Synthesis of isotopically labelled oligoprenyl diphosphates and their application in mechanistic investigations of terpene cyclases

Article abstract of DOI:10.1002/ejoc.201403002

A flexible, efficient and robust method for the synthesis of isotopically labelled oligoprenyl diphosphates was developed. The method makes use of just a few building blocks (acetone, triethyl phosphonoacetate, and ethyl acetoacetate) from which several i

Full text of DOI:10.1002/ejoc.201403002

FULL PAPER
DOI: 10.1002/ejoc.201403002
Synthesis of Isotopically Labelled Oligoprenyl Diphosphates and Their
Application in Mechanistic Investigations of Terpene Cyclases
[a]
[a]
[a]
[b]
[b]
Christian A. Citron, Patrick Rabe, Lena Barra, Chiaki Nakano, Tsutomu Hoshino,
and Jeroen S. Dickschat*[
a]
Keywords: Synthetic methods / Biosynthesis / Terpenoids / Isotopic labelling / Phosphorylation
A flexible, efficient and robust method for the synthesis of
isotopically labelled oligoprenyl diphosphates was devel-
oped. The method makes use of just a few building blocks
reagents were used for the introduction of deuterium label-
ling. Furthermore, the synthesis of [14- H]geranylgeranyl di-
phosphate is reported. The material was used for a stereo-
chemical analysis of the cyclisation reaction catalysed by
2
(
acetone, triethyl phosphonoacetate, and ethyl acetoacetate)
1
3
from which several isotopomers with deuterium or C-label- tuberculosinyl diphosphate synthase from Mycobacterium
ling are commercially available or can be easily obtained by
synthesis. Besides these building blocks, a few deuterated
tuberculosis.
terpene biosynthesis[12–15] are stereospecifically labelled pre-
Introduction
2
cursors such as (1S)- and (1R)-[1- H]FPP that have been
The biosynthesis of terpenes starts with the formation of
the universal C building block dimethylallyl diphosphate
[12]
made available by enzymatic reactions. All these isotopi-
5
cally labelled terpene precursors can be efficiently used in
incubation experiments with recombinant terpene cyclases.
Alternatively, for feeding experiments with actively growing
cultures, isotopomers of the monomeric building blocks
mevalonolactone or deoxyxylulose, depending on which
pathway to terpenes is present in the particular organism,
can be used, because these molecules are, in contrast to the
oligoprenyl diphosphates, able to pass the cell membrane.
For investigations on various problems of terpene biosyn-
thesis via feeding experiments, we have recently developed
flexible methods for the synthesis of various deuterated and
(DMAPP) and isopentenyl diphosphate (IPP) that arise
either via the mevalonate or the deoxyxylulose phosphate
pathway. These monomers are subsequently fused by oligo-
prenyl diphosphate synthases to yield the linear precursors
geranyl diphosphate (GPP, C ), farnesyl diphosphate (FPP,
1
0
C ) and geranylgeranyl diphosphate (GGPP, C ) with all-
1
5
20
E configurations. These linear precursors are then cyclised
by terpene cyclases in complex reaction cascades via cat-
ionic intermediates, usually into terpene hydrocarbons or
[
1]
terpene alcohols. Isotopomers of the linear terpene pre-
2
13
3
cursors labelled with stable ( H, C) or radioactive ( H,
13
[16]
C-labelled isotopomers of mevalonolactone
and de-
1
4
C) isotopes have been frequently used to follow the stereo-
[17]
oxyxylulose, including stereospecifically deuterated mev-
alonolactone isotopomers. In an examplary investigation
we have fed isotopically labelled [6,6,6- H ]mevalono-
[2,3]
chemical course of terpene cyclisations,
the regioselectiv-
[18]
[
4,5]
ity of deprotonation steps,
or other mechanistic aspects
For these purposes, numerous
2
3
of terpene biosynthesis.[
6,7]
lactone to Fusarium fujikuroi. This compound is trans-
synthetic approaches to isotopomers of the linear terpene
2
formed via the mevalonate pathway into [ H ]GGPP with
12
precursors DMAPP,[
8]
GPP,
[2,4,6,9,10]
FPP,
[5,7,11]
and
deuterated methyl groups as shown in Scheme 1 (a). The
stereochemical course of its conversion into ent-copalyl di-
phosphate 1 by the ent-copalyl diphosphate synthase (CPS)
with respect to the geminal methyl groups was demon-
[
3]
GGPP have been described, aiming at the introduction of
isotope labels at specific and well-defined positions. Particu-
larly interesting in addressing stereochemical problems of
[
19]
strated.
