Two new convenient syntheses of 14C-squalene from turbinaric acid

Article abstract of DOI:10.1002/jlcr.3672

Carbon-14 labeled (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-hexaene, also known as squalene, was synthesized as a tool for pharmacokinetic studies. Two simple and efficient labeling approaches were developed to give [2-¹⁴

Full text of DOI:10.1002/jlcr.3672

Two new convenient syntheses of 14C-squalene from turbinaric acid
Authors information
Nicolas Philippea*, Luc Rivrona, Béatrice De Bruina, Joseph Schofieldb and Sébastien Roya
a Isotope Chemistry, Integrated Drug Discovery, Sanofi-Aventis R&D, Vitry-sur-Seine,
France
b Pharmacokinetics, Dynamics and Metabolism, Translational Medicine and Early
Development, Sanofi-Aventis R&D, Alfortville, France
*Correspondence to: Nicolas Philippe, Isotope Chemistry, Integrated Drug Discovery, Sanofi-
Aventis R&D, 13, quai Jules Guesde 94403 Vitry-sur-Seine, France, E-mail:
nicolas.philippe-ICMS@sanofi.com
Abstract
Carbon-14 labelled (6E,10E,14E,18E)-2,6,10,15,19,23-hexamethyltetracosa-2,6,10,14,18,22-
hexaene, also known as squalene, was synthesized as a tool for pharmacokinetic studies. Two
simple and efficient labelling approaches were developed to give [2-14C]-Squalene and [3-
14C]-Squalene from a halogenated precursor derived from turbinaric acid. They were obtained
in 13.5% radiochemical yield in 6 steps and in 38% radiochemical yield in 3 steps
respectively from carbon-14 labelled potassium cyanide with a radiochemical purity higher
than 98% in both cases.
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Introduction
Squalene 1 is a triterpenoid aliphatic hydrocarbon, naturally produced by all plants and
animals including humans (figure 1), for which it is an essential biosynthetic precursor of
cholesterol. It is also used for cosmetic purposes, as a dietary supplement and as an adjuvant
in vaccines. In addition, 1 shows protective and preventive effects in cancer and in the
treatment of other diseases1. For potential pharmaceutical applications, carbon-14 labelled
squalene would be a useful radioactive probe for pharmacokinetic studies.
To our knowledge, only a very few syntheses of isotopically labelled squalene and its
derivatives have been published: the synthesis of [11, 12-14C2]-squalene and of [1-14C]-2,3-
oxidosqualene was reported by Pichard et al.2,3 in 1958 and in 1973, the preparation of
[24,30-14C]-2,3-oxidosqualene was described by Prestwich et al.4 in 1991, a convenient
synthesis of [3-3H]-squalene was published by L.Cattel et al.5 in 1994 and the preparation of
-di-(trideuteromethyl)-squalene was reported by D. Desmaële et al.6 in 2016. We now
describe simple syntheses of [14C]-squalene 2 and 3, labelled at two specific positions (C-2)
and (C-3) respectively, starting from turbinaric acid7 4, readily available in high purity within
our company. The latter 4 was obtained in 4 steps from commercially available squalene 1
according to the synthesis described by J. Mann et al.8. The synthesis of [2-14C]-squalene 2
was performed using a novel approach whereas the synthetic route leading to [3-14C]-
squalene 3 was developed using a Wittig reaction at the last step, as described in the
references above.
