Convenient synthesis of deuterium labelled sesquiterpenes

Article abstract of DOI:10.1016/j.tetlet.2016.08.079

Sesquiterpenes are an important class of molecules, with roles ranging from pollination and signalling to defense mechanisms. Despite their apparent importance, the limited number of commercial standards has hindered their study and precise quantification. Herein, we report the syntheses of fourteen labelled sesquiterpenes with a high level of deuterium incorporation (>95%) for applications in MS-based studies.

Full text of DOI:10.1016/j.tetlet.2016.08.079

Tetrahedron Letters 57 (2016) 4496–4499
Contents lists available at ScienceDirect
Tetrahedron Letters
journal homepage: www.elsevier.com/locate/tetlet
Convenient synthesis of deuterium labelled sesquiterpenes
a,
Nina Duhamel a, Damian Martin b, Roberto Larcher c, Bruno Fedrizzi a, , David Barker
⇑
⇑
a School of Chemical Sciences, University of Auckland, 23 Symonds St, Auckland, New Zealand
b The New Zealand Institute for Plant & Food Research Limited, 85 Budge Street, Blenheim 7240, Marlborough, New Zealand
c FEM-IASMA Fondazione Edmund Mach – Istituto Agrario di San Michele all’Adige, via E. Mach 1, 38010 San Michele all’Adige (TN), Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Sesquiterpenes are an important class of molecules, with roles ranging from pollination and signalling to
defense mechanisms. Despite their apparent importance, the limited number of commercial standards
has hindered their study and precise quantification. Herein, we report the syntheses of fourteen labelled
sesquiterpenes with a high level of deuterium incorporation (>95%) for applications in MS-based studies.
Ó 2016 Elsevier Ltd. All rights reserved.
Received 26 July 2016
Revised 24 August 2016
Accepted 26 August 2016
Available online 28 August 2016
Keywords:
Sesquiterpenes
Isotopic labelling
Deuterium
Terpenoids
Sesquiterpenes are an important member of the terpenoid fam-
ily. Like other members of this family they are made of isoprene
labelled species that could have potentially been formed. Similarly,
the total syntheses of sesquiterpenes have been widely reported in
the literature,14 but these have not proven useful in terms of iden-
tifying and especially quantifying these species in plant tissues as
no labelled sesquiterpenes were also synthesised.
The availability of pure, isotopically labelled, sesquiterpene
analogues could prove pivotal to study the formation and evolution
of these molecules in complex biological matrices, as vastly proven
in other fields.15 Therefore instead of introducing labelled
sesquiterpene building blocks, which can lead to the formation of
a number of sesquiterpenes without the possibility of resolving
them, we have proposed synthetic strategies that allowed us to
obtain new isotopically labelled sesquiterpenes. These molecules
could then be used for analytical purposes in MS-based Standard
Isotopic Dilution Analysis (SIDA) approaches.
The general approach was to obtain commercially available
sesquiterpenes or sesquiterpenoids, and carry out efficient syn-
thetic chemistry on these, in order to introduce deuterium atoms.
The introduced deuterium atoms are required to be sufficiently
stable to be retained in some of the fragments formed during ion-
isation in MS or MS/MS analyses. The use of deuterium labelling
was chosen over the use of 13C labelled compounds as this allowed
commercially available non-labelled compounds to be utilised as
starting materials. 13C labelled sesquiterpenes have recently been
reported for use in mechanistic studies on this class of compounds,
however, they require de novo synthesis from more expensive
labelled precursors.16
units, three in this case, and have an empirical formula of C15H24
.
More than 5000 sesquiterpenes have been identified and their
roles range from plant growth regulators1 to plant signalling
species,2 to herbivore-induced plant defences.3 Some plant-derived
sesquiterpenoids have also been identified as anti-inflammatory
and anti-carcinogenic species.4
Their biogenesis is strictly linked to that of monoterpenes for
which the current knowledge is much more extended.5 In reality,
sesquiterpene biogenesis is less understood, even if some crucial
steps, such as the importance of the DOXP pathway and of the
mevalonic acid pathway have been pinpointed.6,7 These molecules
possess a hydrocarbon backbone and have been grouped into a
number of skeletal types according to their structure.8 The highly
diverse and complex skeletons have made the unique identifica-
tion and quantification extremely challenging. Even in the most
advanced and recent studies9–11 significant limitations on their
chemical detections are encountered, with several sesquiterpenes
only tentatively identified and with almost no quantitative detec-
tion due to the lack of suitable standards.
Isotopic labelled precursors have been previously used to study
the biogenesis of sesquiterpenes as well as the enzymes responsi-
ble for the huge range of cyclised products.7,12,13 However, these
studies fall short in the quantification and identification of other
⇑
Corresponding authors. Tel.: +64 9 373 7599; fax: +64 64 373 7422.
E-mail addresses: b.fedrizzi@auckland.ac.nz (B. Fedrizzi), d.barker@auckland.ac.
The first set of standards were created using hydroxyl
containing sesquiterpenoids. The first structure used was the
nz (D. Barker).
http://dx.doi.org/10.1016/j.tetlet.2016.08.079
0040-4039/Ó 2016 Elsevier Ltd. All rights reserved.

