Synthesis of 25-hydroxy[26,27-3H]vitamin D3 with high specific activity

ANALYTICAL

BIOCHEMISTRY

Synthesis

96, 481-488 (1979)

of 25-Hydroxy[26,27-3H]Vitamin Specific Activity

D, with High

JOSEPH L. NAPOLI, MARY A. FIVIZZANI, ALAN J. HAMSTRA, HEINRICH K. SCHNOES, AND HECTOR F. DELUCA Depurtment

of Biochemistry, Wisconsin-Madison,

College of Agricultural and Life Sciences, University 420 Henry Mall, Madison, Wisconsin 53706

of

Received December 6, 1978 A synthesis of radiochemically pure 25-hydroxy[26,27-3H]vitamin D, with a specific activity of 160 Wmmol is reported. The structure and biological activity of the radiolabeled compound was verified by comigration on high-pressure liquid chromatography with synthetic 2Shydroxyvitamin D, to constant specific activity, and by conversion in vitro to la,25-dihydroxy[26,27-3H]vitamin D, with the chick kidney la-hydroxylase.

The availability of a variety of radiolabeled vitamin D derivatives (l-8) was essential to the demonstration that vitamin D3 is a prohormone that is sequentially metabolized to the hormone, la,25-dihydroxyvitamin D3 (la,25-(OH),D,)’ (9- 11). Current insight into the nature of vitamin D action and the manner by which its metabolism is regulated also was contingent upon the accessibility of labeled vitamin D derivatives. However, with the sole exception of 25-hydroxy[23,24-3H]vitamin Da, the radiolabeled compounds used were of modest specific activity (0.2 to 10.6 Ci/mmol). But 25-hydroxy[22,23-3H]vitamin D, (78 Ci/mmol)2 was obtained by a fairly lengthy and relatively difficult synthesis which reI Abbreviations used: la,25-(OH)2D,, la,25-dihydroxyvitamin DS; 25-OH-D,, 25-hydroxyvitamin D,; hplc, high-pressure liquid chromatography; buffer A, 150 mM Tris-HCI, 300 mM KCI, 1.5 mM EDTA, and 0.5 mM dithiothreitol, pH 7.4; PPO, 2,5-diphenyloxazole; POPOP, 1,4-bis-2-(4-methyl-5-phenyl-oxazolyl)benzene. 2 A subsequent preparation of 25-hydroxy-[23,24-3H]vitamin D,, by a method very similar to that reported in (8) gave material with a specific activity of 92 Ci/ mmol (see, Muccino, R. R., Vemice, G. G., Cupano, J., Oliveto, E. P., and Liebman, A. A., (1978) Steroids 31, 645-652).

quired seven synthetic steps after introduction of the tritium (8). Thus, there existed a need for a more convenient route to radiolabeled 25-hydroxyvitamin D3 (25-OH-D3) of high specific activity. This paper reports a short, facile synthesis of 25-hydroxy[26,27-3H]vitamin D3 with a specific activity of 160 Ci/mmol, and demonstrates its conversion in vitro to la,25-dihydroxy[26,273H]vitamin D,. The very highly radioactive vitamin D3 metabolites obtained by this route should be useful in continued investigations of vitamin D, function, bindingprotein studies, target-tissue receptor isolation and characterization, autoradiographic studies, and further investigation into vitamin D, metabolism. METHODS General procedures. Ultraviolet absorbance (uv) spectra were taken in ethanol with a Beckman Model 24 recording spectrophotometer. Nuclear magnetic resonance (nmr) spectra were obtained in CDC13 with a Bruker WH-270 spectrometer. Mass spectra were obtained at 100°C above ambient with an MS-9 mass spectrometer coupled to a DS-50 data system. High-pressure liquid chromatography (hplc) was done 481

0003-2697/79/100481-08$02.00/O Copyright All rights

0 1979 by Academic Press, Inc. of reproduction kn any form reserved.

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NAPOLI

with a Waters Associates Model ALCl GPC-204 liquid chromatograph. Irradiations were done in a quartz reaction vessel with a 125 watt Hanovia 8A36 lamp fitted with a Corex filter. Radioactivity was measured with a Packard Model 3255 liquid scintillation counter, except for the binding-protein experiment for which a Beckman LS-1OOC liquid scintillation counter was used. Homocholenic acid methyl ester 3-benzoate was a gift from the Upjohn Company, Kalamazoo, Michigan. Crystalline 25OH-D3 and lc~,25-(OH)~D~ were gifts from the Hoffmann-LaRoche Company, Nutley, New Jersey. Sephadex LH-20 was purchased from Pharmacia Chemicals, Piscataway, New Jersey. Lipidex 5000 is a 50% saturated hydroxyalkoxypropylation product of Sephadex with an average alkoxy group chain length of 15 carbons. It was purchased from Packard Instruments Inc., Downers Grove, Illinois.

