Synthesis of d8-geranyl diphosphate

Article abstract of DOI:10.1002/jlcr.1027

Multiply labelled d₈-geranyl diphosphate (3-methyl-7- ²H₃-methyl-[1,1,8,8,8]-²H₅-2E,6- octadienyl diphosphate) was synthesised from geraniol in 8 steps. Geraniol was converted to [1,1]-²H₂-geraniol by a three step oxidation-reduction sequence in 38% yield. Selective epoxidation of [1,1]- ²H₂-geranyl acetate gave 6,7-epoxy-[1,1]- ²H₂-geranyl acetate, which, on oxidative cleavage of the epoxide and Wittig elaboration with d₆-isopropyl triphenylphosphorane, gave d₈-geraniol (14% yield) which was, in turn, converted to the title compound. Copyright

Full text of DOI:10.1002/jlcr.1027

JOURNAL OF LABELLED COMPOUNDS AND RADIOPHARMACEUTICALS
J Label Compd Radiopharm 2006; 49: 47–54.
Published online in Wiley InterScience (www.interscience.wiley.com). DOI: 10.1002/jlcr.1027
Research Article
Synthesis of d -geranyl diphosphate
8
Daniel J. Comeskey*, Daryl D. Rowan and Adam J. Matich
The Horticulture and Food Research Institute of New Zealand Ltd., Private Bag 11 030,
Palmerston North, New Zealand
Summary
2
Multiply labelled d -geranyl diphosphate (3-methyl-7- H -methyl-[1,1,8,8,8]- H -
2
8
3
5
2
E,6-octadienyl diphosphate) was synthesised from geraniol in 8 steps. Geraniol
2
was converted to [1,1]- H -geraniol by a three step oxidation–reduction sequence in
2
2
2
3
8% yield. Selective epoxidation of [1,1]- H -geranyl acetate gave 6,7-epoxy-[1,1]- H -
2 2
geranyl acetate, which, on oxidative cleavage of the epoxide and Wittig elaboration
with d -isopropyl triphenylphosphorane, gave d -geraniol (14% yield) which was, in
6
8
turn, converted to the title compound. Copyright # 2005 John Wiley & Sons, Ltd.
Key Words: geraniol; geranyl diphosphate; synthesis; deuterium
Introduction
Geraniol (3,7-dimethyl-2E,6-octadienol) 1 is a widespread plant volatile and
aroma compound which, as its diphosphate, is a biosynthetic precursor for the
1
,2
numerous mono and higher terpenes produced by plants. In the course of
mechanistic studies in sesquiterpene biosynthesis, we required a doubly
labelled form of geranyl diphosphate.
Deuterated precursors and GCMS analysis have been used extensively in the
3,4
study of terpene biosynthesis and several relevant syntheses of deuterium
5
labelled mono- , sesqui- and di- terpenes have been reported. The reported
6
7
2
syntheses of d -geraniol (3-methyl-7- H -methyl-[8,8,8]- H -2E,6-octadienol)
2
8
6
3
3
6
and d -a-farnesene seemed particularly relevant. We report here the synthesis
6
2
of multiply labelled d8-geranyl diphosphate (3-methyl-7- H -methyl-
3
2
1,1,8,8,8]- H -2E,6-octadienyl diphosphate).
[
5
*Correspondence to: Daniel J. Comeskey, The Horticulture and Food Research Institute of New Zealand
Ltd., Private Bag 11 030, Palmerston North, New Zealand. E-mail: DComesky@hortresearch.co.nz
Contrant/grant sponsor: NZ Foundation for Research Science and Technology; contract/grant number:
C06X0403
Copyright # 2005 John Wiley & Sons, Ltd.
Received 11 October 2005
Accepted 31 October 2005