N. Duhamel et al. / Tetrahedron Letters 57 (2016) 4496–4499
4497
commercially available sesquiterpene (E,E)-farnesol 1. (E,E)-Far-
H
H
H
H
nesol 1 and its derivatives (E,E)-farnesyl acetate and (E,E)-methyl
farnesoate are all known volatile compounds, though their contri-
bution to wine aroma has not been explored. Addition of an acetate
group via reaction of the primary alcohol with acetyl chloride pro-
ceeded in a straight forward manner to give (E,E)-farnesyl acetate.
Replacing acetyl chloride with (D3)-acetyl chloride provided a sim-
ple method for deuterated methyl group introduction. However,
though easy to introduce, these acetyl deuterium labels can be
easily lost to fragmentation during mass spectrometry resulting
in the majority of the standards’ fragments being identical to the
target compounds’. In a revised synthesis, (E,E)-farnesol was
oxidised to farnesal, then to (E,E)-farnesoic acid 2 using a Pinnick
oxidation.17 Reduction of the acid using lithium aluminium deu-
teride, gave (D2)-farnesol 3. Acetylation as above, using (D3)-acetyl
chloride, was then carried out, giving (D5)-farnesyl acetate 4.
The second standard was made from (E,E)-farnesoic acid 2 via
esterification using (D3)-methanol and 1-ethyl-3-(3-dimethy-
laminopropyl)carbodiimide (EDC) to form (D3)-methyl farnesoate
5 in 63% yield (Scheme 1).
O
a
O
O
c
b
O
H
D
H
H
H
OH
OH
O
7
D
(-)-Caryophyllene oxide
7a 58%
82%
8 57%
d
H
H
c
H
D
H
O
D
10 71%
11a 59%
H
H
H
H
H
H
H
H
H
H
H
H
a
c
b
H
H
H
O
H
HO
D
D
OH
(+)-Aromadendrene
6
79%
9
100%
6a 71%
The next set of standards were made from two commercially
available cyclic sesquiterpenes, (+)-aromadendrene 6 and (À)-
caryophyllene oxide 7. These sesquiterpenes contain a single
terminal alkene, and no other functional groups, other than the
epoxide on the (À)-caryophyllene oxide 7. The designed approach
to deuterium labelling was the oxidation of the terminal alkene
groups to the ketone derivatives, which would then undergo reac-
tion with a deuterium labelled Wittig reagent, resulting in the orig-
inal sesquiterpene where the terminal protons are replaced with
deuteriums.
Ozone was initially explored for the synthesis of the ketone
derivatives, but this resulted in significant amounts of side-prod-
ucts. However, use of a two-step procedure of dihydroxylation fol-
lowed by diol cleavage gave the desired norsesquiterpene ketones
8 and 9 in good yield with little by-product formation. Another
sesquiterpene was prepared via a de-epoxidation reaction, using
an iodohydrin intermediate, of (À)-caryophyllene oxide ketone
derivative (kobusone) 8 to form the b-caryophyllene ketone
derivative 10 (Scheme 2). The de-epoxidation reaction gave a
5.5:1 ratio of the trans isomer, over the cis isomer
(isocaryophyllene).
Scheme 2. Synthesis of deuterated alkenes. (a) OsO4 (0.01 equiv), NMO (3 equiv), t-
BuOH/H2O 3:1, 70 h; (b) sodium metaperiodate (1.2 equiv), MeOH/H2O 3:1, 4 h; (c)
CD3PPh3Br (2 equiv), n-BuLi (1.5 equiv), THF, À78 °C to rt, 24 h; (d) Zn (5.7 equiv),
NaI (1.7 equiv), NaOAc (1.03 equiv), AcOH, 48 h, rt.
H
H
H
b
a
O
O
O
H
H
H
D
D
D
D
D
O
O
D
8a
58%
8
7b 79%
H
H
H
H
H
H
H
H
H
D
b
a
D
H
D
D
D
D
O
O
D
D
9a
6b 71%
9
90%
H
H
H
H
H
a
b
The deuterated Wittig reagent was freshly prepared by stirring
methyl triphenylphosphonium bromide in excess deuterium oxide
with sodium deuteroxide for 4 h.18 The reaction time and the
H
D
D
O
D
O
D
D
D
10
10a 98%
48%
11b
Scheme 3. Synthesis of
penes. (a) D2O (16 equiv), NaOD (1 equiv), dioxane, reflux 100 °C, 40 h; (b) CD3-
PPh3Br (2 equiv), n-BuLi (1.5 equiv), THF, À78 °C to rt, 24 h.
a
-deuterated ketones and highly substituted sesquiter-
D
OH
OH
D
(D2)-Farnesol 3
(
)-Farnesol 1
E,E
c
amount of sodium deuteroxide was optimised to maximise the
percentage of deuterium exchange, while minimising the forma-
tion of the ylide. Although ultimately ylide formation is desired
for the Wittig reaction, its formation during the deuterium
exchange step is undesired, as the reactive ylide degrades with
prolonged storage. The optimum stirring time, for the preparation
of 1 g of deuterated reagent was 4 h, using 0.50 mL of sodium
deuteroxide (30 wt.% in D2O) in 10 mL of D2O. Standard Wittig
reaction conditions were then successfully employed with ketones
8, 9 and 10 using the labelled Wittig reagent to give D2-labelled
products 6a, 7a, 11a in 48–79% yields with approximately 95% deu-
terium incorporation.19
d
a,b
D
O
O
OH
D
D
D
O
D
(D5)-Farnesyl acetate 4
2
Farnesoic acid
e
D
O
D
O
D
(D3)-Methyl farnesoate 5
Scheme 1. Synthesis of farnesol based standards. (a) 1. DMP (1.5 equiv), CH2Cl2, rt,
4 h; (b) NaClO2 (9 equiv), NaH2PO4 (7 equiv), t-BuOH/2-methyl-2-butene 4:1, rt,
48 h. 92% (over two steps); (c) LiAlD4 (2 equiv), Et2O, 1 h, 94%; (d) (D3)-acetyl
chloride (1.2 equiv), pyridine (1.2 equiv), CH2Cl2, 23 h, 55%; (e) EDC (10 equiv),
DMAP (16 equiv), CH2Cl2, CD3OH (50 equiv), 63%.
Considering the possible EI-MS structural lability of the deuter-
ated methylene groups from these terminal alkenes, it was decided
to introduce additional deuterium labels to the carbon adjacent to

