) dried by azeotropic distillation from benzene to give (3RS,6S)- [6-'H1 ,6-3H, ,6-14C]mevalonolactone I4C = 10.89. The product was chemically and radiochemically pure as determined by analysis on thin(49 mg, 377 pmol, 5.09 pCi I4C, 20.1 "/, overall yield based on [2-'4C]acetic acid, ,H/14C = 11.36). The layer chromatography in system I (RF 0.13), coincident with authentic 3-hydroxy-3-methylglutaric acid. product was analysed by gas-liquid chromatography

G . T. Phillips and K. H.Clifford

(system B) which indicated the presence of 97% of mevalonolactone, and by mass spectrometry when the molecular ions ( M 130 and 131) showed the presence of 13% of 'HI species. The mevalonolactone was chemically and radiochemically pure when it was chromatographed in thin-layer system 11. ( 3RS,6 R) -[6-' Hl,6-3Hl,6-'4C]Mevalonolactone (Mevalonolactone B) . The synthetic route described above, starting with sodium [2-14C]acetate(0.1 mmol, = 25 pC1) and potassium R-[2Hl,3Hl]acetate (13 6 mmol, 254 pCi 3H) gave (3RS,6R)-[6-2H,,6-3H1, 6-'4C]mevalonolactone (51 mg, 392 pmol, 3.68 pCi 14C, 16.3% overall yield based on 22.6 pCi [2-14C]acetic acid, 3H/'4C = 11.93). The product was analysed on gas-liquid chromatography (system B) which indicated the presence of 96 % of the mevalonolactone ; M + 130,131 (12 % 'HI determined by mass spectrometry). The mevalonolactone, R, 0.43, was chemically and radiochemically pure when analysed by thinlayer chromatography in system 11. (3RS)-[6-3Hl,6-'4C ]Mevalonolactone (Mevalonolactone C) . Sodium [2-3Hl,2-'4C]acetate trihydrate (277 mg, 2.03 mmol) containing 214 pCi 14C with 3H/'4C = 12.55 was converted into (3RS)-[6-3H,, 6-'4C]mevalonolactone (30 mg, 231 pmol, 24.6 pCi I4C, 11.5% overall yield based on the 14C content of acetic acid, 3H/'4C = 11.88) by the procedure already described. This mevalonolactone R, 0.47, was pure as estimated by thin-layer chromatography in system I1 and 98 % pure by gas-liquid chromatography (system B). +

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benzhydrylamide (30 mg) and recrystallised to constant 3H/'4C ratio. The results of the assays are shown in Tables 1, 2 and 3. Muhienzyme Systemfrom Rat Liver for the Synthesis of Cholesterol

Rat liver homogenates were prepared according to the Popjak [45] modification of the procedure of Bucher and McGarrahan [46]. The livers (= 28 g) from five rats were minced and homogenised for 2.5 min at 4 "C with 50 ml of 0.1 M potassium phosphate buffer pH 7.5 containing 5 mM MgCl, and 30 mM nicotinamide. The homogenate was spun at 350 x g for 20 min and the supernatant was spun at 10000 x g for 30 min. The supernatant (S10) was diluted to 70ml with the above buffer and used immediately for the synthesis of cholesterol. The mevalonolactones that were used for the synthesis of cholesterol were converted into the potassium salts by hydrolysis with five equivalents of 0.2 N KOH at 38 "C for 1 h. Each incubation mixture contained 0.1 M potassium phosphate buffer pH 7.5, 2 mM ATP, 2.7 mM MgCl,, 0.61 mM NADPH, 21.4 mM nicotinamide, 0.51 mM potassium (3RS)-mevalonate and 3.5ml of S10 supernatant in a final volume of 4.90 ml. The incubation mixtures in 25-ml stoppered conical flasks were flushed with oxygen at zero time and after 10 min and were shaken mechanically at 38 "C for 2.5 h. Cholesterol from Mevalonolactone A . Potassium (3RS,6S)-[6-2H,,6-3H,,6-'4C]mevalonate was conConfirmation of 3H/'4C Ratios verted into cholesterol in four separate batches, where in Mevalonolactones each S10 preparation was prepared from the livers of ten rats. In a typical procedure potassium (3RS,6S)The accurate confirmation of the 3H/'4C ratios in the mevalonolactones was obtained as previously [6-2Hl,6-3H1,6-14C]mevalonate (92 pmol, 1.24 pCi 14C,3H/14C= 11.36) was incubated in 40 flasks where described [lo] whereby the mevalonolactones were the components and conditions were as described in converted into the benzhydrylamides which were the preceding paragraph. The incubation mixtures recrystallised to constant 3H/'4C ratio. were combined, added to ethanol (100 ml) and 10 N MevalonolactoneA . (3RS,6S)-[6-2H,,6-3H1,6-14C]KOH (80 ml) and saponified at 80 "C for 30 min. Mevalonolactone (0.98 mg, 225600 dis I4C . min-' The solution was cooled, extracted with n-hexane with 3H/'4C = 11.36) was converted into the benzhydrylamide (38 %) which was diluted with mevalonyl (3 x 200 ml) and the extract was dried (Na2S04) and benzhydrylamide (21.3 mg) and recrystallised five evaporated to dryness. The sterols (352 mg) from four sets of incubations were combined and chromatotimes from benzene. graphed on a column (75 cm x 1.7 cm) of Celite 5451 MevalonolactoneB. (3RS,6R)-[6-ZH,,6-3Hl,6-14C]Mevalonolactone (1.02 mg, 163400 dis 14C . min-' silicic acid (1/2) in benzene as described by Frantz et a!. [47]. Fractions of 3 ml were taken at a flow rate with 3H/'4C = 11.93) was similarly converted into the benzhydrylamide (25 %) which was diluted with mevof 15 ml . h-'. The eluates in fraction 115- 190 were alonyl benzhydrylamide (21.3 mg) and recrystallised combined and evaporated to give cholesterol (96 mg, 1186000dis 14C . min-', 32.3% based on 14C in 3Ras described above. Mevalonolactone C . (3RS)-[6-3H,,6-'4C]Mevamevalonate, with 3H/'4C = 11.15), M + 386. A part lonolactone (0.12 mg, 217600 dis I4C . min-' with of the cholesterol (5.8 mg, 71 200 dis 14C min-') 3H/'4C = 113 8 ) was mixed with (3RS)-mevalonowas diluted with cholesterol (44.2 mg) and purified lactone (1.1 mg) and converted into the benzhydrylvia the dibromide [38] as described by Cornforth et amide (61.7 %) which was diluted with mevalonyl al. [lo]. The recovered cholesterol (26 mg, 52%) was I

