Synthesis of a new deuterium-labeled phytol as a tool for biosynthetic studies

Article abstract of DOI:10.1055/s-0031-1289689

An efficient and stereoselective synthesis of a new deuterium-labeled phytol as a useful tracer in biosynthetic processes is presented. Georg Thieme Verlag Stuttgart · New York.

Full text of DOI:10.1055/s-0031-1289689

862
SHORT PAPER
Synthesis of a New Deuterium-Labeled Phytol as a Tool for Biosynthetic
Studies
D
euterium-Labe
o
led Phytol urdes Muñoz, Angel Guerrero*
Department of Biological Chemistry and Molecular Modeling, IQAC (CSIC), 08034 Barcelona, Spain
Fax +34(93)2045904; E-mail: angel.guerrero@iqac.csic.es
Received 24 November 2011; revised 3 January 2012
Abstract: An efficient and stereoselective synthesis of a new deu-
terium-labeled phytol as a useful tracer in biosynthetic processes is
presented.
18OH
1
T
Key words: deuterium-labeled compounds, phytol, terpenoids,
conjugate addition, natural products, biosynthesis
OH
OH
OH
2
13CH3
Phytol [(E)-3,7,11,15-tetramethylhexadec-2-en-1-ol] is
3
an acyclic diterpene alcohol that occurs as a side chain of
chlorophyll and is therefore abundantly present in nature,
not only in plants but also in the animal kingdom. On be-
CD3
D
D
ing digested, the phytol moiety is released and can then be
4
converted into other important metabolites, such as phy-
tanic acid (3,7,11,15-tetramethylhexadecanoic acid),
whose accumulation in human blood and tissues has been
D
D
OH
5
linked to neurological disorders, peroxisomal biogenesis
Figure 1
diseases, and apoptosis.1 In addition, phytol can be used as
3
a precursor of vitamins E2 and K1 and its balsamic prop-
The strategy for the preparation of 5 was based on a
straightforward approach by the regioselective opening of
3,4-epoxyisoprene with the appropriate labeled Grignard
reagent (Scheme 1). 3,4-Epoxyisoprene can be obtained
in a single step and with reasonable yield from meth-
acrolein by selective 1,2-addition of dimethylsulfonium
methylide.8 For preparation of the other labeled building
block 10, farnesol (6) was initially treated with hydrogen
and 10% palladium-on-carbon to give the corresponding
hexahydrofarnesol (7), but the hydrogenolysis product
farnesane was recovered almost exclusively. However,
successful hydrogenation was achieved by using plati-
num-on-carbon as catalyst to provide the fully hydroge-
nated compound in quantitative yield.9 Oxidation of
compound 7 under standard conditions (CrO3, H2SO4)
furnished acid 8, which was quantitatively reduced to the
corresponding deuterated alcohol 9 with lithium alumi-
num deuteride (LiAlD4). In this process, no trace of the
mono- or non-labeled alcohol was detected, as demon-
strated by the absence of the characteristic signal at d =
erties are greatly appreciated in the fragrance industry,
where it is used in approximately 0.1–1.0 metric tons per
year.4 Therefore, the preparation of new isotopically la-
beled phytol derivatives is important because they could
be potentially useful in a number of relevant biological
processes.5
In the course of our investigations aimed at the structural
characterization of a bioactive natural compound (to be
published), phytol appeared to be the most plausible bio-
synthetic precursor. Therefore, synthesis of a labeled ana-
logue of phytol with the labeling far enough from the
oxygenated function to avoid it being affected in the bio-
synthetic process was required. As is well known, isotopic
labeling is a very useful technique for tracking the trans-
formations undergone by a certain molecule or the mech-
anisms involved in many biochemical processes.6
Few labeled phytols (1–4) have been described so far in
the literature (Figure 1).5,7 Most of these derivatives, par-
ticularly 1 and 2, contained the label at positions that can
be also affected in the biosynthetic processes, and com-
pound 4 was part of a mixture of differently labeled com-
pounds.7c For these reasons, a more suitably labeled
phytol, compound 5 with two deuterium atoms at C-5,
away from the functionalized end of the molecule, was
considered for our study.