[
a] Kekulé-Institut für Organische Chemie und Biochemie,
Rheinische Friedrich-Wilhelms-Universität Bonn,
Gerhard-Domagk-Straße 1, 53121 Bonn, Germany
E-mail: dickschat@uni-bonn.de
A recent example from our Japanese laboratories of
using isotopically labelled oligoprenyl diphosphates for in-
cubation experiments with purified recombinant terpene
http://www.chemie.uni-bonn.de/oc/forschung/arbeitsgruppen/
ak_dickschat
cyclases is shown in Scheme 1 (b). Incubation of [16,16,16-
2
H ]GGPP with tuberculosinyl diphosphate synthase
3
[b] Department of Applied Biological Chemistry, Faculty of
Agriculture and Graduate School of Science and Technology, (Rv3377c) from Mycobacterium tuberculosis allowed us to
Niigata University,
follow the stereochemical course of the cyclisation reaction
Nishi-ku, Ikarashi 2-8050, Niigata 950-2181, Japan
to tuberculosinyl diphosphate (2) via a chair-chair transi-
Supporting information for this article is available on the
[
3,20]
WWW under http://dx.doi.org/10.1002/ejoc.201403002.
tion state (Scheme 1, b).
Usually for the synthesis of
7684
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 7684–7691

Isotopically Labelled Oligoprenyl Diphosphates
oligoprenyl diphosphates 8. This route was previously used
by us for the synthesis of unlabelled FPP and GGPP start-
ing from 4b, thus proving its practicability over two elong-
ation cycles with an overall yield of 3% via 12 steps for the
synthesis of GGPP (yields for each step are summarised in
[
21]
Table 1). A peculiar problem of the HWE reactions with
c and 1d is the formation of E- and Z-stereoisomers, thus
1
requiring rigorous product purification by repeated column
chromatography (3c: dr = 4:1, 3d: dr = 11:1, ratios based
on isolated diastereomerically pure material).
Scheme 1. Stereochemical course of cyclisation of GGPP a) to ent-
copalyl diphosphate (1) by Fusarium fujikuroi ent-copalyl diphos-
phate synthase (CPS), and b) to tuberculosinyl diphosphate (2) by
Mycobacterium tuberculosis tuberculosinyl diphosphate synthase
(Rv3377c). Asterisks indicate completely deuterated methyl groups.
isotopically labelled oligoprenyl diphosphates with labelling
in specific positions, a large number of steps is required,
and a different strategy for the introduction of labelling for
each position has to be developed. We now present a gen-
erally applicable approach for the synthesis of deuterium
13
or C-labelled oligoprenyl diphosphates (GPP, FPP, and
GGPP) that can be used for the introduction of labelling
into every position and all combinations of positions from
cost-effective commercially available labelled building
2
blocks. The compound [14- H]GGPP was synthesised and Scheme 2. Synthetic strategy towards oligoprenyl diphosphates.
used in an incubation experiment with tuberculosinyl di-
phosphate synthase, giving further insights into the stereo-
chemical course of the reaction.
Table 1. Isolated yields of reactions according to Scheme 2.
Compd.[a]
a
b
c
d
1
n. a.[b]
18% ( H14
2
)
)
93%[c]
88%[c]
2
2
Results and Discussion
93% ( H
1
)
88% ( H
1
)
2
2
[c]
[c]
3
4
72% ( H
7
)
45% ( H15
55%
56%
2
2
Our strategy for the synthesis of labelled oligoprenyl di-
phosphates with flexible introduction of stable isotope
labelling is shown in Scheme 2. Starting from acetone (1a)
we pursued a sequence of Horner–Wadsworth–Emmons
32% ( H
1
)
46% ( H
1
)
2
[c]
[c]
79% ( H
9
)
68%
85%
2
2
7
5% ( H
1
)
72% ( H
1
)
5
7
(crude product was used without purification)
2
[c]
[c]
38% ( H13
)
78%
76% ( H )
83%
65%
n. a.
2
2
(
HWE) olefination with triethyl phosphonoacetate (2) to
ethyl 3,3-dimethylacrylate (3a), DIBAL reduction to prenol
4a), transformation with PBr3 into the corresponding
58% ( H )
1
1
[
c]
[c]
38%[c]
8
76%
2
3
1
0% ( H )
(
bromide 5a, alkylation with ethyl acetoacetate (6) to the β- [a] Alphanumerical compound identifiers are according to bold
numbers in Table lines to be combined with letters in Table col-
umns. [b] n. a.: not applicable. [c] For comparison, the yields for
keto ester 7a, and saponification with spontaneous decarb-
oxylation to sulcatone (1b). In summary, this five-step pro-
cedure results in chain elongation by one isoprene unit.
We aimed at the higher homologs of 4a, i.e. geraniol (4b),
farnesol (4c), and geranylgeraniol (4d) by repeated analo-
unlabelled materials as reported previously were taken from refer-
[21]
ence.