Results and discussion
For the synthesis of [2-14C]-Squalene 2 (scheme 1), turbinaric acid 4 was reduced to the alcohol
5 with lithium aluminium hydride in 90% yield. Conventional iodination of 5 with iodine in the
presence of triphenylphosphine and imidazole resulted in the corresponding halogeno
intermediate 6 in good yield. The latter was submitted to cyanation with carbon-14 potassium
cyanide in DMSO to give the [14C]-nitrile 7 in 94% radiochemical yield. 7 was then treated with
an excess of methyl lithium to give the ketone 8 in only 25% yield although during unlabelled
development work, this step proceeded in an excellent 90% yield. 8 was transformed to tertiary
alcohol 9 with methylmagnesium iodide in 90% radiochemical yield. At this point, an alternative
synthetic route was developed to increase the overall yield of 9 from the [14C]-cyano compound
7. The first step consisted of hydrolyzing 7 under strong alkaline conditions to obtain the
corresponding acid 10 in 94% yield. 10 was then activated with 1,1'-carbonyldiimidazole and
reacted with methanol to give methyl ester 11 in 92% yield. 11 was finally treated with an
excess of methylmagnesium iodide to give 9 in 75% radiochemical yield over 3 steps. In the last
step, 9 was dehydrated with Burgess reagent to give both expected elimination products: [2-
14C]-squalene 2 and terminal ethylenic isomer 12. Proton NMR analysis showed that the 2/12
ratio was disappointingly poor (55/45). So, different methods to remove the unwanted isomer 12
were tested such as its degradation by 9-BBN9 or purification on classical semi-preparative or
10
specific Silica-gel-AgNO3 columns. Only the latter allowed us to efficiently recover over half
of the radioactivity of [2-14C]-squalene 2 with a radiochemical purity higher than 98% after
multiple chromatographic runs. NMR analysis revealed a contamination of [2-14C]-squalene 2
with significant amounts of butylated hydroxytoluene (BHT) coming from solvents used during
the synthesis, however this was easily removed through a final purification on a RP18 column to
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give the [2-14C]-squalene 2 in 13.5% overall radiochemical yield from carbon-14 potassium
cyanide.
A second approach, quicker and more convenient, was developed to obtain [3-14C]-squalene 3
from turbinaric acid 4 as depicted in scheme 2. 4 was bromodecarboxylated under Barton’s
conditions11 to give the bromo derivative 13 in 34% yield. 13 was converted into the radioactive
nitrile 14 with carbon-14 potassium cyanide in dimethylsulfoxide in 58% yield then reduced with
diisobutylaluminium hydride solution to aldehyde 15. Finally, 15 was reacted with the ylide of
isopropyltriphenylphosphonium bromide using the conditions described by L. Cattel et al.5 to
give [3-14C]-squalene 3 in 65% radiochemical yield in 2 steps and a radiochemical purity higher
than 98%.
Experimental details
General:
Carbon-14 labelled potassium cyanide (specific activity 2109MBq/mmol) was purchased from
Tjaden Bioscience, (Burlington, IA, USA). Pure turbinaric acid was obtained from the
Chemical Development department of Sanofi R&D. All reagents and solvents were purchased
from commercial suppliers and used without further purification.
All experimental procedures were optimized using unlabeled materials. Air and moisture
sensitive reactions were conducted under an inert atmosphere of nitrogen and were magnetically
stirred. Reactions were monitored by HPLC and thin layer chromatography (TLC) which was
performed on 60 F254 silica gel plates. Radiolabeled products were compared with authentic
materials. Merck silica radio-TLC plates were analyzed and quantified by electronic
autoradiography using a Packard Instant imager. Plates were exposed to iodine vapor to show
the correspondence between radiolabeled and unlabeled products. Quantification of radioactivity
was determined using Ultima Gold as liquid scintillation cocktail on a Perkin Elmer TRI-CARB
® 2900TR liquid scintillation counter.
Proton NMR spectra were recorded on a Bruker Avance 600 spectrometer in the stated
deuterated solvent.
HPLC analyses were performed on a Shimadzu Prominence UFLC system equipped with a
diode array UV detector and a Packard Radiomatic 500TR series flow scintillation analyser. The
following chromatographic conditions were used: Akzo Nobel Kromasil C18 (5µm particles –
250mm length x4.6mm internal diameter) column, isocratic elution at room temperature, mobile
phase: acetonitrile with 5% water, flow rate: 1ml/min, UV wavelength: 220nm.
12
Preparation of the Silica – AgNO3
:
900g of silica gel (Art Merck 9385) were dried in an oven at 200°C for 8 hours. 150g of this
silica were poured in amber 250ml round bottom flasks and 30g of AgNO3 in 40ml of water
were added slowly with vigorous stirring between each addition. Place the flask in the rotary
evaporator and rotate for 1 hour under atmospheric pressure and room temperature. The silica–
AgNO3 gel thus obtained was stocked in amber flasks under argon in darkness.