ET AL.

methanolic potassium hydroxide (5 ml). After 2.5 h at room temperature, water and ether were added, and the organic phase was separated and washed repeatedly with water. The solvent was evaporated and the residue was chromatographed on a silica gel column (1 x 15 cm) eluted with 25% ethyl acetateihexane to give 2 (0.08 g, 0.2 mmol): uv 294, 282, 272, 264 (shoulder) nm: nmr 6 0.67 (s, 18-CH,), 1.01 (d, J = 6.5 Hz, 21CH& 1.04 (s, 19-CH& 3.72 (s, -CO,CH,), 5.43, 5.64 (m, 6H, 7H). Methyl 26,27-dinor-9,10-secocholesta5,7,10(19)-trien-25oate (4). A solution of

2 (28 mg) in 20% ethanol/benzene (150 ml), under nitrogen, cooled in an ice bath, was irradiated for 7.5 min. The solvents were evaporated and the residue was purified by hplc (0.6 x 25-cm microparticulate silica gel column developed with 1.5% 2-propanol/ hexane) to give 5,7-diene 2 (23 mg) and previtamin 3 (5 mg): uv h,,, 260, hmfn 233 Methyl 3/$Hydroxy-25-homo-5,7-cholnm, E,,,/E,in 1.5. adien-25oate (2). Methyl 3P-hydroxy-25Previtamin 3 was heated at reflux under homo-5-cholen-25-oate 3-benzoate (0.5 g, nitrogen in ethanol for 4.5 h to give cor0.99 mmol), sodium bicarbonate (0.55 g, responding vitamin 4: uv A,,, 264, Amin 228, Emax/Emm 1.8; nmr 0.54 (s, 18-CH3), 0.94 6.5 mmol), and 1,3-dibromo-5,5-dimethylhydantoin (0.16 g, 0.56 mmol) in hexane (d, J = 6.2 Hz, 21-CH,), 3.67 (s, -CO&H,), (10 ml) were heated under nitrogen for 20 3.94 (m, 3a-H), 4.82, 5.05 (2 m, 19-H’s), min at 80°C. The reaction mixture was cooled 6.03, 6.23 (ABq, J = Hz, 6H, 7H); mass and filtered. The residue obtained after spectrum m/e (relative intensity) 400 (0.85, evaporating the solvent from the filtrate was M+), 382 (0.07, M+ -H,O), 369 (0.09, M+ -OCH,), 367 (0.35, M+ -H,O-CH,), 341 dissolved in a solution of 2,4,6-trimethylpyridine (1 ml) in xylene (10 ml) and was (0.10, M+ -CO&H,), 271 (0.16, M+ -side heated at reflux under nitrogen for 1.5 h. chain), 253 (0.21, M+ -H,O-side chain), 166 (0.31), 158 (0.76), 136 (l.OO), 118 (0.91). The reaction mixture was cooled, diluted Compound 4 was homogeneous in hplc. with benzene, and washed successively 25-Hydroxy[26,27-3H]vitamin D, (5). with 1 N HCI, dilute NaHCO,, and water. Methyl ester 4 (1 .O mg, 2.5 mmol) was treated The solvent was removed, and the residue with C3H3MgBr (80 Ci/mmol) in the laborawas dissolved in a solution of p-toluenesulfonic acid (0.06 g) in dioxane (12 ml), tories of New England Nuclear, Boston, Massachusetts. After work-up,3 the radioand heated at 70°C for 40 min. After cooling the reaction mixture, water and ether were labeled materials (3 14 mCi) were purified by added. The phases were separated, and the 3 The actual Grignard reaction and its workup were organic phase was washed with dilute sodium performed in the laboratories of New England Nuclear, bicarbonate, water, and brine. The solvent Boston, Mass. Purification of 25-OH-[26,27-3H]vitamin was removed and the residue was dissolved D, and all other reactions and experiments were in a mixture of ether (3.0 ml) and 0.4 M conducted in our laboratories.