A major advantage of the route presented in Scheme 2 is
gous reactions. Their corresponding halides 5 were planned the possibility to incorporate 13C-labellings into every posi-
to be subjected to nucleophilic substitution with tris(tetra- tion of the oligoprenyl diphosphates from commercially
butylammonium) hydrogen diphosphate to yield the desired available building blocks: All three compounds used to
Eur. J. Org. Chem. 2014, 7684–7691
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7685

J. S. Dickschat et al.
FULL PAPER
build the carbon frameworks, i.e. 1a, 2, and 6, can be ob-
1
3
tained with a single C-labelling in each (incorporated) po-
sition or as multiply labelled compounds with various com-
1
3
13
binations of C-labellings (Figure 1). The C-labelling at
the ester carbonyl carbon of 6 as it is present in two com-
mercial isotopomers will be lost in the saponification/de-
carboxylation step from intermediates 7 to 1. However, the
respective isotopomers without a labelling of this lost
carbon are not available from standard suppliers of fine
chemicals. Therefore, the usage of the isotopomers as sum-
marised in Figure 1 offers the most practical solution, if a
synthesis aims at an introduction of the additional label-
lings at C-2 or at all three other carbons of 6.
2
2
2 5
Scheme 3. Synthesis of [ H ]-2 and [ H ]-6.
2
2
ation of [ H ]-6 with [ H ]-5a proceeded with complete loss
5
9
2
of the acidic deuterium at the α-carbon to yield [ H ]-7a
instead of the desired [ H ]-7a, even if the reaction was
12
2
13
performed in deuterated acetone as solvent. The most likely
2
1
explanation for this finding is a H/ H-exchange with sil-
anol groups of the silica gel in the chromatographic purifi-
cation of deuterated 7a. The lost deuterium could, however,
be reintroduced in the subsequent deethoxycarbonylation
Figure 1. Commercially available deuterated and 13C-labelled isoto-
pomers of acetone (1a), triethyl phosphonoacetate (2), and ethyl
acetoacetate (6).
2
2
2
2
with KO H in H O/EtO H to [ H ]-1b (herein, it is also
2
12
While the introduction of 13C-labellings, although not crucial to use HCl in the acidic workup). The yields for
2
performed in this study, is an obvious possibility, a special the overall procedure are summarised in Table 1 (cf. com-
2
2
2
2
difficulty during the synthesis of analogous deuterated com- pounds [ H ]-3a, [ H ]-4a, [ H ]-7, and [ H ]-1b). In sum-
7
9
13
14
2
1
pounds arises by the danger of deuterium losses in H, H- mary, this work demonstrated that the route of Scheme 2
exchange reactions, e.g. during aqueous workup. Therefore, can be used to incorporate deuterium into any of the posi-
we wanted to prove the operability of the route for the syn- tions of the oligoprenyl diphosphates with high incorpora-
thesis of all kinds of isotopomers of deuterated oligoprenyl tion rates. However, in contrast to the HWE reaction from
2
2
2
2
diphosphates by the completion of one cycle from [ H ]-1a [ H ]-1a with [ H ]-2 to [ H ]-3a, the corresponding conver-
6
6
2
7
2
2
2
2
to [ H ]-1b. While [ H ]-1a is commercially available, the sion of [ H ]-1b into [ H ]-3b showed a 66% loss of deu-
1
4
6
14
15
2
2
other two required building blocks [ H ]-2 and [ H ]-6 were terium at the olefinic C-2 carbon. This loss of deuterium
made available by synthesis (Scheme 3). Deuterated [ H ]-2 was observed even under aqueous workup with H O, dem-
2
5
2
2
2
2
with a high deuterium content (Ͼ99%) was obtained from onstrating that the HWE reaction is somewhat unreliable in
1
2
2
unlabelled 2 by basic H, H-exchange with K CO in H O. terms of deuterium introduction.
2
3
2
2
The synthesis of [ H ]-6 was possible from Meldrum’s acid
A major disadvantage of the transformations from 1a to
5
2
(
9), which can likewise be converted to [ H ]-9 by stirring in 1b as shown in Scheme 2 is the volatility of all products
H O and K CO . Its acylation with commercially available within this first elongation cycle which engenders signifi-
H ]acetyl chloride yielded [ H ]-10, if H O and HCl cant losses of isotopically labelled material during product
2
2
2
2
3
[2
2
2
2
3
4
2
were used for aqueous workup of the reaction. The crude isolation. To overcome this problem, an easier-to-handle al-
2
product was stirred in boiling EtO H (deuterium content ternative approach for the introduction of labellings into
2
Ͼ99%) to yield the desired [ H ]-6. All three deuterated the C-terminal portion of 4b was developed (Scheme 4; a
5
2
2
2
building blocks [ H ]-1a, [ H ]-2, and [ H ]-6 were sub- similar approach was reported by Stratakis and co-
6
2
5
[
22]
sequently used to aim at a synthesis of fully deuterated workers ). The synthetic route starts from 4b that was
2
2
2
[
[
H ]-1b. Starting from [ H ]-1a, the HWE reaction with protected with TBDPSCl to yield the silyl ether 11. Epoxid-
14 6
2
2
2
[23]
H ]-2 to [ H ]-3a, the reduction with LiAl H to [ H ]-4a ation to 12 and periodate cleavage resulted in 13, which
and the conversion into [ H ]-5a all resulted in completely was converted with KOH and iodine in methanol into the
2
7
4
9
2
9
deuterated materials. Surprisingly, the subsequent alkyl- methyl ester 14. This reaction proved to be low-yielding on
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Eur. J. Org. Chem. 2014, 7684–7691

Isotopically Labelled Oligoprenyl Diphosphates
2
sequently converted into [14- H]-8d by the methodology of
Scheme 2. The obtained yields of this procedure are also
summarised in Table 1.