4498
N. Duhamel et al. / Tetrahedron Letters 57 (2016) 4496–4499
12a. The rearrangement occurs under harsh conditions upon reac-
H
H
H
tion of aromadendrene 6 with potassium metal on Al2O3 in hex-
anes, but no mechanistic information has been reported.23 It was
discovered by undertaking the rearrangement on (D2)-labelled aro-
madendrene 6a, that the deuteriums remain on the exocyclic
methyl (C-4), with no loss of deuterium at this position; thus
suggesting that proton transfer from C-1 and C-2 to C-3 and C-4
occurs without scrambling of the protons at the terminal C-4 posi-
tion (Scheme 4).
H
H
1
1
a
2
2
3
3
H
D
4
4
D
D
D
(D2)-(+)-Aromadendrene 6a
(D2)-Isoledene 12a
Scheme 4. Rearrangement of labelled aromadendrene 6a to isoledene 12a.
(a) K/Al2O3, hexanes, 12 h, rt, 50%.
Preliminary studies in grape and wine were based on gas chro-
matography tandem mass spectrometry (GC–MS/MS). Optimisa-
tion of the MS/MS conditions allowed us to focus on the
molecular ion and exploit the 3–5 Da m/z shift caused by the stable
isotopic labelling. The standards prepared were used as internal
standards in stable isotopic dilution (SID) MS/MS studies, to accu-
rately quantify sesquiterpene levels in biological samples. Figure 1
shows the EI-MS/MS scan spectra obtained for some labelled and
unlabelled sesquiterpenes studied.
In conclusion the methods presented herein provide a robust
route for the synthesis of deuterated sesquiterpenes with various
amounts of deuteration as required. The use of these labelled
sesquiterpenes in the quantitative analysis of plant and fruit matri-
ces will be reported in due course.
the terminal alkene. This was possible by exchange of the acidic a-
protons of the previously prepared ketone intermediates. Using a
method reported by Tkachov et al.,20 deuteration of ketones 8–10
was found to be efficient giving deuterated ketones 8a–10a.21
Deuteration of ketones 8 and 10 resulted in exchange of the a-pro-
tons on the 9 membered ring, but not of the proton at the bridging
carbon with the cyclobutane ring. However, with ketone 9 deu-
terium substitution occurred at both the
a-carbons, resulting in
(D3)-ketone 9a (Scheme 3). It should be noted that the labelled
ketones 8a–10a are also useful SIDA MS/MS analysis standards,
as these norsesquiterpenes have all been identified as either natu-
ral products or, for 9a, of interest as a synthetic starting material.22
With these (D2) and (D3) labelled ketones in hand, the Wittig
reaction was undertaken on ketones 8a–10a to give (D4) and (D5)
labelled sesquiterpenes 6b, 7b and 11b, now containing deuterium
labels on both cyclic and exocyclic carbons (Scheme 3).
Acknowledgements
This work was funded by grants to B.F. from the New Zealand
Ministry of Business, Innovation and Employment, New Zealand.
Finally an additional deuterated sesquiterpene was prepared
via the rearrangement of (D2)-aromadendrene 6a to (D2)-isoledene
Figure 1. Examples of MS experiment with labelled and unlabelled sesquiterpenes fragmentation.