1
3.67 ppm in the H NMR spectrum. The alcohol 9 was
transformed into the corresponding bromide 10 in 95%
yield by treatment with triphenylphosphine–N-bromo-
succinimide10a in dichloromethane. Reaction of 10 with
freshly activated magnesium turnings in tetrahydrofu-
ran,10a,b followed by treatment with a catalytic amount of
copper(I) iodide, and finally with 3,4-epoxyisoprene at
–78 °C yielded the expected compound 5 stereoselective-
ly as the E isomer (E/Z = 96:4), the isomer corresponding
to natural phytol, in a reasonable 45% yield. We have no-
SYNTHESIS 2012, 44, 862–864
x
x
.x
x
.2
0
1
2
Advanced online publication: 03.02.2012
DOI: 10.1055/s-0031-1289689; Art ID: Z110311SS
© Georg Thieme Verlag Stuttgart · New York

SHORT PAPER
Deuterium-Labeled Phytol
863
H2, Pt/C
EtOH
CrO3, H2SO4
OH
OH
OH
99%
6
8
7
9
72%
LiAlD4
Et2O
Ph3P, NBS
CH2Cl2
O
D
D
D
99%
OH
95%
1) Mg, THF
2) CuI
D
D
D
O
3)
OH
Br
10
5
45% (E/Z = 96:4)
Scheme 1
tered on Celite and the solvent evaporated to yield the saturated al-
cohol 7 as a colorless oil, pure enough for the next step of the
synthesis without need of further purification.
ticed that an excess of magnesium is required in this reac-
tion to avoid the homocoupling reaction. It is worthy of
note that this synthetic strategy ensures the introduction of
two deuterium atoms in a predefined position of the mol-
ecule, avoiding problematic mixtures of differently la-
beled compounds.
Yield: 5.13 g (99%).
IR (film): 3349, 2955, 2927, 2869, 1463, 1378, 1056, 908, 735 cm–1.
1H NMR (400 MHz, CDCl3): d = 3.67 (m, 2 H), 1.55 (b, 3 H), 1.01–
1.38 (b, 14 H), 0.86 (m, 12 H).
13C NMR (100 MHz, CDCl3): d = 61.2 (CH2), 40.0 (CH2), 39.3
(CH2), 37.5 (CH2), 37.4 (CH2), 37.3 (CH2), 32.8 (CH), 29.5 (CH),
27.9 (CH), 24.8 (CH2), 24.4 (CH2), 22.7 (CH3), 22.6 (CH3), 19.7
(CH3), 19.6 (CH3).
MS (EI, 70 eV): m/z (%) = 210 [M – 18]+ (1), 182 (13), 140 (28),
125 (88), 111 (84), 97 (91), 83 (91), 69 (100), 57 (97), 43 (82).
In conclusion, we have accomplished the synthesis of a
new labeled phytol with two deuterium atoms at C-5, far
enough from the functionalized end of the molecule,
which makes it an attractive candidate for a variety of bio-
synthetic studies. The synthesis is short, stereoselective,
and with a good overall yield (16.6%) from readily avail-
able starting materials.
3,7,11-Trimethyldodecanoic acid (8)12
All reactions involving air- or moisture-sensitive materials were
carried out under argon. All solvents were dried and distilled ac-
cording to standard procedures. Locations of deuterium atoms and
purity of the labeled compounds were determined by elemental
analysis, MS, and NMR. NMR spectra were recorded at 400 MHz
for 1H and 100 MHz for 13C on a Varian Mercury 400 MHz spec-
trometer. IR spectra were recorded on a Nicolet Avatar 360 FT-IR
spectrometer. MS was carried out in EI mode (70 eV) on a Fisons
MD 800 instrument. Elemental analyses were determined on a
Carlo Erba 1108 and on a Metrohm Titrando 808 analyzer. HRMS
was run on a UPLC Acquity (Waters, USA) coupled to a mass spec-
trometer LCT Premier XE (Waters, USA).