2
The synthetic compound [14- H]-8d was subsequently
used in an incubation experiment with the tuberculosinyl
diphosphate synthase from M. tuberculosis to investigate
the stereochemical course of the reaction (Scheme ). The
product obtained was dephosphorylated with acid phos-
phatase from potato to tuberculosinol (19) and then ana-
1
lysed by H NMR spectroscopy. In unlabelled 19, the signal
2
2
7
Scheme 4. Synthesis of [ H]-4b and [ H ]-4b.
a large scale, and therefore, 13 was converted into 14 in ten
separate ca. 20 mmol portions. Introduction of deuterium
2
was performed by reduction with LiAl H to the alcohol
4
2
[
H ]-15 which was followed by a sequence of tosylation
2
and two subsequent nucleophilic substitutions with iodide
2
and triphenylphosphane to the Wittig salt [ H ]-18. A Wit-
2
2
tig reaction either with unlabelled 1a or [ H ]-1a gave access
6
2
2
to the TBDPS ethers [ H]-11 and [ H ]-11. A final deprotec-
7
tion with TBAF yielded the deuterated geraniol isotopo-
2
2
mers [ H]-4b and [ H ]-4b in 19% and 16% overall yield,
7
2
respectively, via 10 steps. The compound [ H]-4b was sub-
1
1
Scheme 5. Stereochemical course of cyclisation of GGPP to tuber-
Figure 2. a) H NMR spectrum of 19, b) H NMR spectrum of
2 2
culosinyl diphosphate (2) by Mycobacterium tuberculosis tuberculo-
(R)-[3- H]-19 obtained from the incubation of [14- H]-8d with tu-
berculosinyl diphosphate synthase and dephosphorylation, and c)
2
sinyl diphosphate synthase (Rv3377c). Incubation of [14- H]GGPP,
1
1
2
dephosphorylation by potato acid phosphatase, and H NMR
H NMR spectrum of (S)-[3- H]-19 obtained from the incubation
analysis of the product (Figure 2) demonstrated that protonation
at C-14 proceeds from the Si side.
of 8d with tuberculosinyl diphosphate synthase in deuterium oxide
and dephosphorylation.
Eur. J. Org. Chem. 2014, 7684–7691
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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7687

J. S. Dickschat et al.
FULL PAPER
at δH = 1.39 exhibited a ddd splitting pattern (J = 12.5, out using Merck silica gel 60 (70–200 mesh). 1H NMR and 13
C
NMR spectra were recorded with Bruker DRX-400 (400 MHz) or
1
2.5, 5.4 Hz), indicating that this signal can be assigned to
H-3β, while the signal at δH = 1.53 was assignable to H-3α
Figure 2, a). The latter signal overlapped with that of H-
1, but these assignments were unambiguously confirmed
AV III-400 (400 MHz) spectrometers, and were referenced against
1
TMS (δ = 0.00 ppm) for H NMR and CDCl
C-NMR spectroscopy. IR spectra were recorded with a Bruker
3
(δ = 77.01 ppm) for
(
1
1
3
Tensor 27 ATR (attenuated total reflectance) instrument. GC–MS
analyses were carried out with a HP 6890 gas chromatograph con-
nected to a HP 5973 inert mass detector fitted with a BPX-5 (25 m,
2
by the HSQC spectrum. Feeding of [14- H]-8d resulted in a
missing signal for the 3-pro-R hydrogen (H-3α) in the H
NMR spectrum (Figure 2, b), indicating that [14- H]-8d is
converted into (R)-[3- H]-19, consistent with a protonation 0.25 μm film) fused silica capillary column. Instrumental param-
of the substrate from the Si side at C-14. This result was eters were (1) inlet pressure, 77.1 kPa, He flow 23.3 mLmin , (2)
corroborated by a complementary experiment in which un- injection volume, 2 μL, (3) transfer line, 300 °C, and (4) electron
labelled 8d was incubated with the tuberculosinyl diphos-
phate synthase in deuterium oxide followed by dephosphor-
ylation. The signal for the 3-pro-S hydrogen (H-3β) was
1
2
0.25 mm i. d., 0.25 μm film) or HP5-MS (30 m, 0.25 mm i. d.,
2
–
1
energy 70 eV. The GC was programmed as follows: 5 min at 50 °C
–
1
increasing at 10 °C min to 320 °C, and operated in split mode
–
1
(
20:1, 60 s valve time). The carrier gas was He at 1 mLmin . Re-
tention indices (I) were determined from a homologous series of n-
alkanes (C –C38).
1
missing in the H NMR spectrum of the enzyme product
2
8
(Figure 2, c), establishing its structure as (S)-[3- H]-19
(E)-tert-Butyl[(3,7-dimethylocta-2,6-dien-1-yl)oxy]diphenylsilane
11): A solution of geraniol (4b) (51.1 g, 331 mmol, 1.0 equiv.) in
which is in full agreement with the previous finding.