SYNTHESIS

OF 25-HYDROXY[26,27-3H]VITAMIN

D,

483

successive chromatography through col- spectively), suspended in buffer A was umns of Sephadex LH-20 (2 x 50 cm) de- added with good stirring. After 10 min the veloped with chloroformlhexane (1: l), and tubes were centrifuged at 2300g for 10 min. Lipidex 5000 (1 x 50 cm) developed with A 0.5-ml aliquot of the supernatant was determination in chloroform/hexane (1: 1) to give 32 mCi of used for radioactivity scintillation fluid consisting of 1.32 liter 25-hydroxy[26,27-3H]vitamin D,. An aliquot of the 25-hydroxy[26,27-3H]vitamin D3 (160 Triton X-100, 8.0 g 2,5-diphenyloxazole Ci/mmol) thus purified was found to be at (PPO), and 0.2 g 1,4-bis-2-(4-methyl-5phenyl-oxazolyl)benzene (POPOP) per least 96% pure by hplc. la,25-Dihydroxy[26,27-3H]vitamin D3 4 liters toluene. Counting efficiency was 26%. (6). 25-Hydroxy[26,27-3H]vitamin D3 5 RESULTS AND DISCUSSION (3 mCi, 8 pg) was treated with a vitamin D-deficient chick kidney homogenate (1 ml The synthesis of 25-hydroxy[26,27-3H]homogenatelpg substrate) as previously vitamin D3 started with homocholenate 1 described (12,13). Incubations were carried (Fig. 1). Diester 1 was brominated at carbon out for 1 h at 37°C. After the usual workup, 7 with dibromatin in a modified Hunzikerthe radiolabel recovered was purified by Miillner procedure (16) and dehydrobromichromatography on a Sephadex LH-20 nated with 2,4,6-collidine to give a mixture column (1 x 60 cm) developed with chloroof 4,6- and 5,7-diene diesters. To facilitate formlhexane (65:35). la,25-dihydroxychromatographic separation of the two [26,27-3H]vitamin D3, 6 (1.5 mCi) eluted dienes, the crude reaction mixture was from 160 to 200 ml. Its radiochemical purity treated with p-toluenesulfonic acid which and identity were established by high-prescaused elimination of the 3-ester from the sure liquid cochromatography of 6 and an allylic 4,6-diene, but not from the homoauthentic sample of synthetic la,25-(OH)*D,. allylic 5,7-diene.” Selective hydrolysis of Further evidence for the structure of 6 was the 3-ester in the 5,7-diene provided a crude provided by the observed competition of reaction mixture containing 2, and the 6 and synthetic la,25-(OH),D, for the elimination product of the 4,6-diene which la,25-(OH),D-specific, chick-intestinal-cywere easily separated by silica gel chromatosol binding protein. tography . Thus, provitamin-like structure 2 Binding protein experiment. Competitive could be obtained from 1 in four relatively displacement of 1a,25-dihydroxy[26,27-3H]easy steps with minimal workup and no vitamin D3 by synthetic la,25-(OH)*D3 intermediate purification. The uv spectrum from the la,25-(OH),D,-specific, chickof 2 was typical of steroidal 5,7-dienes with intestinal cytosolic binding protein was a series of peaks at 264,272,282, and 294 nm. observed by a previously described experiThe structural assignment was further supmental protocol (14,15) with modifications ported by the appearance of two multiplets introduced by Dr. William S. Mellon. Unin the nmr spectrum at 65.43 and 5.64 relabeled lcu,25-(OH),D3 and la,25-dihydroxysulting from the resonance of the 6 and [25,26-3H]vitamin 4 (8400 dpm) were added 7 protons. to glass tubes (12 x 75 mm) in 50 ~1 ethanol. Brief irradiation of 2 provided a mixture Chick-intestinal cytosolic protein was added of 2 and previtamin-like structure 3, which in buffer A (150 mM Tris-HCI, 300 mM was easily resolved by hplc. By limiting KCl, 1.5 mM EDTA, and 0.5 mM dithiophotolysis time, over-irradiation of 3 was threitol, pH 7.4) to make the final incubation avoided, and 2 reclaimed from the irradiavolume 0.5 ml. After incubation, assay tubes were placed on ice and dextran-coated ’ Thomas Narwid, Hoffmann-La Roche, Inc., charcoal (0.25 ml, 0.05 and 0.5% w/v, re- N utley , N J , personal communication.

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ET AL.