A soln of Jones reagent, prepared from CrO3 (3.19 g), concd H2SO4
(2.6 mL), and H2O (9 mL) until the reaction mixture turned red, was
added to a soln of alcohol 7 (3.00 g, 13.0 mmol) in acetone (75 mL)
at 0 °C. After the mixture had stirred at 0 °C for 4 h, propan-2-ol (10
mL) was added to stop the reaction, the solvent was evaporated, and
the residue was purified by column chromatography (silica gel, hex-
ane–Et2O, 99:1); this gave acid 8.
Yield: 2.28 g (72%); pale yellow oil.
IR (film): 3084, 2955, 2927, 2678, 1709, 1463, 1298, 937 cm–1.
1H NMR (400 MHz, CDCl3): d = 2.37 (m, 1 H), 2.14 (m, 1 H), 1.96
(m, 1 H), 1.52 (m, 1 H), 1.05–1.38 (br, 13 H), 0.97 (d, J = 8.0 Hz, 3
H), 0.86 (d, J = 8.0 Hz, 6 H), 0.84 (d, J = 8.0 Hz, 3 H).
13C NMR (100 MHz, CDCl3): d = 179.7 (C), 41.6 (CH2), 39.3
(CH2), 37.3 (CH2), 37.1 (CH2), 37.0 (CH2), 32.7 (CH), 30.2 (CH),
28.0 (CH), 24.8 (CH2), 24.3 (CH2), 22.7 (CH3), 22.6 (CH3), 19.7
(CH3), 19.6 (CH3).
3,4-Epoxy-2-methylbut-1-ene8
Methacrolein (2.4 mL, 28.5 mmol) was added in 4 portions over 20
min to a stirred suspension of methyl trimethylsulfonium sulfate
(6.14 g, 32.63 mmol) and NaOH (7.10 g, 0.18 mol) in anhyd CH2Cl2
(70 mL). After the mixture had stirred for 4 h at r.t., H2O (100 mL)
was added and the organic layer was separated and dried (MgSO4).
After filtration, the crude product was purified by distillation at at-
mospheric pressure; the material with bp 90 °C was collected as
pure 3,4-epoxy-2-methylbut-1-ene.
MS (EI, 70 eV): m/z (%) = 242 [M+] (3), 227 (2), 180 (30), 157 (57),
139 (39), 111 (45), 97 (79), 87 (100), 71 (84), 57 (87), 43 (68).
[1,1-2H2]-3,7,11-Trimethyldodecan-1-ol (9)
A soln of 8 (1.58 g, 6.5 mmol) in anhyd Et2O (10 mL) was slowly
added to a suspension of LiAlD4 (0.41 g, 9.8 mmol) in anhyd Et2O
(10 mL) at 0 °C. The reaction mixture was then refluxed until com-
plete consumption of 10 (3 h). The mixture was cooled to 0 °C and
quenched with H2O (0.4 mL), 15% aq NaOH (0.4 mL), and H2O
(1.2 mL). The salts were filtered and the solvent was removed under
vacuum to provide alcohol 9 as a pale yellow oil, pure enough for
the next step of the synthesis without need of further purification.
Yield: 1.32 g (55%); colorless liquid.
1H NMR (400 MHz, CDCl3): d = 5.16 (m, 1 H), 5.01 (m, 1 H), 3.36
(m, 1 H), 2.85 (m, 1 H), 2.71 (m, 1 H), 1.61 (m, 3 H).
13C NMR (100 MHz, CDCl3): d = 141.3 (C), 114.5 (CH2), 54.4
(CH), 46.6 (CH2), 16.0 (CH3).
3,7,11-Trimethyldodecan-1-ol (7)11
A soln of farnesol (6; 5.00 g, 22.52 mmol) in EtOH (25 mL) was
subjected to hydrogenation at atmospheric pressure in the presence
of 10% Pt/C (200 mg) as catalyst.9 After the mixture had stirred at
r.t. for 12 h, the reaction was complete. The crude product was fil-
Yield: 1.5 g (99%).
IR (film): 3335, 2955, 2926, 2869, 1463, 1378, 969, 908, 736 cm–1.