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EXPERIMENTAL PROCEDURE
Radiosynthesis of [2-14C]-squalene (2)
(4E,8E,12E,16E)-4,8,13,17,21-pentamethyldocosa-4,8,12,16,20-pentaenol (5)
A stirred solution of turbinaric acid 4 (1.60g, 4mmol) in diethyl ether (250ml) was treated
slowly with LiAlH4 (0.25g, 6.6mmol) at 0°C and the mixture was stirred for 1 hour at room
temperature. After cooling to 0°C, a saturated aqueous solution of Na2SO4 was added to destroy
excess hydride. The organic solution was poured into a separating funnel and extracted twice
with diethyl ether (150ml). The combined organic solution was dried over MgSO4, filtered and
concentrated under vacuum. The oil was dissolved in a mixture of n-pentane/diethyl ether (1:1,
v/v) (4ml) and purified by chromatography on a silica gel column eluted with a mixture of n-
pentane/diethyl ether (6:4, v/v) to afford 5 (1.39g, 90%) as colorless oil. TLC (SiO2, diethyl
ether/n-pentane (6:4, v/v), Rf = 0.35).
1H-NMR (600 MHz, CDCl3) δ 5.08 to 5.20 (m, 5H), 3.64 (t, J=6.42 Hz, 2H), 2.04 to 2.13 (m,
8H), 1.96 to 2.04 (m, 10 H), 1.69 (s, 3H), 1.68 (m including J=6.40 Hz, 2H), 1.63 (s, 3H),
1.61 (m, 12H)
(6E,10E,14E,18E)-22-iodo-2,6,10,15,19-pentamethyldocosa-2,6,10,14,18-pentaene (6)
A stirred solution of 5 (1.35g, 3.5mmol) in dichloromethane (200ml) was treated with imidazole
(0.255g, 3.75mmol), triphenylphosphine (0.984g, 3.75mmol) and iodine (0.965g, 3.8mmol). At
the end of the addition, the reaction was allowed to stir at room temperature for 1 hour. Water
(100ml) was added and the organic layer separated, washed again with water (100ml), dried
over MgSO4, filtered and concentrated under vacuum. The residue was taken up in a minimum
of pentane and purified by chromatography using a silica gel column eluted with n-pentane to
afford 6 (1.56g, 90%). TLC (SiO2, diethyl ether/n-pentane (15:85, v/v), Rf = 0.85).
1H-NMR (600 MHz, CDCl3) δ5.09 to 5.21 (m, 5H), 3.15 (t, J=7.02 Hz, 2H), 2.05 to 2.13 (m,
8H), 1.97 to 2.05 (m, 10H), 1.92 (quint, J=7.02 Hz, 2H) 1.70 (s, 3H), 1.62 (m, 3H), 1.61 (s,
9H), 1.60 (s, 3H)
[1-14C]-(5E,9E,13E,17E)-5,9,14,18,22-pentamethyltricosa-5,9,13,17,21-pentaenenitrile (7)
A stirred suspension of [14C]-Potassium cyanide (3.7GBq, 1.75mmol) in dimethylsulfoxide
(15ml) was treated with a solution of 6 (1g, 2mmol) in dimethylsulfoxide (5ml) and the mixture
stirred at room temperature overnight. Water (100ml) was added and the product was extracted
twice with diethyl ether (150ml). The combined organic solution was dried over MgSO4, filtered
and concentrated under vacuum. The residue was taken up in a minimum of diethyl ether/n-
pentane (1:1, v/v) and purified by chromatography using a silica gel column eluted with diethyl
ether/n-pentane (15:85, v/v) to afford 7 (3.478GBq, 94% radiochemical yield) as colorless oil.
TLC (SiO2, diethyl ether/n-pentane (15:85, v/v), Rf = 0.80).
[2-14C]-(6E,10E,14E,18E)-6,10,15,19,23-pentamethyltetracosa-6,10,14,18,22-pentaen-2-
one (8)
A stirred solution of 7 (3.14 GBq, 1.5 mmol) in anhydrous diethyl ether (50ml) was treated
slowly with a 1.6M solution of methyl lithium in diethyl ether (1.5ml, 2.4mmol) at 0°C. The
mixture was stirred at room temperature for 2h. The flask was cooled in an ice bath and treated
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with a saturated aqueous solution of NH4Cl (20ml). The product was extracted twice with
diethyl ether (20ml). The combined organic layers were dried over MgSO4, filtered and
concentrated. The residue was taken up in a minimum of pentane and purified by
chromatography using a silica gel column eluted with diethyl ether/n-pentane (25:75, v/v) to
afford 8 (787.5 MBq, 25% radiochemical yield) as a colorless oil. TLC (SiO2, diethyl ether/n-
pentane (25:75, v/v), Rf = 0.75)
[2-14C]-(6E,10E,14E,18E)-2,6,10,15,19,23-Hexamethyltetracosa-6,10,14,18,22-pentaen-2-ol (9)
From Ketone compound (8)
A stirred solution of 8 (787.5 MBq, 0.37mmol) in anhydrous diethyl ether (20ml) was treated
slowly with a 3M methyl magnesium iodide solution in diethyl ether (0.3 ml, 0.9mmol) at 0°C.