4
8
D. J. COMESKEY ET AL.
Results and discussion
2
,7-Dimethyl-[1,1]- H -2E,6-octadienol (d -geraniol) 4 was prepared by a three
3
2 2
step oxidation-reduction sequence (Figure 1) in 38% overall yield. Oxidation
of geraniol with Cr(VI) gave an 82% yield of geranial 2 with c. 21% double
9
bond isomerisation to neral. Oxidation of 2 with MnO via the cyanohydrin
2
gave methyl ester 3 which was reduced with LiAlD to give d -geraniol 4. The
4
2
1
H NMR of 4 showed H-2 as a broad singlet at d ¼ 5:42 ppm. GCMS analysis
gave a molecular ion at m=z ¼ 156 with characteristic fragment ions at m=z ¼
+
6
9 (C H ), 95 (C H D ), 123 (C H ) and 138 (M -H O) confirming the
5 9 7 7 2 9 15 2
expected pattern of deuterium incorporation (Figure 2). The product
contained c. 20% of d -nerol, as determined by NMR.
2
Conversion of d -geraniol 4 to d -geraniol 8 (14% yield) followed the
8
2
6
8
methods of Fielder and Rowan and of Luan et al. Protection of 4 as the
t-butyldimethylsilyl ether did not provide a derivative suitable for subsequent
reactions. Alternatively acetylation of 4 with lipase under anhydrous
conditions in vinyl acetate and pentane gave a quantitative yield of d -geranyl
2
acetate 5. Epoxidation of 5 with m-chloroperbenzoic acid at 08C gave
selectively the 6,7-epoxide 6 in 70% yield which was cleaved with periodic acid
to give aldehyde 7 in 72% yield. Aldehyde 7 showed a characteristic low field
1
aldehydic triplet (J ¼ 1:6 Hz) at d ¼ 9:78 ppm in the H NMR spectrum as
well as a single allylic methyl group (d ¼ 1:71 ppm) showing a distinctive long-
range coupling (J ¼ 1:3 Hz) to the broad singlet of H-2 (d ¼ 5:35 ppm).
Aldehyde 7 readily trimerised on standing and was used immediately
in reaction with d -isopropyl triphenylphosphorane 9 (prepared from
6
d -isopropyl iodide), with in situ hydrolysis of the acetate ester to give
7
1
d -geraniol 8 in 29% isolated yield. The H NMR showed a single allylic
8
methyl group (d ¼ 1:69 ppm), again with distinctive long-range coupling
(
J ¼ 1:3 Hz), and two olefinic signals, a broad singlet (H-2) and a triplet (H-6,
1
3
J ¼ 6:7 Hz) at d ¼ 5:43 and 5.11 ppm, respectively. The C NMR showed 4
olefinic and 3 alkyl signals. No additional methyl resonances, or resonances
for H-1 and C-1, were observed. GCMS analysis of 8 gave a molecular ion
O
O
D
D
OH
OH
(
i)
H
(ii)
OMe (iii)
1
3
2
4
Figure 1. Synthesis of d -geraniol 4. Reagents and conditions: (i) Dipyridinium
2
chromium (VI) oxide, DCM. (ii) NaCN, MnO , AcOH, MeOH. (iii) LiAlD ,
2
4
Et O, reflux
2
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 47–54

SYNTHESIS OF d
8
-GERANYL DIPHOSPHATE
49
Figure 2a. GCMS EI (70 eV) MS of geraniol (a, from NIST library); d2-
geraniol (b) and d -geraniol (c)
8
m=z ¼ 162:1853 (D ¼ 4:3 ppm) with characteristic fragment ions at m=z ¼ 75
(C H D ), 95 (C H D ) and 129 (C H D ) (Figure 2) confirming the expected
5 3 6 7 7 2 9 9 6
pattern of deuterium incorporation. The final product 8 also contained 5%
d8-nerol resulting from double bond isomerisation in the initial step of the
synthesis.
d -Geraniol 8 was converted to d -geranyl diphosphate in 32% yield,
8
8
1
0
following the method of Keller and Thompson. The use of this compound in
biosynthetic studies will be reported elsewhere.
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 47–54

5
0
D. J. COMESKEY ET AL.
D
D
D
D
D
O
D
OH
OAc
(
i)
OAc
(
ii)
4
5
6
(iii)
D
D
D
D
OH
OAc
(iv)
O
D C
3
CD3
8
7
Figure 2b. Synthesis of d -geraniol 8. Reagents and conditions: (i) Lipase, vinyl
8
acetate, pentane. (ii) mCPBA, DCM, 08C. (iii) H IO , THF, Et O. (iv)
5
6
2
2
[
1,1,1,2,3,3,3- H ]-isopropyl triphenylphosphonium iodide, n-BuLi, THF,
7
MeOH, 08C
Experimental
General
All reagents were of commercial grade and were used as received unless
otherwise stated. Lipase AK ‘amano’ 20 was gifted from Amano Enzyme Inc.
1
Nagoya, Japan. H, C and P NMR spectra were recorded on a Bruker
13
31
Avance 400 MHz spectrometer in CDCl , except for d -geranyl diphosphate
3
8
31
which was dissolved in D O. P NMR were recorded with proton and carbon
2
decoupled and the signals referenced to phosphoric acid. GCMS and high
resolution MS were recorded on a Waters GCT Time of Flight mass
spectrometer at 70 eV ionisation energy.
3
,7-Dimethyl-2E,6-octadienal (geranial) 2. Chromium (VI) oxide (40 g,
00 mmol) was added in one portion to a solution of pyridine (63 g, 796 mmol)
4
in DCM (1 l) at 58C. This was stirred and allowed to warm to room
temperature over one hour. To this solution was added a solution of geraniol
(10 g, 64.9 mmol) in DCM (100 ml). After stirring for 30 min the reaction
mixture was washed sequentially with 200 ml 5% NaOH, 5% HCl, 5%
NaHCO3 and water. The organic layer was dried over MgSO and
4
1
concentrated in vacuo to give 2 as a colourless oil (8.13 g, 82%). H NMR:
d (ppm) 9.99 (1H, d, J ¼ 8:1 Hz), 5.88 (1H, dq, J ¼ 8:1; 1:3 Hz), 5.08 (1H, m),
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 47–54