(
DMF (300 mL) was treated with imidazole (49.5 g, 729 mmol,
.2 equiv.). After the imidazole was dissolved, the solution was co-
oled to 0 °C and TBDPS-Cl (100 g, 365 mmol, 1.1 equiv.) was
2
Conclusions
In summary, we have presented an efficient, flexible and added dropwise. The reaction mixture was stirred for 3 h at room
temperature and afterwards quenched by the addition of H
aqueous layer was extracted three times with Et O, and the com-
bined organic layers were washed with a satd. aqueous solution
of NaHCO . After drying with MgSO and concentration under
reduced pressure, the product 11 (129 g, 329 mmol, 99%) was ob-
tained as a yellow oil. R = 0.89 [hexane/EtOAc (5:1)]. GC (BPX-
): I = 2550. H NMR (400 MHz, CDCl , TMS): δ = 7.72–7.69
m, 4 H, 4ϫ CH), 7.43–7.23 (m, 6 H, 6ϫ CH), 5.40–5.37 (m, 1 H,
2
O. The
robust protocol for the synthesis of oligoprenyl diphos-
phates with stable isotope labelling from just a few cost-
effective, commercially available building blocks and rea-
gents. Many of the reactions summarised in Table 1 have
been performed several times in our laboratories, but not
always with isotopically labelled material. Since yield op-
timisation with labelled compounds would be too expens-
2
3
4
f
1
5
(
3
3
ive, the optimised yields for each step as they were obtained CH), 5.12–5.08 (m, 1 H, CH), 4.23 (d, J
= 6.3 Hz, 2 H, CH ),
H,H
2
4
with unlabelled material[21] are also included in Table 1. 2.09–2.04 (m, 2 H, CH
), 1.60 (s, 3 H, CH
) ppm. 13C NMR (100 MHz, CDCl
), 135.6 (4ϫ CH), 134.1 (C ), 131.5 (2ϫ C ), 129.5 (2ϫ CH),
27.6 (4ϫ CH), 124.1 (CH), 124.0 (CH), 61.2 (CH ), 39.5 (CH ),
6.8 (3ϫ CH ), 26.4 (CH ), 25.7 (CH ), 19.2 (C ), 17.7 (CH ), 16.3
) ppm. MS (EI, 70 eV): m/z (%) = 392 (Ͻ1) [M] , 335 (27),
), 1.99–1.96 (m, 2 H, CH
), 1.43 (s, 3 H, CH
): δ = 137.0
2
2
), 1.68 (d, JH,H
These data show which yields along the route are possible. = 0.6 Hz, 3 H, CH
3
3
3
), 1.05
(
(
s, 9 H, 3ϫ CH
3
3
We have shown that deuterium labelling can be efficiently
introduced in any position of the isoprenoid precursors
C
q
q
q
1
2
2
2
using the presented chemistry. Only the Horner–Wad-
2
3
2
3
q
3
sworth–Emmons reaction with [ H ]-2 proved to be some-
2
+
(
CH
3
what unreliable and proceeded in single cases with partial
257 (3), 199 (100), 181 (5), 135 (5), 69 (3), 41 (3). IR (ATR): ν˜ =
loss of deuterium labelling. The prepared compound [14-
3
071 (w), 3050 (w), 2961 (w), 2930 (w), 2857 (w), 1428 (w), 1108
2
H]GGPP was used in an incubation experiment with the
–1
(
s), 1056 (m), 822 (m), 738 (m), 700 (s), 610 (m) cm . UV/Vis
–
1
–1
tuberculosinyl diphosphate synthase from M. tuberculosis, (CH Cl ): λ
(ε, Lmol cm ) = 271 (495), 265 (699), 260 (689),
2
2
max
1
followed by dephosphorylation. The H NMR analysis of 228 (6229) nm.
the enzyme’s product demonstrated that the substrate was
(
E)-tert-Butyl{[5-(3,3-dimethyloxiran-2-yl)-3-methylpent-2-en-1-yl]-
protonated from the Si face at C-14, which was further cor-
oxy}diphenylsilane (12): Compound 11 (129 g, 329 mmol,
roborated by the complementary experiment of GGPP in- 1.0 equiv.) was dissolved in CHCl (1000 mL) and cooled to 0 °C.
3
cubation in deuterium oxide and dephosphorylation. These In one portion, mCPBA (88.9 g, 362 mmol, 1.1 equiv.) was added,
and similar experiments using other isotopically labelled and the reaction mixture was stirred for 1 h at 0 °C. The mixture
was then washed with a satd. solution of NaHCO
The organic layer was dried with MgSO
reduced pressure. Column chromatography with hexane/EtOAc
10:1) yielded the desired epoxide 12 (80.5 g, 197 mmol, 60%) as a
colorless oil. R = 0.58 [hexane/EtOAc (5:1)]. GC (BPX-5): I =
675. H NMR (400 MHz, CDCl , TMS): δ = 7.73–7.68 (m, 4 H,
4ϫ CH), 7.43–7.35 (m, 6 H, 6ϫ CH), 5.42 (tq, JH,H = 6.4, JH,H
3
, brine, and H
and concentrated under
2
O.
oligoprenyl diphosphates prepared via our route are cur-
rently being performed in our laboratory.