StereochemistTy of a Biological Methyl-Group ReaTTangCmCtIt

276

and concentrated to 1.6 ml and chromatographed on 16 thin-layer plates (20 cm x 20 cm) in system 111. The androsta-1,4-diene-3,17-dione (RF 0.16) was detected by ultraviolet fluorescence and by scanning the plates for radioactivity. The gel containing androsta1,4-diene-3,17-dione was extracted with ethyl acetate (250 ml) which was dried (Na'SO,) and the solvent removed to give 57 mg containing 381 200 dis I4C . min-' (66.9%) with 'H/14C = 10.94. Conversion oj' (18R, 19R,21R,26R)-[ 18,19,21, 26 - 'H1 ,18,19,21,26 - 'Hl, 18,19,21,26 - 14C]Cholesterol ('from Mevalonolactone B ) . Cholesterol (104 mg) containing 1008000 dis 14C . min-' with 3H/'4C = 11.70 was mixed with unlabelled cholesterol (6 mg) and dissolved in dimethylformamide (1 1 ml) containing 2,2'-dipyridyl (44 mg). The microbiological transformation of the cholesterol and the isolation of the products were as described above and gave androsta1,4-diene-3,17-dione (56.6 mg) containing 356400 dis 14C . min-' (70.7 %) with 'H/I4C = 11.42. Conversion of' (18,19,21 ,26-3H , ,18,19,21,26''C]Cholesterol (jrom Mevalonolactone C ). Cholesterol (23 mg) containing 604100 dis 14C . min-' with 'H/14C = 11.58 was mixed with unlabelled cholesterol (7 mg), dissolved in dimethylformamide (3.0 ml) containing 2,2'-dipyridyl (24 mg) and transformed as described above. The androsta-l,4-diene-3,17-dione (6 mg) contained 57900 dis 14C . min-'. The cholesterol that was not transformed (327300 dis I4C * min- ') was recovered, mixed with unlabelled cholesterol (to 40 mg) and dissolved in dimethylformamide Microbiological Transformation of Cholesterol (4.0 ml) containing 2,2'-dipyridyl(l6 mg). This cholesinto Androsta-l,4-diene-3,17-dione terol was transformed into androsta-l,4-diene-3,17dione which was combined with the previous sample. The labelled cholesterol derived from mevalonate was transformed into androsta-l,4-diene-3,17-dione The combined samples of androsta-l,4-diene-3,17by a suspension culture of Mycobacterium phlei dione (22.5 mg) contained 124900 dis 14C . min-' (CBS 49466). The conditions for the maintenance (41.4%). and growth of M.phlri have been described [lo] and were used throughout this investigation. Androsta-l,4-dien-3-one-l7-eth~~kne Ketal Conversion of (18S,I 9 5 2 1 S,26S)-[18J9,21,26(18S,19S) - [18,19 - 2H,,18,19 - 'HH,,18,19-14Cj 2 H l , 18, 19,21,26-3H,, 18, 19,21,26-14C]Cholesterol (jrom Mevulonoluctone A ) . Cholesterol (87 mg) conAndrosta-I ,4-dien-3-one-l7-ethylene Ketal (from Mevalonolactone A ) . A solution of (18S,19S)-[18,19taining 1 139000 dis I4C . min with 'H/14C = 11.15 'H, ,18,19 - 'HI ,18,19 - 14C]androsta- 1,4 - diene - 3,17 was mixed with unlabelled cholesterol (1 3 mg) and dione (57.0 mg, 381200 dis 14C . min-', 'HH/ dissolved in dimethylformamide (10 ml) containing I4C = 10.94), ethanediol (0.02 mi) and toluene-p2,2'-dipyridyl (40 mg). The solution was added to sulphonic acid (4.5 mg) in benzene (4.1 ml) was cells of M . phlei suspended in 0 . 4 x NaCl solution heated under reflux with a Dean-Stark trap (volume (10 x 30 ml) contained in ten 250-ml conical flasks 1.6 ml) for 1 h. The solution was cooled and diethyl and incubated on a rotary shaker at 30 "C for 48 h. ether (10 ml) containing a few drops of pyridine was The origin and quantity of cells in each flask were as added, and the ethereal solution was washed with a previously described [lo]. The cell suspension was saturated solution of NaCl (3 x 5 ml), dried (Na'SO,) extracted with dichloromethane (5 x 150 ml) which and evaporated. The extract in benzene (0.5 ml) was was evaporated to leave a residue which was dissolved chromatographed on neutral alumina (Woelm, grade I, in ethyl acetate (100 ml). The extract in ethyl acetate was washed with 1 N HCl (1 x 50 ml) which was 1 cm x 10 cm) prepared in n-hexane. The column was eluted with benzene (25 ml) followed by benzene/ backwashed with ethyl acetate (2 x 50 mi). The comethyl acetate (97/3, v/v, 250 mi). The latter fractions bined ethyl acetate extracts were dried (Na,S04)