SYNTHESIS OF d
8
-GERANYL DIPHOSPHATE
51
1
3
2
.23 (4H, m), 2.16 (3H, d, J ¼ 1:3 Hz), 1.68 (3H, s), 1.61 (3H, s). C NMR: d
ppm) 191.3, 163.90, 132.9, 127.4, 122.5, 40.6, 25.7, 25.7, 17.7, 17.6. MS m/z
(
+
relative intensity %) 152.1223 (15, M , C H O requires 152.1201), 137 (22),
(
10
l6
1
23 (18), 109 (23), 94 (33), 84 (55), 69 (78), 41 (100).
Methyl 3,7-dimethyl-2E,6-octadienoate 3. Sodium cyanide (9.52 g, 194 mmol)
followed by manganese dioxide (50 g, 575 mmol) and acetic acid (3.4 ml, 3.5 g,
5
8.8 mmol) was added to a solution of 2 (5.6 g, 36.8 mmol) in MeOH (500 ml)
at room temperature. After stirring for 20 hours, the reaction mixture was
filtered through celite, concentrated in vacuo, then taken up in 100 ml water
and extracted twice with 200 ml diethyl ether. The organic phase was dried
over MgSO4 and the crude product purified by flash chromatography
(
petroleum ether/diethylether 9:1) to give 3 as a colourless oil (3.7 g, 55%).
1
H NMR: d (ppm) 5.68 (1H, br s), 5.09 (1H, m), 3.69 (3H, s), 2.16 (7H, m),
13
1
.69 (3H, s), 1.61 (3H, s). C NMR: d (ppm) 167.3, 160.2, 132.5, 122.9, 115.8,
+
0.8, 40.9, 25.7, 18.8, 17.7. MS m/e (relative intensity %) 182.1327 (3, M ,
5
C H O requires 182.1307), 151 (11), 139 (13), 123 (47), 114 (48), 83 (25), 69
1
1
18
2
(100), 41 (41).
2
,7-Dimethyl-[2,2]- H -2E,6-octadienol (d -geraniol) 4. A solution of 3
3
3
2
(3.7 g, 20.3 mmol) in diethyl ether (20 ml) was added to a stirred slurry of
lithium aluminium deuteride (1.1 g, 25.4 mmol) in diethyl ether (60 ml), at a
rate sufficient to maintain reflux. The reaction mixture was stirred for a further
hour before dropwise quenching with 15% NaOH and further dilution with
water. This was then extracted twice with 50 ml diethyl ether, dried over
MgSO , concentrated in vacuo and purified by flash chromatography
4
(
petroleum ether/ethyl acetate 9:1) to give 4 as a colourless oil (2.7 g,
1
1
7.3 mmol, 85%). H NMR: d (ppm) 5.42 (1H, br s), 5.10 (1H, m), 2.08
1
3
(
4H, m) 1.69 (3H, s), 1.68 (3H, s), 1.61 (3H, s). C NMR: d (ppm) 139.9,
1
1
9
31.8, 123.9, 123.2, 39.6, 26.4, 25.7, 17.7, 16.3. MS m/e (relative intensity %),
+
56.1513 (4, M , C H D O requires 156.1483), 138 (17), 123 (49), 113 (13),
10
16
2
5 (100), 81 (30), 69 (48), 53 (16), 41 (92).
2
,7-Dimethyl-[2,2]- H -2E,6-octadienyl acetate (d -geranyl acetate) 5. Lipase
3
3
2
AK ‘Amano’ 20 Lot No. LAKY0950502 (5 g), followed by vinyl acetate
(6.4 ml, 69.2 mmol), was added to a solution of 4 (2.7 g, 17.3 mmol) in pentane
(300 ml) at room temperature. The reaction mixture was stirred for 2 hours
before removal of the enzyme by filtration. Concentration followed by flash
chromatography (petroleum ether/diethyl ether 9:1) gave 5 as a colourless oil
1
(3.24 g, 95%). H NMR: d (ppm) 5.34 (1H, br s), 5.09 (1H, m), 2.10 (2H, m),
2
.06 (3H, s), 2.05 (2H, m), 1.71 (3H, d, J ¼ 1:3 Hz), 1.69 (3H, s), 1.61 (3H, s).
C NMR: d (ppm) 171.6, 132.2, 124.1, 123.9, 118.5, 39.9, 32.6, 26.7, 26.1,
1
3
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 47–54