4
(
f
Experimental Section
1
2
3
3
4
General Methods: Chemicals were purchased from Acros Organics
3
(
Geel, Belgium) or Sigma Aldrich Chemie GmbH (Steinheim, Ger-
many) and used without further purification. All nonaqueous reac-
tions were performed under an inert atmosphere (N ) in flame-
= 1.2 Hz, 1 H, CH), 4.23 (d, JH,H = 6.3 Hz, 2 H, CH
2
), 2.70 (t,
), 1.68–1.57 (m,
), 1.26 (s, 3 H,
) ppm. 13C NMR (100 MHz, CDCl
):
), 135.6 (4ϫ CH), 133.9 (2ϫ C ), 129.5 (2ϫ CH),
), 58.3 (C ), 36.1
), 19.1 (C ), 18.7
3
J
H,H = 6.3 Hz, 1 H, CH), 2.15–2.03 (m, 2 H, CH
2 H, CH ), 1.46 (s, 3 H, CH ), 1.30 (s, 3 H, CH
CH ), 1.04 (s, 9 H, 3ϫ CH
δ = 136.1 (C
2
2
2
3
3
dried flasks. Solvents were purified by distillation and dried accord-
ing to standard methods. Thin-layer chromatography was per-
3
3
3
q
q
formed with 0.2 mm precoated plastic sheets Polygram® Sil G/ 127.6 (4ϫ CH), 124.6 (CH), 64.0 (CH), 61.0 (CH
2
q
UV254 (Machery–Nagel). Column chromatography was carried
(CH
2
), 27.1 (CH
2
), 26.8 (3ϫ CH
3
), 24.9 (CH
3
q
7688
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
Eur. J. Org. Chem. 2014, 7684–7691

Isotopically Labelled Oligoprenyl Diphosphates
(
CH
,
3
), 16.3 (CH
351 (2), 273 (6), 199 (100), 181 (7), 135 (24), 107 (5), 77 (4). IR
2 f
ATR): ν˜ = 3071 (w), 3049 (w), 2959 (w), 2931 (w), 2891 (w), 2857 pound [1,1- H ]-15 (48.8 g, 131 mmol, 91%) as a yellowish oil. R
3
) ppm. MS (EI, 70 eV): m/z (%) = 408 (Ͻ1) [M] times with EtOAc, and the combined organic layers were dried with
+
MgSO and concentrated under reduced pressure to give com-
4
2
(
(
–
1
1
w), 1428 (w), 1110 (m), 821 (m), 739 (m), 701 (s), 608 (m) cm . = 0.26 [hexane/EtOAc (3:1)]. GC (BPX-5, MSTFA): I = 2580. H
): λmax (ε, Lmol–1 cm ) = 271 (461), 265 (686), 260
–1
NMR (400 MHz, CDCl
7.42–7.34 (m, 6 H, 6ϫ CH), 5.42 (tq, JH,H = 6.3, JH,H = 1.3 Hz,
UV/Vis (CH
2
Cl
2
3
, TMS): δ = 7.71–7.68 (m, 4 H, 4ϫ CH),
3
4
(686), 253 (641), 228 (6823) nm.
3
2 H,H
JH,H = 6.3 Hz, 2 H, CH J =
), 2.03 (t, 3
1
7
CH
H, CH), 4.22 (d,
.3 Hz, 2 H, CH ), 1.81 (s, 1 H, OH), 1.62 (t, JH,H = 7.9 Hz, 2 H,
(
E)-6-[(tert-Butyldiphenylsilyl)oxy]-4-methylhex-4-enal (13): Epox-
3
2
ide 12 (80.5 g, 197 mmol, 1.0 equiv.) was dissolved in THF
4
2
), 1.45 (d, JH,H = 0.8 Hz, 3 H, CH
3
), 1.05 (s, 9 H, 3ϫ CH
3
)
(400 mL) and cooled to 0 °C. Within 90 min, a solution of H
5 6
IO
ppm. 13C NMR (100 MHz, CDCl
3
): δ = 136.8 (C
q
), 135.5 (4ϫ
(53.9 g, 236 mmol, 1.2 equiv.) in THF (400 mL) was added drop-
CH), 134.0 (2ϫ C ), 129.5 (3ϫ CH), 127.5 (4ϫ CH), 124.3 (CH),
q
wise. After complete addition, the reaction mixture was stirred for
an additional 2 h at 0 °C. The reaction was extracted with Et O,
dried with MgSO and concentrated under reduced pressure to give
aldehyde 13 (69.9 g, 191 mmol, 97%) as a pale yellow oil. R = 0.44
1
2
6
(
1.7 (quint., JC,D = 21.6 Hz, C H
CH ), 26.8 (3ϫ CH ), 19.1 (C ), 16.1 (CH
MSTFA): m/z (%) = 385 (5) [M] , 370 (Ͻ1), 271 (98), 199 (100),
81 (11), 135 (10), 97 (53), 75 (11). IR (ATR): ν˜ = 3358 (br), 3071
w), 2931 (w), 2857 (w), 1428 (w), 1109 (m), 1050 (m), 822 (w), 700
2
), 61.0 (CH
2 2
), 35.6 (CH ), 30.3
2
2
3
q
3
) ppm. MS (EI, 70 eV,
4
+
f
1
1
[hexane/EtOAc (5:1)]. GC (BPX-5): I = 2493. H NMR (400 MHz,
(
(
(
3
CDCl
3
, TMS): δ = 9.73 (t, JH,H = 1.8 Hz, 1 H, CHO), 7.73–7.67
–1
–1
–1
2 2
s) cm . UV/Vis (CH Cl ): λmax (ε, Lmol cm ) = 271 (481), 265
677), 260 (670), 230 (3961) nm.