crystalked (acetone) to constant specific radioactivity (see Table 3). Cholesterol fiom Mevalonolactone B. Potassium (3RS,6R)- [6-'H ,6-'H, ,6-'4C]mevalonate (382 pmol, 3.59 pCi 14C, 'H/14C = 11.93) was converted into sterols as described above. The sterols were separated by chromatography which gave cholesterol (109 mg) : M + 386 with 1050000 dis 14C . min-' (39.7 based on I4C in 3R-mevalonate) and 'H/14C = 11.47. A sample of cholesterol (39900 dis I4C . min-' in 50 mg) was purified as described, recovered (20 mg, 40 and recrystallised four times from acetone. The results are in Table 2. Cholesterol from Mevalonoluctone C. Potassium (3RS)-[6-3H1,6-14C]mevalonate (9.24 pmol, 1.96 pCi 14C,'H/I4C = 1I .88) was converted into sterols in an S10 preparation prepared from the livers of 10 rats in which each of 40 incubations contained 0.1 M potassium phosphate buffer p H 7.5,22.4 mM nicotinamide, 0.47 mM ATP, 3.73 mM MgCl,, 0.75 mM NADPH and 0.13 mM potassium mevalonate. The sterols were separated by chromatography, which gave cholesterol (25.5 mg) containing 690400 dis 14C . min-' (47.6 o/, conversion of 3R-mevalonate), 'H/I4C = 11.58. A sample of cholesterol (51 800 dis I4C . min-' in 50 mg) was purified as the dibromide, recovered (33 mg, 66.0 %) and recrystallised to constant specific radioactivity and 3H/14C ratio. The results are shown in Table 1.

x)

G. T. Phillips and K. H. Clifford

277

were combined and after evaporation gave androstawas mixed with unlabelled material (13 mg) and 1,4-dien-3-one-l7-ethylene ketal (43 mg). Further elurecrystallised from n-hexane. The recovered ketal tion with the same solvent gave a mixture of androstashowed a 3H/14C= 11.61. The results are shown in 1,4-dien-3-one-I7-ethyleneketal and androsta-1,4Table 1. diene-3,17-dione which were separated by chromatography on two thin-layer plates (20 cm x 20 cm) in Conversion system 111. The gel containing the androsta-1,4-dienKetal of Androsta-l,4-dien-3-one-l7-ethylene 3-one-17-ethylene ketal (RF 0.25) was eluted with into Oestrone ethyl acetate (5 x 10 ml) which was dried (by filtration through phasc-separating paper), combined with the This transformation was performed by the method previous sample and evaporated to give androstaof Dryden et al. [36]. Dry nitrogen which was free of 1,4-dien-3-one-17-ethylene ketal(47.9 mg, 75.5 %, M + all traces of oxygen was found to be essential for the 328 on mass spectrometry), containing 284000 dis success of this reaction and was prepared by passing 14C . min-' (74.5%) with 3H/14C= 11.18. The comcommercial nitrogen through a series of wash bottles pound was chemically and radiochemically pure as containing concentrated H2S04, KOH pellets and a judged by analysis on two thin-layer chromatography solution of lithium aluminium hydride (0.5 g) and systems with RF 0.25 and RF 0.52 in system 111 and benzopinacolone (10 g) in pyridine (50 ml) [48]. system IV respectively. A sample of the solution of labelled androsta-1,4-dien-3-one-17-ethyleneketal (2.4 mg, 14200 dis 14C . min-', 3H/'4C = 11.18) was 18s-[18-2H1,18-' H l , 18-'4C]Oestrone mixed with unlabelled androsta-l,4-diene-3-one-l7(from Mevalonolactone A ) ethylene ketal (1 5 mg) and was recrystallised (nhexane) to radiochemical purity. The results are shown Lithium wire (220 mg, 31.5 mg atom) was washed under dry toluene and cut into sections (0.5 cm in Table 3. x 0.2 cm) before being added to a stirred solution of (18R,19R) - [I8,19- 2H,,18,19- 3H1,18,19-l4C] biphenyl (3.31 g, 21.5 mmol) and diphenylmethane Androsta-1,4-dien-3-one-l7-ethylene Ketal (from (1.85 g, 11 .O mmol) in dry tetrahydrofuran (30 ml) at Mevalonolactone B ) . (18R,19R)-[18,19-2H1,18,19-3H,, 18,19-'4C]Androsta-l,4-diene-3,17-dione (56.6 mg, 20 "C, under dry oxygen-free nitrogen gas. A blue356400 dis I4C . min-l, 3H/14C = 11.42) was congreen colour was produced within 2 min. The solution was stirred at 20 "C (bath temperature) for 1 h, then at verted as described above into (1SR,19R)-[18,19-2HI, 18,19-'Hl,18,19-14C]androsta-1,4-dien-3-one-17-ethy-50 "C for 1 h and last at 65 "C for 30 min. The temperalene ketal (50 mg, 75.6%, M + 328 on mass spectroture was reduced to about 60 "C and 1 ml of the solution containing lithium biphenyl was transferred with a metry) containing 269000 dis 14C . min-' (75.5 syringe into a reaction vessel, maintained at 60 "C, with 3H/'4C = 11.53. The ketal was chemically and radiochemically pure as judged by analysis by thinwhich was continuously flushed with oxygen-free dry layer chromatography in system 111 and system IV. nitrogen. A solution of (18S, 19S)-[18,l9-'H1 ,18,19A sample of a solution of androsta-1,4-dien-3-one-17- 3H1,18,19-14C]androsta- 1,4-dien-3-one- 17-ethylene ethylene ketal (2.5 mg, 13500 dis 14C min-') was ketal (45.5 mg, 139 pmol, 269800 dis 14C . min-', mixed with unlabelled androsta-1,4-dien-3-one-17- 'H/I4C = 11.18) in dry tetrahydrofuran (0.5 ml) was ethylene ketal(l5 mg) and was recrystallised to radioadded dropwise from a syringe, over 15 min, into the chemical purity. The results are shown in Table 2. stirred solution of lithium biphenyl (1 ml). The reac/18,19- 3 H 1,18,19- ''CjAndrosta- 1,4-dien - 3 -onetion mixture was maintained at 60 "C for another 17-ethylene ketal (from Mevalonolactone C ) . [18,1915 min, warmed to 85 "C for 20 min and heated under 3HH,,18,19- 14C]Androsta - 1,4 - diene - 3,17 - dione reflux for 15 min. The solution was cooled in ice, (22.5 mg, 124900 dis I4C . min-') was mixed with methanol (0.14 ml), water (0.35 ml) and concentrated unlabelled material (77.5 mg) in benzene (7 ml) conHCl (0.2 ml) were added consecutively and the mixture was stirred for 2 h and extracted with chlorotaining ethanediol (0.06 ml) and toluene-p-sulphonic acid (5.0 mg) and was heated under reflux with a form (3 x 5 ml). The chloroform extract was concenDean-Stark (2.4 ml) trap for 1.5 h. The reaction trated (2 ml) and chromatographed on Sephadex mixture was treated as previously described and the LH-20 (2 cm x 75 cm, 4.5-ml fractions) which was product was isolated after chromatography on alupacked and eluted with chloroform. The eluates were mina in the usual way to give [18,19-3H1,18,19-'4C]- assayed by thin-layer chromatography in system 111. androsta-1,4-dien-3-one-l7-ethyleneketal (74.0 mg, Fractions 37- 55 were combined and the solvent 64.3%, RF 0.52 in system IV) which was crystallised was removed to give oestrone which was dissolved in from n-hexane and gave 58.9 mg (51.2%) m.p. 168ethanol (100 ml), and assayed spectrophotometrically 170 "C (cj: [39] 171- 172 "C) (55 190 dis I4C . min-' 282 nm, E 2118 M-I . cm-'). The solution with 'H/14C = 11.6). The radiolabelled ketal (5 mg) 89 100 dis 14C contained oestrone (21.4 mg, 57.1