5
2
D. J. COMESKEY ET AL.
+
1.5, 18.1, 16.9. MS m/e (relative intensity %) 156.1491 (4, M -CH CO,
2
2
C H D O requires 156.1483), 138 (42), 123 (47), 109 (10), 95 (100), 81 (26),
2
1
0
16
6
9 (99), 43 (26), 41 (31).
2
,7-Epoxy-3,7-dimethyl-[2,2]- H -2E,6-octadienyl acetate (d -6,7-epoxygera-
6
3
2
nyl acetate) 6. m-Chloroperoxybenzoic acid (mCPBA) (3.2 g, 18.2 mmol) in
DCM (35 ml) was added over 10 min to a solution of 5 (3.2 g, 16.2 mmol) in
DCM (60 ml) cooled to 08C. After stirring for an hour at 08C the reaction
mixture was treated with portions of mCPBA until the starting material
disappeared as indicated by thin-layer chromatography. The reaction mixture
was filtered and the filtrate washed sequentially with 50 ml saturated NaHCO3
solution, water and brine and then dried over MgSO . The solution was
4
concentrated in vacuo and the crude product purified by flash chromatography
(
petroleum ether/ethyl acetate 17:3) to give 6 as a colourless oil (2.42 g, 70%).
1
H NMR: d (ppm) 5.37 (1H, br s), 2.71 (1H, t, J ¼ 6:3 Hz), 2.20 (2H, m), 2.05
3H, d, J ¼ 0:5 Hz), 1.72 (3H, d, J ¼ 1:5 Hz), 1.66 (2H, m), 1.30 (3H, s), 1.26
(
(
1
3
3H, s). C NMR: d (ppm) 171.5, 141.8, 119.1, 64.4, 58.9, 36.6, 27.4, 25.2,
+
2
1.4, 19.1, 16.8. MS m/e (relative intensity %) 154.1336 (15, M -CH COOH,
3
C H D O requires 154.1327), 139 (12), 127 (8), 111 (34), 96 (30), 85 (94), 83
1
0
14
2
(83), 71 (78), 59 (46), 43 (100).
2
-Acetoxy-4-methyl-[2,2]- H -4-hexenal 7. A solution of periodic acid (2.8 g,
6
3
1
2.4 mmol) in THF (40 ml) was slowly added to a vigorously stirred solution of
(2.4 g, 11.3 mmol) in diethyl ether (40 ml) at room temperature. After an
6
hour the reaction mixture was quenched with 40 ml ice water. The organic
layer was separated and the aqueous layer extracted with 50 ml diethyl ether.
The combined organic fractions were washed sequentially with 50 ml sat.
NaHCO solution, water and brine then dried over MgSO , concentrated in
3
4
vacuo and purified by flash chromatography (petroleum ether/diethyl ether
1
7
:3) to give 7 as a yellow oil (1.4 g, 72%), H NMR: d (ppm) 9.78 (1H, t,
J ¼ 1:6 Hz), 5.35 (1H, br s), 2.58 (2H, m), 2.38 (2H, t, J ¼ 7:6 Hz), 2.05
13
ð3H; sÞ, 1.71 (3H, d, J ¼ 1:3 Hz). C NMR: d (ppm) 202.1, 171.5, 140.6,
19.5, 42.1, 31.8, 21.5, 17.0. MS m/e (relative intensity %) 127.0745
+
1
(
40, M -CH CDO, C H DO requires 127.0744), 112 (25), 110 (28), 97 (15),
3 7 9 2
8
5 (96), 83 (68), 69 (36), 55 (18), 43 (100).
2
1,1,1,2,3,3.3- H ]-Isopropyl triphenylphosphonium iodide 9. A mixture of
[
7
2
triphenylphosphine (0.77 g, 2.94 mmol) and 2-iodo-[1,1,1.2,3,3,3- H ]-propane
7
(0.5 g, 2.82 mmol) was heated at 1408C for 3.5 hours in a thick walled tube
sealed with a screw cap. The crude product was recrystallised from ethyl
acetate/ethanol 2:1 to give 9 as white crystals (925 mg, 75%). m.p.
1
11
94.5–1988C (lit. 194–1978C for the unlabelled compound).
Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 47–54