N. Duhamel et al. / Tetrahedron Letters 57 (2016) 4496–4499
4499
13. Citron, C. A.; Rabe, P.; Barra, L.; Nakano, C.; Hoshino, T.; Dickschat, J. S. Eur. J.
Org. Chem. 2014, 34, 7684–7691.
Winegrowers and Plant and Food Research and the FEMIASMA
Fondazione Edmund Mach—Istituto Agrario di San Michele all’A-
dige. We also acknowledge the University of Auckland for addi-
tional funding.
14. (a) Fraga, B. M. Nat. Prod. Rep. 2013, 30, 1226–1264; (b) Kuwahara, S.; Hamade,
S.; Leal, W. S.; Ishikawa, J.; Kodama, O. Tetrahedron 2000, 56, 8111–8117.
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Supplementary data
Supplementary data (full experimental details, characterisation
data and NMR spectra for all compounds) associated with this arti-
cle can be found, in the online version, at http://dx.doi.org/10.
1016/j.tetlet.2016.08.079.
16. (a) Rabe, P.; Dickschat, J. S. Beilstein J. Org. Chem. 2016, 12, 1380–1394; (b) Rabe,
P.; Barra, L.; Rinkel, J.; Riclea, R.; Citron, C. A.; Klapschinski, T. A.; Janusko, A.;
Dickschat, J. S. Angew. Chem., Int. Ed. 2015, 54, 13448–13451.
17. Bal, B. S.; Childers, W. E., Jr; Pinnick, H. W. Tetrahedron 1981, 37, 2091–2096.
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19. General procedure for deuteration of sesquiterpene ketones: To
methyltriphenylphosphonium bromide or (D3)-methyltriphenylphosphonium
bromide (0.5 mmol) in THF (1.0 mL) under nitrogen at À78 °C was added n-
BuLi (0.36 mmol) dropwise. The resulting mixture was stirred at 0 °C for
10 min, then cooled to À78 °C before adding a solution of ketone (0.24 mmol)
in THF (1 mL) dropwise. The mixture was warmed to room temperature and
stirred for a further 24 h. Water (5 mL) was slowly added, and the resulting
mixture extracted with diethyl ether (25 mL). The organic layer was washed
with 2 M HCl (5 mL), then 4 M NaOH (5 mL). The combined basic aqueous
washes were further extracted with diethyl ether (3 Â 20 mL). The combined
organic extracts were dried (MgSO4) and the solvent was removed in vacuo.
The crude product was purified using flash chromatography (petroleum ether)
to give the desired alkenes.
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´