3
(m, 4 H, 4ϫ CH), 7.41–7.35 (m, 6 H, 6ϫ CH), 5.38 (tq, JH,H
=
2
),
4
3
6
2
CH
.2, JH,H = 1.3 Hz, 1 H, CH), 4.22 (d, JH,H = 6.3 Hz, 2 H, CH
.50–2.46 (m, 2 H, CH ), 2.30–2.26 (m, 2 H, CH ), 1.44 (s, 3 H,
), 1.04 (s, 9 H, 3ϫ CH ):
) ppm. 13C NMR (100 MHz, CDCl
δ = 202.1 (CHO), 135.6 (4ϫ CH), 134.8 (C ), 133.9 (2ϫ C ), 129.5
2ϫ CH), 127.6 (4ϫ CH), 125.0 (CH), 60.9 (CH ), 41.8 (CH ),
) ppm. MS (EI,
0 eV): m/z (%) = 366 (Ͻ1) [M] , 309 (9), 231 (24), 213 (6), 199
2
(
E)-[1,1- H
2
]-6-[(tert-Butyldiphenylsilyl)oxy]-4-methylhex-4-en-1-yl
2
2
2
4-Methylbenzenesulfonate (16): Alcohol [1,1- H ]-15 (48.8 g,
2
3
3
3
131 mmol, 1.0 equiv.) was dissolved in pyridine (165 mL) and co-
q
q
oled to 0 °C. TsCl (36.1 g, 190 mmol, 1.5 equiv.) was added in small
portions, and stirring was continued for 3 h at 0 °C. The reaction
mixture was poured onto ice and extracted three times with EtOAc.
The combined organic layers were washed with a satd. solution of
(
3
7
2
2
2 3 q 3
1.4 (CH ), 26.8 (3ϫ CH ), 19.1 (C ), 16.4 (CH
+
(100), 183 (10), 139 (14), 105 (4), 77 (9). IR (ATR): ν˜ = 3071 (w),
aqueous CuSO
4 2 4
, 2 m HCl and H O. After drying with MgSO and
3
049 (w), 2957 (w), 2931 (w), 2891 (w), 2857 (w), 1725 (w), 1428
2
–
1
concentration under reduced pressure, tosylate [1,1- H ]-16 (64.0 g,
(w), 1110 (s), 1069 (m), 822 (m), 740 (m), 702 (s), 611 (m) cm .
2
Cl
2
): λmax (ε, Lmol–1 cm ) = 271 (354), 265 (687), 260
–1
122 mmol, 93%) was obtained as a yellow oil. R = 0.56 [hexane/
UV/Vis (CH
2
f
1
EtOAc (3:1)]. H NMR (400 MHz, CDCl
3
, TMS): δ = 7.79–7.77
(649), 230 (4042) nm.
(
6
=
m, 2 H, 2ϫ CH), 7.69–7.65 (m, 4 H, 4ϫ CH), 7.44–7.35 (m, 6 H,
Methyl (E)-6-[(tert-Butyldiphenylsilyl)oxy]-4-methylhex-4-enoate
3
4
ϫ CH), 7.33–7.31 (m, 2 H, 2ϫ CH), 5.30 (tq, JH,H = 6.3, JH,H
(
14): Aldehyde 13 (7.3 g, 20.0 mmol, 1.0 equiv.) was dissolved in
MeOH (200 mL) and cooled 0 °C. Subsequently, solutions of KOH
2.9 g, 52. 0 mmol, 2.6 equiv.) in MeOH (67 mL) and I (6.6 g,
6.0 mmol, 1.3 equiv.) in MeOH (34 mL) were added, and stirring
3
1.3 Hz, 1 H, CH), 4.16 (d, JH,H = 6.3 Hz, 2 H, CH
2
), 2.42 (s, 3
3
3
H, CH
3
), 1.97 (t, JH,H = 7.1 Hz, 2 H, CH
), 1.37 (s, 3 H, CH ), 1.03 (s, 9 H, 3ϫ CH
NMR (100 MHz, CDCl ): δ = 144.6 (C ), 135.5 (4ϫ CH), 135.1
), 129.8 (2ϫ CH), 129.5 (2ϫ CH),
2
), 1.71 (t, JH,H = 8.1 Hz,
(
2
2
13
2
H, CH
2
3
3
) ppm.