The mixture was stirred at room temperature for 1h. The flask was cooled in an ice bath and
treated with a saturated aqueous solution of NH4Cl (10ml). The product was extracted twice
with diethyl ether (15ml). The combined organic phases were dried over MgSO4, filtered and
concentrated. The residue was dissolved in a minimum of n-pentane and purified by
chromatography using a silica gel column eluted with diethyl ether/n-pentane (from 1:1 to 8:2)
to afford 9 (708.8 MBq, 90% radiochemical yield) as a colorless oil. TLC (SiO2, ethyl acetate/n-
pentane (7:3, v/v), Rf = 0.30)
From ester compound (11)
A stirred solution of 11 (3.071GBq, 1.4mmol) in anhydrous diethyl ether (80ml) was treated
slowly with a 3M methyl magnesium iodide solution in diethyl ether (1.5ml, 4.5mmol) at 0°C.
The mixture was stirred at room temperature overnight. The flask was cooled in an ice bath and
treated with a satured aqueous solution of NH4Cl (50ml). The product was extracted twice with
diethyl ether (100ml). The combined organic phases were dried over MgSO4, filtered and
concentrated. The residue was dissolved in n-pentane and purified by chromatography using a
silica gel column eluted with diethyl ether/n-pentane (from 1:1 to 8:2, v/v) to afford 9
(2.664GBq, 86.7% radiochemical yield) as a colorless oil. TLC (SiO2, ethyl acetate/n-pentane
(7:3, v/v), Rf = 0.30)
[1-14C]-(5E,9E,13E,17E)-5,9,14,18,22-pentamethyltricosa-5,9,13,17,21-pentaenoic acid (10)
A stirred solution of 7 (3.478 GBq, 1.65mmol) in ethanol (35ml) was treated slowly with a
solution of potassium hydroxide (14g, 0.24mol) in water (35ml). The mixture was heated under
reflux for 6h. The reaction was monitored by HPLC (Rt acid: 9.7 min - Rt amide: 10.6 min
according to the above conditions).The reaction was allowed to cool to room temperature
overnight and concentrated under vacuum. The residue was dissolved in water (150ml) and
acidified by the addition of citric acid to pH= 5. The mixture was extracted twice with ethyl
acetate (150ml). The combined organic solution was dried over MgSO4, filtered and
concentrated in vacuum.
The residue was dissolved in ethyl acetate (4ml) and purified by chromatography using a silica
gel column eluted with a mixture of ethyl acetate/n-pentane (from 5:5 to 8:2, v/v) to afford 10 as
a colorless oil. Radio-TLC analysis showed 94% radiochemical yield. TLC (SiO2, ethyl
acetate/n-pentane (6:4, v/v), Rf = 0.35). HPLC (Rt acid: 9.7 min)
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[1-14C]-(5E,9E,13E,17E)-5,9,14,18,22-Pentamethyltricosa-5,9,13,17,21-pentaenoic acid methyl ester
(11)
N,N-Carbonyl diimidazole (0.324g, 2mmol) was added to
a
solution of 10
(3.304GBq,1.56mmol) in tetrahydrofuran (50ml). The mixture was refluxed under stirring for
1h. The reaction was monitored by HPLC (Rt amide: 12.6min according to above conditions).
When no more acid was detected by HPLC, methanol (20ml) was added and the reflux
maintained for 4h. The esterification was monitored by HPLC (Rt amide: 12.6min - Rt ester:
14.6min according to above conditions). The reaction was allowed to cool to room temperature
and concentrated in vacuum. Diethyl ether (200ml) was added and washed twice with water
(150ml). The organic solution was dried over MgSO4, filtered and concentrated in vacuum. The
residue was taken up in a minimum of pentane and purified by chromatography using a silica gel
column eluted with n-pentane to afford 11 (3.071GBq, 92% radiochemical yield) as oil. TLC
(SiO2, diethyl ether/n-pentane (85:15, v/v), Rf = 0.75).