x)

-

x,

Stereochemistry of a Biological Methyl-Group Rearrangement

278

. min-' (66.1 %) with 3H/'4C = 10.23, RF 0.49 in system 111). The oestrone (10.7 mg, 44500 dis 14C . min-') was purified by sublimation (160 "C at 0.01 mm). The recovered material was dissolved in ethanol (50 ml), assayed spectrophotometrically 282 nm) and contained oestrone (10.7 mg, 45640 dis I4C . min-' with 3H/'4C = 10.46). The 18S-[18-'H1,1 8-3H,,18-'4C]oestrone, M 270 (by mass spectrometry) analysed as a single component, with the same retention time as authentic oestrone on gas-liquid chromatography (system C).

Oxidation of Oestrone to Acetic Acid The degradation of oestrone to acetic acid was based on the method of Cornforth et al. [30]. S-[2Hl,3Hl ,2-'4C]Acetic Acid from 18S-[18-'H1 ,1tb3H,,18-14C]Oestrone

+

18R-[18-' H, ,m3 Hl ,18-'4C]Oestrone (from Mevalonolactone B ) (18R,19R) - [18,19- 2H1,18,19- 3H1,18,19 - 14C]Androsta-l,4-dien-3-one-17-ethyleneketal (33.0 mg, 122 pmol) containing 174000 dis 14C . min-' with 3H/'4C = 11.53 was converted in the same way to give oestrone (19.63 mg, 72.2%) containing 64160 dis 14C . min-' (73.7%) with 3H/'4C = 10.78. A part of this sample (9.81 mg, 32060 dis I4C . min-') was sublimed and gave 18R-[ 18-2H1,18-3H1,18-I4C]oestrone (9.75 mg, determined spectrophotometrically, 2::" 282 nm, M + 270 by mass spectrometry) containing 32870 dis 14C - min-' with 3H/14C= 10.87. The oestrone (RF 0.49) was pure when analysed by thinlayer chromatography in system I11 and showed the same retention time as an authentic sample on analysis by gds-liquid chromatography in system C. [18-3H , ,18-14C]Oestrone

(from Mevalonolactone C )

The 18S-[18-2H,,18-3H1,18-14C]oestrone(10.5 mg, 38.9 pmol, 44790 dis I4C . min-' , 3H/ l4C = 10.46) was coated on the bottom of a flask and heated with a solution of chromium trioxide (5.5 g) in water (5.5 ml) at a bath temperature of 105 "C for 22 h. The solution was cooled, water (20 ml) and H3P04 (1.3 ml) were added and the acetic acid was recovered by steam distillation. The distillate (90 ml) was brought to pH 8.2 with 0.01 N KOH and the quantity of potassium acetate was determined by radioactive counting and from the specific radioactivity of oestrone (1152 dis I4C . min-' pmol-'), The potassium S[2Hl,3H,,2-'4C]acetate (13.9 pmol, 35.7 %) contained 16013 dis 14C . min-' with 3H/'4C = 11.61. The potassium acetate was purified by partition chromatography [49]. Potassium S-[2HI,3H1,2-l4C]acetate (13.9 pmol, 16013 dis 14C min-') was dissolved in 0.5 N H2S04(0.5 ml), mixed with silicic acid (0.75 g) and applied to a column of silicic acid/0.5 N H2S04 (1 cm x 30 cm; 16 g silicic acid and 11.0 ml of 0.5 N H2S04) packed in butan-1-ol/chloroform (5/95, v/v, equilibrated with 0.5 N H2S04) which was also the eluting solvent. The column was developed under pressure (16-cm Hg or 21 kPa) and fractions of 2.5 ml were taken. The elution volume of acetic acid was determined by radioactive monitoring of the fractions and from the previously determined elution volume of [3H]acetic acid on an identical column. S-[2H,,3Hl,2-'4C]Acetic acid was recovered in fractions 25 - 35 which were combined and extracted with 0.01 N KOH (10 ml, 100 pmol). The extract contained potassium S-[2H,,3H,,2-'4C]acetate (13.4 pmol, 15480 dis I4C min-' with 3H/'4C = 12.21).