SYNTHESIS OF d
8
-GERANYL DIPHOSPHATE
53
2
-Methyl-7-[ H ]-methyl-[2,2,8,8.8- H ]-2E,6-octadienol (d -geraniol) 8.
2
3
3
5
8
n-Butyl lithium (1.6 M in hexanes, 0.66 ml, 1.05 mmol) was added dropwise to
a solution of 9 (460 mg, 1.05 mmol) in THF (5 ml) cooled to 08C. The reaction
mixture was stirred for 30 min at 08C before the addition of 7 (180 mg, 1.05 mmol)
in THF (5 ml). This was stirred for a further hour at 08C before quenching with
5
ml MeOH. The reaction mixture was concentrated in vacuo, taken up in 10 ml
water, extracted with petroleum ether (4 ꢀ 20 ml), dried over MgSO and
4
concentrated in vacuo. The crude product was purified by flash chromatography
1
petroleum ether/ethyl acetate 4:1) to give 8 as a colourless oil (49 mg, 29%). H
(
NMR: d (ppm) 5.43 (1H, br s), 5.11 (1H, t, J ¼ 6:7 Hz), 2.08 (4H, m), 1.69 (3H,
13
d, J ¼ 1:3 Hz). C NMR: d (ppm) 140.3, 132.0, 124.3, 123.6, 39.9, 26.7, 16.7. MS
m/e (relative intensity) 162.1853 (1, M , C H D O requires 162.1860), 144 (6),
+
10
10
8
129 (11), 126 (8), 113(6), 95 (47), 81 (15), 75 (100), 55 (12), 44 (59).
2
2
3
nyl diphosphate). d -Geraniol 8 (42 mg, 0.26 mmol) was taken up in
-Methyl-7-[ H ]-methyl-[2,2,8,8,8- H ]-2E,6-octadienyl diphosphate (d -gera-
3 5 8
8
trichloroacetonitrile (1.14 ml) in a 30 ml polypropylene Oak Ridge centrifuge
tube with a magnetic stirrer bar, and heated to 378C. Concentrated phosphoric
acid (2.5 ml) was added to CH CN (9.5 ml) to produce solution A. Et N
3
3
(
(
11 ml) was added to CH CN (10 ml) to produce solution B. Solution A
3
9.1 ml) was added, dropwise, to solution B (15 ml) to create the bis-
triethylammonium phosphate (TEAP) solution, and immediately thereafter
TEAP (0.76 ml) solution was added to the d -geraniol solution. Two more
8
additions of TEAP solution were made at 5 min intervals. The reaction
mixture was stored at ꢁ188C overnight. d -Geranyl diphosphate was
8
separated from the mono and triphosphate by flash chromatography on silica
using i-PrOH/conc NH /H O, 6:2.5:0.5. Fractions (15 ml) were collected after
3
2
elution of a yellow pigment. d -Geranyl diphosphate was identified by TLC
8
(i-PrOH/conc NH /H O, 6:3:2) against an authentic sample of geranyl dipho-
3 2
sphate and was visualised using vanillin. The eluent volume was reduced to
2
0 ml in vacuo at 308C, before freeze drying to yield the title compound as a
1
white solid (31.5 mg, 0.08 mmol, 32%). H NMR: d (ppm) 5.32 (1H, br s), 5.08
3
l
(
1H, t, J ¼ 6:8 Hz), 2.00 (4H, m), 1.59 (3H, s). P NMR: d (ppm) ꢁ9.29
d, J ¼ 53 Hz), ꢁ9.94 (d, J ¼ 51 Hz).
(
Acknowledgements
We wish to thank Martin Hunt for high resolution GCMS, Massey University
for NMR and the NZ Foundation for Research Science and Technology for
financial support (C06X0403).
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[
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Copyright # 2005 John Wiley & Sons, Ltd.
J Label Compd Radiopharm 2006; 49: 47–54