[2-14C]-(6E,10E,14E,18E)-2,6,10,15,19,23-Hexamethyltetracosa-6,10,14,18,22-hexaene (2)

A stirred solution of 9 (2.664GBq, 1.26mmol) in diethyl ether (25ml) was treated with
Burgess reagent (0.476g, 2mmol, CAS Number : 29684-56-8) at -20°C for 15mn. The
reaction was monitored by HPLC (Rt alcohol: 14.9 min - Rt squalene + isomer: 30.4 min
according to above conditions). After 14 hours, the mixture was diluted with diethyl ether
(50ml) and a brine solution (10ml) was added. The product was extracted twice with diethyl
ether (150ml). The combined organic solution was dried over MgSO4, filtered and
concentrated. The residue was dissolved in pentane (5ml) and purified by chromatography
using a silica gel column eluted with diethyl ether/n-pentane (1:9, v/v) to afford a mixture
of two hexaenes in
a
[2-14C]-squalene 2/(6e,10e,14e,18e)-1,6,10,15,19,23-
Hexamethyltetracosa-6,10,14,18,22-[2-14C]-hexaene 12 ratio of 55%/45% as oil (2.62GBq,
98% yield ). The ratio was determined by proton NMR spectrum integration of ethylenic
signals (characteristic isolated doublet at 4.63 ppm belonging to the unwanted isomer 12).
Purification of [2-14C]-squalene (2)
The oily residue from the above experiment (1.78GBq) was purified by chromatography, successively
through three columns of silica gel-AgNO3 (200g) eluted with n-pentane/ethyl acetate (1:1, v/v) to give
555 MBq of crude 2 with a stereoisomeric purity of 98,7% measured by proton NMR. A final
purification on a RP18 column, using a mixture of acetonitrile - tetrahydrofuran (freshly distilled) was
necessary to eliminate traces of BHT to afford 495MBq of [2-14C]-squalene 2 as a colorless oil (27%
1
yield), 99.9% radiochemical purity determined by HPLC according to above conditions). H-NMR
(600 MHz, CDCl3) δ 5.09 to 5.19 (m, 6H), 2.04 to 2.12 (m, 8H), 1.96 to 2.04 (m, 12H), 1.69 (s, 6H), 1.61
(m, 18H)
Radiosynthesis of [3-14C]-squalene (3)
(6E,10E,14E,18E)-21-Bromo-2,6,10,15,19-pentamethylhenicosa-2,6,10,14,18-pentaene (13)
A stirred solution of turbinaric acid 4 (700mg, 1.175mmol) in tetrahydrofuran (15ml) was
treated with 1,1’-carbonyldiimidazole (370mg; 2.28mmol) in one portion. The mixture was
heated at 60°C for 1 hour. The reaction was monitored by TLC (after esterification of a sample
in methanol) on silica plates eluted with diethyl ether/n-pentane (3:7, v/v) (methyl ester: Rf = 1).
2-Mercaptopyridine N-oxide sodium salt hydrate (440mg, 2.68mmol, 2.28eq.) and 4-
dimethylaminopyridine (50mg, 0.41mmol, 0.35eq.) were added and the mixture was stirred at
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60°C for 1 hour. Bromotrichloromethane (30ml) was added and the mixture was stirred at 115°C
for 45 min. The reaction was monitored by TLC (SiO2, diethyl ether/n-Pentane (3:7, v/v),
product: Rf = 1). After cooling to room temperature, the mixture was diluted with diethyl ether
(30ml) and brine solution (50ml). The mixture was poured into a separating funnel and extracted
three times with diethyl ether (60ml). The combined organic solution was dried over MgSO4,
filtered and concentrated under vacuum. The residue was purified by chromatography on a silica
gel column eluted with diethyl ether/n-pentane (5:95, v/v) to afford 13 (250mg, 34%) as a
yellowish oil. TLC (SiO2, diethyl ether/n-pentane (5:95, v/v), Rf = 0.95).