The synthesis of [18-3H,,18-'4C]oestrone was carried out in conditions slightly different from those already described. A solution of lithium biphenyl was prepared from biphenyl (3.08 g, 20 mmol) and lithium (300 mg, 42.9 mmol) in tetrahydrofuran (30 ml) at 20°C for 2 h under an atmosphere of oxygen-free nitrogen. The solution of lithium biphenyl (1.3 ml) was added to diphenylmethane (50 mg, 0.30 mmol) and [18,19-3Hl,18,19-'4C]androsta-1,4-dien-3-one-17ethylene ketal (50 mg, 0.152 mmol, 46850 dis 14C R-[2Hl,3Hl,2-14C]Acetic Acid . min-' with 3H/'4C = 11.67). The reaction mixture from 18R-[18-2H1 H1,18-'4C]Oestrone was stirred at 20 "C for 2 h, 2 N HCl(3 ml) and ethanol (6 ml) were added and the stirring continued for 5 h. 18R-[18-'H1, 18-3H1,18-14C]Oestrone (9.5 mg, 35.2 pmol, 32700 dis I4C min-', 3H/'4C = 10.87) Solid KHC03 was added (to pH 7.0) and the solution was filtered and the residue was washed with ethanol was oxidised with chromium trioxide (1.12 g) in water (50 ml). The combined filtrates were evaporated to (2.8 ml) at a bath temperature of 101 "C for 22 h. The acetic acid (7.83 pmol, 22.2%, 7130 dis I4C . min-', dryness and the product was dissolved in chloroform (3 ml) and chromatographed on Sephadex LH-20 as 3H/14C= 11.16) was isolated from the oxidation described. The [18-3H,,18-'4C]oestrone (14 mg, mixture in the usual way, purified by partition chromatography and extracted with 0.008 N KOH 34.1 %; 282 nm) contained 6905 dis I4C . min-' (10 ml, 80 pmol). The potassium R-[2H1?H1,2-14C](29.5 "/,> with 3H/'4C = 11.23 and co-chromatographacetate (6.3 pmol) contained 5853 dis 14C . min-' ed with an authentic sample (RF0.48) on thin-layer with 3H/14C= 11.44. chromatography in system 111.

G. T. Phillips and K. H. Clifford

[3Hl,2-'4C]AceticAcid from [18-3Hl,18-'4C]Oestrone

[18-3Hl,18-'4C]Oestrone (12.8 mg, 47.4 pmol, 6313 dis I4C min-I, 3H/14C = 11.23) and chromium trioxide (1.25 g) in water (3.1 ml) were heated at a bath temperature of 101 "C for 22 h. The acetic acid from the reaction was isolated by steam distillation and the distillate (40 ml) was brought to pH 7.8 with 0.01 N NaOH. The solution of sodium [3Hl,2-'4C]acetate was assayed for radioactivity and contained 3094 dis 14C . min-' (49.0%; 3H/'4C = 12.37) in 23.2 pmol, based on the specific radioactivity of [18-'H1, 18-'4C]oestrone.

-

Confirmation of 3Hl'4C Ratios in Sodium Acetates

Sodium [3Hl,2-'4C]acetate trihydrate (136 mg, 1.0 mmol, 948900 dis I4C min-' , 3H/ l 4C = 12.55) which was used for the synthesis of mevalonolactone C, was stirred withp-bromophenacyl bromide (306 mg, 1.1 mmol) in dimethylformamide (15 ml) at 20 "C for 18 h. The reaction mixture was poured into water (75 ml) and extracted with diethyl ether (4 x 50 ml) which was washed with water (4 x 25 ml) and the ether extract was dried (Na,SO,) and evaporated to dryness. The product was chromatographed on a column of silicic acid (1.5 cm x 30 cm) which was packed and eluted with toluene and gave p-bromophenacyl [3H,, 2-14C]acetate (208 mg, 86.3 %) which was recrystallised from n-pentane m.p. 84°C (cf. [l] 85°C). The results are shown in Table 1. Similarly sodium [3Hl,2-14C]acetate (1.90 mg, 23.2 pmol, 3094 dis I4C . min-', 3H/'4C = 12.37) from the oxidation of [18-3Hl,18-'4C]oestrone was mixed with sodium acetate trihydrate (11.9 mg, 87.5 pmol), converted to the p-bromophenacyl derivative, purified by chromatography and crystallised three times from n-pentane at -20 "C. The results are shown in Table 1. Determination of Chiral Acetates Derived JOestrone ,from 18R-[ 18-'H1 ,18-3H 1,IL?-'~C and 18s-[ 18-'H1 ,18-3H , ,18-'4CJOestrone

The quantities of R-[ZH1,3H1,2-14C]and S-['H,, 3Hl,2-'4C]acetates obtained from the oxidation of the corresponding oestrone samples were determined by a modification of a published procedure [50]. The incubation mixture, in 6.0 ml contained 0.15 M triethanolamine buffer pH 7.6, 10 mM 2S-malic acid, 3.0 mM MgC12, 1.0 mM NAD, 0.17 mM coenzyme A, 14 U malate dehydrogenase, 30 U phosphotransacetylase, 30 U si-citrate synthase and 30 U acetate kinase. The incubation mixture was divided into two cuvettes and the reaction was followed from the increase in absorbance at 340 nm after the addition of standard amounts of potassium acetate (from 1.0 to 2.5 pmol)

219

to one cuvette (3 ml). The initial velocity ( u o ) was proportional to the concentration of potassium acetate. The concentrations of chiral acetates were determined from a direct measurement of u,, and the quantities of R-[ZH,:H,,2-14C]acetate and S-[2H1,3H1,2-14C]acetate were in close approximation to the quantities calculated from the specific radioactivity of the corresponding chiral oestrones. Conversion of Chiral Acetates into Malic Acids and Equilibration with Fumarate Hydratase