FIG. 1. Chemical synthesis of 25-hydroxy[26,27-3H]vitamin dihydroxy[26,27-3H]vitamin D,, 6.

tion mixture could be recycled. Longer photolysis times resulted in a greater initial yield of 3, but also caused the formation of by-products, at the ultimate expense of 2, since a steady-state concentration of 3 was reached. Therefore, a more efficient conversion of 2 to 3 was realized by repeated short irradiations of 2 followed by recovery of 3, rather than by a single long irradiation. The 3 obtained displayed the characteristic previtamin uv spectrum with an absorbance maximum at 260 nm and a minimum at 233 nm. The ratio Emax/Emm of 1.5 indicated that the compound was pure. Thermal rearrangement of 3 provided the vitamin-like structure 4, whose purity was established by hplc. The vitamin D3 analog

Dfr 5; and biological synthesis of la,25

4 showed the usual vitamin D uv absorbance resulting from the cis-triene chromophore with a maximum at 265 nm and a minimum at 228 nm. The ratio of 1.8 for Emax/Emmfurther indicated that 4 was pure. The nmr spectrum of 4 displayed all the expected signals from a vitamin D-like structure, with the exception of those which would be caused by the presence of carbons 26 and 27, of course. Instead an intense singlet at 63.67 caused by the protons on the methyl group of the 25ester were observed. A mass spectrum, with an intense molecular ion at 400 (85% of base peak height), completed the structural assignment. Peaks due to loss of -OCH, (m/e 369) and carboxymethyl (m/e 341) were observed besides the peaks arising

SYNTHESIS

OF 25-HYDROXY[26,27-3H]VITAMIN

D3

485

where 25-hydroxy[23,24-3H]from side-chain cleavage (m/e 271), and fractions fragmentation between carbons 7 and 8. vitamin D3 had been collected previously. The latter fragmentation gave the base peak This material was then chromatographed fragment (m/e 136) corresponding to the on a Lipidex 5000 column which resolved A-ring plus carbons 6 and 7. Loss of water the radioactivity into two major peaks, as well as several minor ones. from m/e 136 produced another major peak The less polar major peak (32 mCi) was (91% of m/e 136) at m/e 118. The two peaks identified as 25-hydroxy[26,27-3H]Vitamin at m/e 136 and 118 are diagnostic for the cis-triene system present in vitamin D-like D3, 5, of at least 96% purity by comigration compounds, whereas the peaks at m/e 400, with authentic 25-OH-D, in hplc (Fig. 2). 369, 341, and 271 indicate the presence of a A purified aliquot of 25-hydroxy[26,27-3H]side-chain ester. vitamin D3 (104,500 dpm) was then diluted The last synthetic step was introduction of with 15 ,ug (quantitated by uv absorbance) the tritium by Grignard reaction of PH,MgBr of 25-OH-D, to give a final specific activity (80 Wmmol) with ester 4. This reaction gave of 6967 dpm/pg. This material was chronot only the desired product 5 with a specific matographed in an hplc system capable of activity of 160 Wmmol since two C3HS- resolving all known vitamin D metabolites groups were incorporated, but a number of and isomers from 25-OH-D, (17). The 25other radiolabeled compounds were not OH-D3 recovered (Fig. 3) had not changed characterized. 25-Hydroxy[26,27-3H]vitain specific activity giving further evidence min D,, 5, was separated from the radiofor the structural assignment and purity active by-products by two chromatographic of the radiolabel. steps. A Sephadex LH-20 column was caliThe bioactivity of, and confirming evibrated with 25-hydroxy[23 ,24-3H]vitamin dence for, the identity of 25-hydroxy[26,27D, (8), and then the crude reaction mixture 3H]vitamin D,, 5, was obtained by the conwas chromatographed. Of the 314 mCi version of S to la,25-dihydroxy[26,27-3H]applied to the column, about 103 mCi were vitamin D3, 6, in vitro by a chick kidney collected as a discrete peak in the same homogenate (Fig. 1). The material obtained

Fraction

number

( I ml each)

FIG. 2. The hplc of 25-hydroxy[26,27-3H]vitamin D, (Q) recovered from the Lipidex 5000 cblumn. A mixture of 25-hydroxy[26,27-3H]vitamin D, and synthetic 25OH-D, were injected together onto a microparticulate silica gel column (0.46 x 25 cm) eluted with 4% 2-propanoYhexane. At least 96% of the radioactivity migrated with the authentic 25-OH-D, (uv at 254 nm ( )).