© Thieme Stuttgart · New York
Synthesis 2012, 44, 862–864

864
L. Muñoz, A. Guerrero
SHORT PAPER
IR (film): 3329, 2954, 2925, 2869, 1463, 1378, 1002 cm–1.
1H NMR (400 MHz, CDCl3): d = 5.40 (m, 1 H), 4.14 (d, J = 8.0 Hz,
2 H), 1.97 (s, 2 H), 1.66 (s, 3 H), 1.52 (m, 1 H), 1.00–1.39 (br, 17
H), 0.85 (m, 12 H).
13C NMR (100 MHz, CDCl3): d = 140.2 (C), 123.1 (CH), 59.4
(CH2), 39.7 (CH2), 39.4 (CH2), 37.4 (CH2), 37.3 (CH2), 37.2 (CH2),
36.5 (CH2), 32.8 (CH), 32.6 (CH), 28.0 (CH), 24.8 (CH2), 24.5
(CH2), 22.7 (CH3), 22.6 (CH3), 19.7 (CH3), 19.6 (CH3), 16.2 (CH3).
1H NMR (400 MHz, CDCl3): d = 1.47–1.61 (br, 3 H), 1.01–1.41 (br,
14 H), 0.87 (m, 12 H).
13C NMR (100 MHz, CDCl3): d = 60.51 (quin, J = 21.0 Hz, CD2),
39.8 (CH2), 39.3 (CH2), 37.4 (CH2), 37.3 (CH2), 37.2 (CH2), 32.8
(CH), 29.4 (CH), 27.9 (CH), 24.8 (CH2), 24.3 (CH2), 22.7 (CH3),
22.6 (CH3), 19.7 (CH3), 19.6 (CH3).
MS (EI, 70 eV): m/z (%) = 230 [M+] (1), 212 (2), 182 (12), 142 (27),
127 (87), 111 (78), 99 (77), 85 (84), 71 (100), 57 (98), 43 (82).
Anal. Calcd for C15H30D2O: C, 78.19; H, 14.87. Found: C, 78.34; H,
14.50.
MS (EI, 70 eV): m/z (%) = 298 [M]+ (1), 280 (6), 139 (18), 125 (52),
111 (47), 97 (56), 84 (58), 71 (100), 57 (58), 43 (56).
HRMS (ESI): m/z calcd for C20H39D2O [M + 1]+: 299.3283; found:
299.3283.
[1,1-2H2]-1-Bromo-3,7,11-trimethyldodecane (10)
NBS (1.31 g, 7.4 mmol) was added portionwise over 30 min to a
soln of 9 (1.50 g, 6.5 mmol) and Ph3P (2.05 g, 7.8 mmol) in anhyd
CH2Cl2 (10 mL) at 0 °C.10a The reaction mixture was stirred for 30
min and then concentrated under reduced pressure. Purification by
column chromatography (silica gel, hexane) afforded 10.
Acknowledgment
We gratefully acknowledge G. Rosell (University of Barcelona) for
performing the HRMS analysis of compound 5, Generalitat de
Catalunya (2009SGR871) for a contract to L.M., and MICINN
(AGL2009-13452-C02-01) for financial support.
Yield: 1.82 g (95%); colorless oil.
IR (film): 3006, 2925, 2860, 1462, 1251, 908, 736 cm–1.
1H NMR (400 MHz, CDCl3): d = 1.87 (m, 1 H), 1.50–1.67 (br, 3 H),
1.08–1.37 (br, 13 H), 0.87 (m, 12 H).
References
13C NMR (100 MHz, CDCl3): d = 39.8 (CH2), 39.4 (CH2), 37.4
(CH2), 37.3 (CH2), 36.8 (CH2), 32.8 (CH), 31.7 (quin, J = 22.0 Hz,
CD2), 31.6 (CH), 28.0 (CH), 24.8 (CH2), 24.2 (CH2), 22.7 (CH3),
22.6 (CH3), 19.7 (CH3), 19.0 (CH3).