The conversion of chiral acetates into 2s-malates was carried out as previously described [l]. A typical incubation (10 ml) at 25 "C for 12 h contained 0.1 M K2CO3/KHCO3 buffer pH 9.5, 1 mM potassium acetate, 2 mM sodium glyoxylate, 4 mM ATP, 4 mM MgCl,, 1 mM Mg'EDTA, 1 mM K3EDTA, 34 U acetate kinase, 100 U phosphotransacetylase, 20 U malate synthase and 0.3 mM coenzyme A which was used to start the reaction. The 2S-malic acids were isolated as previously described [l]. Potassium R[2H,,3H1,'4C]acetdte (6.3 pmol, 5853 dis I4C . min-', 'H/I4C = 11.44) from 18R-[18-'H1, 18-3H1,18-14C]oestrone gave 2S-malic acid (5.72 pmol, 4900 dis 14C . min-', 83.8 %, with 3H/14C = 9.04). Potassium S- ['H, ,3H1,I4C]acetate from 18S-[ 18-'H1, 18-3H1,18'4C]oestrone (10 pmol, 11 550 dis 14C . min-', 'H/ I4C = 12.21) gave 29malic acid (7.98 pmol, 9216 dis I4C . min-', 79.8 %, 3H/14C = 11.87). The 2s-malic acids from the chiral potassium acetates were incubated with fumarate hydratase. 24Malic acid (5.72 pmol, 4904 dis 14C . min-', 3H/'4C = 9.04) from potassium R-['H1 :H,,2-14C]acetate, was mixed with 100 pmol of 2S-[U-14C]malic acid (z 10000 dis . min-') and dissolved in 1.30 ml of 38.5 mM potassium phosphate buffer pH 7.0. Three samples (0.15 ml) were taken and the remaining solution was incubated with fumarate hydratase (14 U, 4p1 of ammonium sulphate suspension) at 40°C. Samples of 0.15 ml were taken at 1, 2, 3 and 4 h and boiled for 3 min. Each sample was treated with 0.2 M barium acetate (0.1 ml) and the supernatant was chromatographed on Dowex-SOW (H', 1 cm x 6 cm) which was eluted with water (2 x 5 ml). The eluates were taken to dryness and the 3H/'4C ratio of the mixture of 2s-malic and fumaric acids was determined by scintillation counting. The results are given in Table 4. 2S-Malic acid (7.98 pmol, 9216 dis I4C . min-', 3H/'4C = 11.87) from potassium S-[2Hl,3Hl,2-'4C]acetate was mixed with 100 pmol of 2S-[U-14C]malic acid (z20000 dis . min-I) and equilibrated with fumarate hydratase to give a mixture of 2s-malic and fumaric acid which were isolated and assayed for radioactivity as described in the previous paragraph. The results are given in Table 4.

Stereocheinistry of a Biological Methyl-Group Rearrangement

280

RESULTS AND DISCUSSION In order to determine the stereochcmistry of thc intramolecular migration of a methyl group between C-15 in (3S)-2,3-oxidosqualene and C-13 in lanosterol (Scheme 1) it is necessary to transform mcvalonate containing a chiral C-6 methyl group of known configuration, into cholesterol viu the sequence : Mevalonate squalene

--

--

squalene lanosterol

(3S)-2,3-oxidocholesterol

(1)

and to determine the configuration of the resulting C-18 chiral methyl group in cholesterol. This was achieved by the synthesis of mevalonates containing chiral methyl groups at C-6, followed by their transformation into cholesterol [lo] in which the C-18 methyl group was isolated by the sequence:

-

androsta-l,4-diene-3,17-dione Cholesterol androsta-l,4-dien-3-one-17-ethylene ketal (2) oestrone acetate.

-

-

-

The chiralities of the acetic acids were determined from the loss of tritium incurred when they were subjected to the procedure [l] : Acetate

-

acetyl-CoA

-

2s-malate

+ fumarate. (3)

The stereochemistry of migration of the methyl group could not be deduced unless it suffered little or no racemisation (which in this context means minimal exchange of hydrogen) during both the synthesis of mevalonate (Scheme 2) from acetate and thc degradation of cholesterol to acetate via oestrone (Scheme 3). Consequently the synthetic route was tested starting with [3H1,2-14C]acetic acid. Reference to the results in Table 1 shows that the 'H/I4C ratios of the p bromophenacyl derivative of the [3Hl,2-14C]acetic acid and of the benzhydrylamide derivative of (3RS)[6-'H1 ,6-'4C]mevalonolactone are essentially the same. Unless there was a large isotope effect against the removal of tritium during the chemical synthesis, no cxchange of hydrogen had occurred. Again (3RS)[6-'HH,,6-'4C]mevalonate was converted into cholesterol which was degraded to acetic acid by the method summarised in Scheme 3. The 3H/14Catomic ratios in thc intermediate compounds are shown in Table 1 and illustrate the point that the 3H/14Cratios of the methyl groups in the intermediates are, as expected, essentially unchanged relative to the 'H/14C ratio in the mevalonyl benzhydrylamide. ln particular the [3H,,2-14C]acetic acid that was isolated from the oxidation of the oestrone was converted into the p-bromophenacyl derivative which gave a 3H/'4C ratio of 1.03 (relative

r

3~-2,3-oxidosqualene

3E2.3-oxidosqualene-lanosterol

cyclase/H+

Protolanosterol

W

1

Lanosterol

Scheme 1. Conversion of (3S)-2,3-oxidosqualene into lano.rtero1 showing ilie postuluted intermediate, protolano.?terol. X is either a positive charge or an enzymic functional group; the ringed methyl groups are derived from C-6 of mevalonate