1H-NMR (600 MHz, CDCl3) δ 5.23 (m, 1H), 5.08 to 5.18 (m, 4H), 3.43 (t, J=7.5 Hz, 2H), 2.53
(t, J=7.5 Hz, 2H), 1.94 to 2.14 (m, 16H), 1.69 (s, 3H) 1.63 (s, 3H), 1.61 (m, 12H)
[1-14C]-(4E,8E,12E,16E)-4,8,13,17,21-Pentamethyldocosa-4,8,12,16,20-pentaenenitrile (14)
A stirred suspension of [14C]-Potassium cyanide (355MBq, 0.16mmol) in dimethylsulfoxide
(5ml) was treated with a solution of 13 (250.00 mg; 0.57mmol; 4.72 eq.) in dimethylsulfoxide
(10ml). The mixture was stirred at room temperature overnight. A brine solution (40ml) was
added. The product was extracted three times with diethyl ether (60ml). The combined organic
solution was dried over MgSO4, filtered and concentrated under vacuum. The residue was
purified by chromatography on a silica gel column eluted with diethyl ether/n-pentane (1:9, v/v)
to afford 14 (199MBq, 58% yield) as a colorless oil. TLC (SiO2, diethyl ether/n-pentane (1:9,
v/v), Rf = 0.85).
[1-14C]-(4E,8E,12E,16E)-4,8,13,17,21-Pentamethyldocosa-4,8,12,16,20-pentaenal (15)
Compound 14 (199MBq, 0.098mmol) was dissolved in a mixture of ether (1ml) and n-pentane
(10ml) under argon. The solution was cooled at -78°C. A solution of 1.0M diisobutylaluminium
hydride in dichloromethane (0.5ml, 0.5mmol) was added dropwise. The mixture was stirred for
3 hours at -78°C. The reaction was monitored by TLC (SiO2, diethyl ether/n-pentane (1:9, v/v),
product Rf = 0.6).The mixture was quenched with diethyl ether (20ml) then with a 0.5M aqueous
solution of HCl (20ml). The mixture was warmed to room temperature and extracted three times
with diethyl ether (30ml). The combined organic solution was dried over MgSO4, filtered and
concentrated under vacuum. The residue was purified by chromatography on a silica gel column
eluted with diethyl ether/n-pentane (1:9, v/v) to afford 15 as a colorless oil, directly used in the
next step. TLC (SiO2, diethyl ether/n-pentane (1:9, v/v), Rf = 0.6).
[3-14C]-(6E,10E,14E,18E)-2,6,10,15,19,23-Hexamethyltetracosa-2,6,10,14,18,22- hexaene (3)
A stirred solution of isopropyltriphenylphosphonium bromide (2.8g, 7.3mmol) in anhydrous
tetrahydrofuran (8ml) was treated dropwise with a 1.6M n-Butyllithium solution in hexane
(4.6ml, 7.4mmol) at -78°C. During the addition, the color progressively turned to dark red. The
mixture was warmed at room temperature then heated to reflux. A solution of 15 from the above
experiment in anhydrous diethyl ether (2ml) was added all at once under reflux. After stirring at
reflux for 20min, a saturated aqueous solution of NH4Cl was poured then the mixture was
cooled to room temperature. The solution was extracted three times with diethyl ether (30ml).
The combined organic layers were washed twice with a brine solution. The organic solution was
dried over MgSO4, filtered and concentrated under vacuum. The residue was purified by
chromatography on a silica gel column eluted with n-pentane to afford 129.9MBq of 3 as a
colorless oil (65% yield over 2 steps, 98,3% radiochemical purity determined by HPLC
according to the above conditions). TLC (SiO2, n-pentane Rf = 0.18)
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Acknowledgement: The authors gratefully acknowledge the help of Dr. Serge Perard for the
NMR analyses and Sanofi Chemical Development Department for providing us with turbinaric
acid.
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Figure 1. Structure of squalene 1
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(4)
(5)
LiAlH4, Et2O
(90%)
PPh3, I2
CH2Cl2
(90%)
K 14CN,
DMSO
(6)
(7)
(94%)
KOHaq 40%, EtOH
(94%)
CH3Li, THF
(25%)
(8)
(10)
THF, CDI, MeOH, reflux
(92%)
CH3MgI, THF
(90%)
(11)
CH3MgI, Et2O
(86%)
(9)
Burgess reagent
Et2O, -20°C
(98%)
(12)
(2)
Analytical ratio:
(2)/(12) =55:45
Purification :
1) Silica gel-AgNO 3 column
(27%)
2) RP18 column
[2- 14C]-squalene (2)
Scheme 1: synthesis of [2-14C]-squalene 2
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Scheme 2: synthesis of [3-14C]-squalene 3
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