MS (EI, 70 eV): m/z (%) = 294 [M + 1]+ (1), 292 [M – 1]+ (1), 279
(3), 277 (3), 209 (36), 207 (38), 181 (28), 179 (29), 167 (27), 165
(28), 153 (44), 151 (46), 127 (62), 113 (79), 99 (42), 97 (44), 85
(72), 71 (100), 57 (99), 43 (43).
(1) van den Brink, D. M.; Wanders, R. J. A. Cell. Mol. Life Sci.
2006, 63, 1752.
(2) Netscher, T. Vitam. Horm. 2007, 76, 155.
(3) Daines, A. M.; Payne, R. J.; Humphries, M. E.; Abell, A. D.
Curr. Org. Chem. 2003, 7, 1625.
(4) McGinty, D.; Letizia, C. S.; Api, A. M. Food Chem. Toxicol.
2010, 48, S59.
(5) (a) Muralidharan, F. N.; Muralidharan, V. B. Biochim.
Biophys. Acta 1986, 883, 54. (b) Poulter, C. D.; Aoki, T.;
Daniels, L. J. Am. Chem. Soc. 1988, 110, 2620.
(6) Hanson, J. R. The Organic Chemistry of Isotopic Labeling;
The Royal Society of Chemistry: Cambridge, 2011, 200.
(7) (a) Emery, V. C.; Akhtar, M. Biochemistry 1987, 26, 1200.
(b) Samuel, O.; El Hachimi, Z.; Azerad, R. Biochimie 1974,
56, 1279. (c) Schulz, S.; Yildizhan, S.; van Loon, J. J. A.
J. Chem. Ecol. 2011, 37, 360. (d) Urano, S.; Matsuo, M.
Heterocycles 1984, 22, 1975.
Anal. Calcd for C15H29D2Br: C, 61.42; H, 11.34; Br, 27.24. Found:
C, 61.36; H, 10.98; Br, 27.03.
[5,5-2H2]-(E)-3,7,11,15-tetramethylhexadec-2-en-1-ol (5)
1,2-Dibromoethane (32 mL, 0.37 mmol) was added to a suspension
of Mg turnings (0.19 g, 7.8 mmol) in anhyd THF (1.5 mL). The mix-
ture was stirred at 50 °C for 15 min, the solvent was removed by sy-
ringe, and the black activated turnings were suspended again in
anhyd THF (1.5 mL). A soln of 10 (0.57 g, 1.9 mmol) in anhyd THF
(1.5 mL) was slowly added and the mixture was stirred at r.t. for 2
h. The Grignard reagent soln of 10 was added at –78 °C via cannula
to a second flask containing a suspension of CuI (15 mg, 97 mmol)
in THF (1.5 mL). The reaction mixture was stirred at –78 °C for 5
min. Then, 3,4-epoxyisoprene (0.19 g, 2.3 mmol) was added and af-
ter additional stirring for 5 min at –78 °C, the mixture was allowed
to warm to r.t. for 1 h. After this time, the solvent was removed un-
der reduced pressure and the crude was directly purified by column
chromatography (silica gel, hexane–Et2O, 4:1); this afforded alco-
hol 5.
(8) Harwood, L. M.; Casy, G.; Sherlock, J. Synth. Commun.
1990, 20, 1287.
(9) MacCarthy, E. D.; Calvin, M. Tetrahedron 1967, 23, 2609.
(10) (a) Hengartner, U.; Chougnet, A.; Liu, K.; Woggon, W. D.
Chem. Eur. J. 2010, 16, 1306. (b) Wang, X.-Z.; Wu, Y.-L.;
Jiang, S.; Singh, G. Tetrahedron Lett. 1999, 40, 8911.
(11) Silva, I.; Rocha, S. M.; Coimbra, M. A.; Marriott, P. J.
J. Chromatogr. A 2010, 1217, 5511.
(12) Havlik, J.; Kokoska, L.; Vasickova, S.; Valterova, I. Flavour
Fragr. J. 2006, 21, 713.
Yield: 0.26 g (45%), E/Z = 96:4, colorless oil.
Synthesis 2012, 44, 862–864
© Thieme Stuttgart · New York