G. T. Phillips and K . H. Clin'ord

28 1

of the compounds listed in Table 2 and Table 3 are in general agreement with the expected values based on the information available on the biosynthesis of cholesterol [35]. The samples of doubly labelled acetic acids from CH, = CHCH,Br, mevalonolactone B and mevalonolactone C were puriMgITHF fied by partition chromatography [49] and analysed for chirality by a previously described procedure [l] whereby they were converted into 2s-malates which were equilibrated with fumarate hydratase to determine the loss of tritium. Authentic samples of Racetate and S-acetate give malates that retain 76.5';;; and 25.3% of their tritium after treatment with fumarate liydratase [9]. When (3RS,6R)-[6-ZH,,6-3Hl,6-'4C](IV) mevalonate was put through the procedure shown in (i)BH, / THF Scheme 3, the resulting acetate gave a 2s-malate (ii) H,O, MeOH that retained 68.6 of its tritium after treatment with fumarate hydratase, from which it follows that the configuration of this acetate was R. Similarly when (3RS,6S)-[6-2H,,6-'H1 ,6-14C]mevalonate was put through the same procedure the resulting acetate Ag, C0,lCelite gave a 2s-malate that retained 31.9% of its tritium on treatment with fumarate hydratase ;thus the configuraCH,OH CH,OH tion of this acetate was S. The results on the configura(V) (VI) tional assay of the acetic acids are shown in Table 4. Scheme 2.Sjwihesis u f chir.a~3RS-mc~ralot~olac~oti~.~. MevalonokdcThe stereochemical results mean thatduringthe biotoile A, R = S-[2H,,3HH,,14C]rnethyl; mevalonolactone B, R = Rsynthesis of cholesterol from mevalonate there has ['HI,'HI ,I4C]methyl; mevalonolactone C, R = ["H,,'4C]methyl been no overall change in the configuration of the methyl group that is attached to C-3 in mevalonate and becomes attached to C-13 in cholesterol. Consequently the intramolecular rearrangement of the to inevalonyl benzhydrylamide), which showed that methyl group from C-15 in (3S)-2,3-oxidosqualene there was no detectable loss of tritium from the C-18 methyl group of oestrone during the oxidation to to C-13 in lanosterol is stereospecific and occurs with acetic acid. Again unless there was a large isotope overall retention of the configuration of the methyl effect against the removal of tritium during the oxidagroup. Since the methyl group at C-18 in lanosterol has arrived there by an intramolecular 1 :2-migration [30] tion, this result was consistent with the expected the stereochemical result is independent of the nature preservation of the chirality of a C-18 chiral methyl group in oestrone, during the isolation of this methyl of any postulated intermediates between (3S)-2,3group as acetic acid. oxidosqualene and lanosterol. As there is indirect (3RS,6S) - [6-2H1,6-3H1,6-14C]Mevalon~lactone evidence [51] for the transient formation of a proto(mevalonolactone A) and (3RS,6R)-[6-2H,,6-2Hl,6lanosterol intermediate (Scheme 1) the chiral methyl ''C]mevalonolactone (mevalonolactone B) were syngroup is envisaged as migrating with retention of thesised from (2S)-[2Hl,'HH,,2-14C] and (2R)-[2Hl,RHI, configuration from C-14 to C-13 in this intermediate. 2-'4C]acetic acids respectively (Scheme 2). The synHowever, the result can be equally well accommodated thetic route was such that the configuration of the by the alternative mode of cyclisation of (3S)-2,3chiral methyl at C-6 in mevalonate was the same as the oxidosqualene which has been proposed by van Tameconfiguration of the chiral methyl in the acetic acid. len et d.[14,52,53] in which the first intermediate is (3RS,6R)-[6-2H,,6-'HH1,6-'4C]Mevalonate (mevapostulated to be a perhydrocyclopentanonapthalene lonolactone B) was converted into cholesterol which carbonium ion that undergoes several rearrangements was degraded to oestrone from which the C-18 methyl to form lanosterol in a way that is consistent with the group was removed as acetic acid (Scheme 3). The detailed information [35] on the migration of the radiochemical data on the intermediates that were hydrogen and methyl groups. 'The details of this isolated during this sequence are tabulated in Table 2. scheme are such that the migrations of the methyl Similarly (3RS,6S)-[6-'H1 ,6-'H1 ,6-14C]mevalonate groups are postulated to occur on a protolanosterol was converted in the same way to give acetic acid. skeleton in which the positive charge is placed on C-13 The radiochemical data on the intermediates of this following the completion of the two hydride migraconversion are shown in Table 3. The 'H/I4C ratios tions.

I6 -

282

Stereochemistry of a Biological Methyl-Group Rearrangement

'k!

3H,j/

'H

,&*'

HO,C (3R,6R)-[6-'Hl

CH'OH

:H, -6-14C1 Mevalonic acid

I

Rat liver enzymes (b) Stereochemial inversion

(a) Stereothemica1retention 3H

'H

'H

3H

Cholesterol

HO

HO I

M. phlei, a,cw'-dipyridyl

Androstadienedione

formation of 17-ethylene ketd Li, biphenyl, diphenylmethane, THF/$

Oastrone

Cr03M,0

R acetic acid

S_ acetic acid

Scheme 3. Formation oj'cholesterolfrom (3R,6R)-[6-2H,,6-3H,,6-'4C]mevalonic acid, show>ingthe configuration of the methyl groups for the case where the C-I8 methyl group has rearranged either ( a ) with retention of configuration or ( b ) with inversion of configuration during the conversion of(3S)-2,3-oxidosqualeneinto lanosterol. The scheme also illustrates the procedure for the isolation of the C-18 methyl group of cholesterol as acetic acid. 0, 14C label

G. T. Phillips and K. H. Clifford

283

Table 1. Radiochemical data on [2-3H,,2-'4C]aceticacid and on samples formed from (3RS)-/6-3H,,6-14C]mevalonale (mevalonolactone C ) Results are given f S.D. Sample

l4c

3H/14C

Atomic ratio

12.5 f 0.0

1.06:l

dis . min-' . mg-' Sodium acetate used for the synthesis of mevalonolactone p-Bromphenacyl acetate, from sodium acetate used for synthesis of mevalonolactone

3392 k 101

11.5 f 0.0)

3417 f I 3402 k 14

11.6& 0.0 11.7k 0.1

'O''

Mevalonolactone Mevalonyl benzhydrylamide, successive recrystallizations

11.9 f 0.1 3970 k 7 3898 f 14

Cholesterol, after chromatography Cholesterol purified via dibromide, successive recrystallizations

924 4 916 k 3 928 5

I

Androsta-l,4-dien-3-one17-ethylene ketal

937 & 3

Androsta-l,4-dien-3-one-17-ethylene ketal, successive recrystallizations

264 f 2

491 & 12 496 f 5

Sodium acetate from oxidation of oestrone p-Bromophenacyl acetate from oxidation of oestrone