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ET AL.

-12

-6

Fraction

number

(I.Oml

each)

FIG. 3. High-pressure liquid cochromatography of25-hydroxy[26,27-3H]vitamin D3 (m) and authentic 25OH-4 ( ). 25OH-D, (15 pg) and 25-hydroxy[26,27-3H]vitamin D, (104,500 dpm; 160 Ci/mmol), to give a final specific activity of 6967 dpm/wg, were coinjected onto a microparticulate silica gel column (0.45 x 25 cm) and eluted with 4% 2-propanohhexane at a flow rate of 1 mumin. Virtually 100% of the radioactivity was recovered in the 25-OH-D3 area. The specific activity of the recovered material was 6903 dpmlpg.

from incubations of 5 with the kidney enzymes was chromatographed on a Sephadex LH-20 column which easily resolved unreacted 5 from product 6. The identity of

Fraction

6, which was obtained established in two ways. graphed with synthetic hplc (Fig. 4). Second,

number

( I.Oml

in 50% yield, was First 6 cochromatola,25-(OH)2D, on 6 competed with

each)

FIG. 4. The hplc of la,25-dihydroxy[26,27-3H]vitamin D, (cpm, m) with authentic la,25-(OH),D, (uv, -). The materials were coinjected onto a microparticulate silica gel column (0.45 x 25 cm) developed with 10% 2-propanol/hexane at a flow rate of 2 mUmin. Greater than 95% of the radiolabeled material migrated with synthetic h1,25-(0H)~D~.

SYNTHESIS

OF 25-HYDROXY[26,27-3H]VITAMlN

la,25Dihydroxyvitomin

D:x

487

D3 (pq)

FIG. 5. Displacement of la,25-dihydroxy[26,27-*HIvitamin D, (160 Ci/mmol) from the chick-intestinal-cytosol binding protein by la,25-(OH)2D,. Percentage specific binding of the tritiated analog (ordinate) is plotted versus log pg of unlabeled lcz,25-(OH),D3 (absissa). The values are mean + SD from triplicate determinations. Linear regression analysis in the 4 to 50 pg range of 1,25-(OH),D$ indicated a very high inverse linear correlation (r > 0.99) between percentage specific binding of labeled hormone and concentration of unlabeled hormone.

synthetic la,25-(OH),D3 for the chick-intestinal binding protein specific for lq25(OH),D, (14,15,18). Figure 5 shows the wellestablished displacement curve produced by competition of increasing concentrations of unlabeled lc~,25-(OH)~D~ for binding sites, on the chick cytosol receptor, previously saturated with a fixed amount of radiolabeled la,25-(OH),D,. This newest route for the synthesis of 25-hydroxy[26,27-3H]vitamin D3 provides the highest specific activity of radiolabeled vitamin D, metabolites known, and offers several advantages over other routes. The main advantage is introduction of the label in the last synthetic step, thereby avoiding the problems inherent in a multistep conversion of radiolabeled intermediates. The synthesis of precursor 4 takes advantage of well-established vitamin D chemistry, and can be accomplished in a very short time. After introduction of the label, two chromatographic steps are sufficient to provide material that is 96% pure. Synthesis of labeled 25-OH-D3 by this route also avoids 3H-scrambling that may have occurred in our previous synthesis of 25-hydroxy[23,243H]vitamin D,. Moreover, the route reported

here is amenable to the introduction of 14C, 2H, and 13C. Although the yield of 32 mCi from 2.5 pmol of precursor is modest, it certainly provides enough radioactivity to support a vast number of studies. The relative ease by which the label is obtained, outweighs the consideration of a modest yield. It is likely that the yield would be greater, if larger amounts of 4 were available. The ester vitamin 4 is also potentially interesting as a vitamin D analog in its own right. This previously unknown compound could represent an intermediate in the sidechain degradation of vitamin D and its metabolites. Biological evaluation of 4 is in progress. ACKNOWLEDGMENTS We thank Dr. Norman Silberman (New England Nuclear, Boston, Mass.) and his colleagues for performing the Grignard reaction, Mr. Sean Hehir for technical assistance with the NMR spectra, and Mr. Mel Micke for technical assistance with the mass spectra. This work was supported by the National Institutes of Health Program Project Grant AM-14881 and Postdoctoral Fellowship DE-07031 and the Harry Steenbock Research Fund.

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NAPOLI

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ET AL.

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