110 f 2 111 f 2 109 1

1

11.6k 0.1

0.98: 1 1.01:l

I

11.8f 0.1 11.7 0.1 11.8 k 0.0 11.8 f 0.0

1.00:1

11.6 f 0.1

0.98: 1

11.5 f 0.0 0.98: 1

923 f 6 11.5 k 0.1 11.7

266 f 3 Oestrone, purified on Sephadex LH20

I

493

*

3

110 f 1

0.99: 1

0.1

I * 1

11.7 f 0.1 11.6k 0.1 11.6f 0.1 11.6f 0.1

0.98: 1

11.3 f 0.21 11.2 0.1 11.2f 0.1

0.95: 1

12.4f 0.0

1.05:1

*

12.3f 0.2 12.2f 0.1 12.2 0.1 12.1 0.1

1.03:l

Table 2.Radiochemical data on samples formed from (3RS,6R)-[6-2H,,6-3Hl,6-14C]mevalonate(mevalonolactone B ) Results are given k S.D.

recrystallizations

1796 f 19 1841 f 1 ,1870f 88 2020 f 1 1821 f 30,

11.7 f 0.2 11.4f 0.1 11.5 11.4f 0.0 11.7 f 0.5,

0.2

1.oo:l

284

Stereochernistry of a Biological Methyl-Group Rearrangement

Tablc 3. Radiochrmicul dutu on samples formed from 13RS,6S)-[6-' HI,6-'HH,,6-14C]n~evalonate (mevalonolactoneA ) Results are given f S.D. Sample

14C

3H/14C

Atomic ratio

dis . min-' . mg-' 1.01: 1

Mevalonolactone Mcvalonyl benzhydrylamide, successive recrystallizations

11.5 f 0 .2 1 11.3 k 0.3 11.2 f 0.1 11.3 11.2 0.2

3676 f 6 3591 f 30 3630 f 42 3670 f 64 3624 k 3 4 ,

*

* 0.1

0.98 : 1

Cholesterol, after chromatography Cholesterol purified via dibromide, successive recrystallizations

1.OO:l

1445 f 8 1 1445 1427 f 21 1407 f 14 1412 f 17 J

' }

} * 0.1

O.l 11.1 11.2 f 0.1 11.0 f 0.3)

0.98 : 1

Androsta-l,4-diene-3,17-dione ex chromatography

10.9 f 0.1

0.97 : 1

Androsta-I,4-dien-3-one-l7-~thylcne kctal, PS chromatography

11.2 f 0.2

0.99: 1

Androsta-l,4-dicn-3-one-17-ethylene ketal, successive recrystallizations

698 f 6

11.3 f 0.0

681 f 13

11.4 f 0.3

1.OO:l

Oestrone, purified on Sephadex LH20

4164

Oestrone, after sublimation

4237

10.2 f 0.5

0.90: 1

10.5 f 0.2

0.93: 1

Potassium acetate after steam distilkation

11.6 f 0.4

1.03:l

Potassium acetate after partition chromatography

12.2 f 0.5

1.08:l

Table 4. Retention of tritium from the pro-% position of labelled mulates after equilibration with fumarute hydrutase The malic acids were incubated with fumarate hydratase and samples were taken at different time intervals. Each sample was purified, dissolved in 1.0 rnl of water and assayed for radioactivity. 0,, 0, and 0, are triplicate determinations prior to the equilibration with furnarate hydratase. Results are given f S.D. Malate source

Time

3H/14Cratios ~~~~

actual

average u./

h

R-Acelate viu 18R-[18-ZHl,18-3H1,18-14C]ocstronefrom (3R.S,6R)-[6-2Hl,6-3HH,,h-'4C]mevalonolactone (mcvalonolactone B)

0, 02 03

1 2 3 4 S-Acetate via 18S-[18-ZHl,18-3HH,,18-'4C]oestronc from (3RS,6S)-[6-'H1 ,6-'H1 ,6-'4C]mevalonolactone (mevalonolactone A)

10

2.757 2.649 2.687

I

1.842 1.826 1.897 I

0' 1 2 3 4

0.791 0.849 0.834 0.862

Although the essential structural requirements within substrates for their transformation into lanostcrol have been delineated [54] it cannot be ascertained

2.698

0.055

100

1.838 1

2.581 2.564 2.704

O1 02

'H retained

I

1.851 i- 0.032

2.616 f 0.076

0.834 f 0.031

68.59

100

31.87

what the role of the cyclase enzyme is in the rearrangement of the methyl groups. It is possible that the enzyme plays no part in the migration of methyl

G. T. Phillips and K. H. Clifford

285

c' /I

(VW

2H'H 4-k\

/

(VIII)

Schenic 4. Configiiruiioii of i n ~ u , ~ i n anon-classrcal r~~ carhoniuni ion /rtrn.sition stnie.y ,fiw u cliirul R-niethj'l group during rearratiRemenl ,from ('-14 10 C-13 in (1rotolaiio.s~er01. Transition state (VII) leads to rctention of configuration of the methyl after migration to C-13 and transition state (VIII) leads t o inversion of configuration of thc methyl after migration to C-13

groups per se, although there is no doubt that it initiates and terminates the reaction of which these migrations are an integral part. If the 1 :2 rearrangements that accompany the cyclisation of (3S)-2,3-oxidosqualene into lanosterol involve the transient formation of nonclassical carbonium ions, then our stereochemical rcsult is consistent with the transition state depicted as (VII) resulting in retention of configuration of the chiral methyl during its transfer from C-14 to C-13 for the conversion of a protolanosterol into lanosterol ; the transition state depicted as (VIII) is ruled out as this would lead to inversion (Scheme 4). The intramolecular migration of a chiral methyl group that accompanies the enzymic synthesis of lanosterol proceeds with the retention of configuration of the methyl group and as such is similar to the retention of configuration that has been observed with asymmetric migrating groups in other carbonium ion rearrangements such as the pinacol rearrangement [ 5 5 , 561 and the Wagner-Meenvein rearrangement [57]. We thank Mrs L. Linton for technical assistancc.

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G. T. Phillips and K. H. Clifford: Stereochemistry of a Biological Methyl-Group Rearrangement

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G. T. Phillips and K. H. Clifford, Milstead Laboratory of Chemical Enzymology, Shell Research Ltd, Broad Oak Road, Sittingbourne, Kent, Great Britain ME9 SAG