C
3
q
was continued for 90 min at 0 °C. The reaction mixture was diluted
with EtOAc (1 L) and washed three times with satd. aqueous
Na
and concentrated under reduced pressure. Column chromatography
with hexane/EtOAc (10:1) yielded product 14 (5.6 g, 14.0 mmol,
7
(
7
=
(
1
2
(
(
1
C
q
), 133.9 (2ϫ C
q
), 133.2 (C
q
1
27.8 (2ϫ CH), 127.6 (4ϫ CH), 125.1 (CH), 69.5 (quint., JC,D
=
S
2
O
3
and with brine. The organic layer was dried with MgSO
4
2
2
2.9 Hz, C H
), 60.9 (CH
), 19.1 (C
w), 2957 (w), 2931 (w), 2891 (w), 2858 (w), 1359 (m), 1175 (m),
), 34.9 (CH
), 26.8 (3 ϫ CH
), 26.6
2
2
2
3
CH
2
), 21.6 (CH
3
q 3
), 16.1 (CH ) ppm. IR (ATR): ν˜ = 3070
1 %) as a yellowish oil. R
BPX-5): I = 2592. H NMR (400 MHz, CDCl , TMS): δ = 7.70–
f
= 0.32 [hexane/EtOAc (10:1)]. GC
–1
2 2
111 (m), 815 (s), 700 (s), 607 (m), 553 (s) cm . UV/Vis (CH Cl ):
max (ε, Lmol cm ) = 265 (1292), 261 (1250), 231 (11676) nm.
1
–1
–1
3
λ
3
.66 (m, 4 H, 4ϫ CH), 7.44–7.35 (m, 6 H, 6ϫ CH), 5.39 (tq, JH,H
2
6.5, 4
H,H = 1.3 Hz, 1 H, CH), 4.21 (d, 3
(E)-[6,6- H
2
]-tert-Butyl[(6-iodo-3-methylhex-2-en-1-yl)oxy]diphenyl-
J
J
H,H = 6.3 Hz, 2 H,
2
silane (17): Tosylate [1,1- H
dissolved in acetone (750 mL) and treated with NaI (34.8 g,
32 mmol, 1.9 equiv.). The reaction mixture was stirred overnight
2
]-16 (64.0 g, 122 mmol, 1.0 equiv.) was
CH
2
2
), 3.66 (s, 3 H, CH
3
), 2.42–2.38 (m, 2 H, CH
2
), 2.31–2.27 (m,
1
3
H, CH
2
), 1.44 (s, 3 H, CH
3
), 1.04 (s, 9 H, 3ϫ CH
3
) ppm.
C
2
NMR (100 MHz, CDCl
), 133.9 (2ϫ C ), 129.5 (2ϫ CH), 127.6 (4ϫ CH), 124.8 (CH),
1.0 (CH ), 51.5 (CH ), 34.3 (CH ), 32.6 (CH ), 26.8 (3ϫ CH ),
9.1 (C ), 16.2 (CH ) ppm. MS (EI, 70 eV): m/z (%) = 396 (Ͻ1)
3
): δ = 173.7 (CO), 135.6 (4ϫ CH), 135.2
at room temperature under exclusion of light. The mixture was
diluted with hexane, filtered and concentrated under reduced pres-
(C
q
q
6
1
2
3
2
2
3
2
sure to give [6,6- H
2
]-17 (57.9 g, 121 mmol, 99%) as a pale red oil.
q
3
1
+
R
(
7
f
= 0.79 [hexane/EtOAc (5:1)]. GC (BPX-5): I = 2812. H NMR
400 MHz, CDCl , TMS): δ = 7.70–7.68 (m, 4 H, 4ϫ CH), 7.44–
.36 (m, 6 H, 6ϫ CH), 5.42 (tq, JH,H = 6.3, JH,H = 1.2 Hz, 1 H,
[M] , 339 (68), 309 (39), 279 (5), 261 (16), 229 (12), 213 (75), 199
3
(
(
(
(
(
100), 181 (16), 153 (9), 135 (11), 123 (15), 105 (5), 81 (10). IR
ATR): ν˜ = 3071 (w), 2954 (w), 2932 (w), 2892 (w), 2857 (w), 1739
m), 1429 (m), 1109 (s), 1056 (m), 822 (m), 740 (m), 701 (s), 609
3
4
3
3
CH), 4.22 (d, JH,H = 6.3 Hz, 2 H, CH
2 H, CH
), 1.87 (t, 3JH,H = 7.5 Hz, 2 H, CH
.04 (s, 9 H, 3ϫ CH
) ppm. 13C NMR (100 MHz, CDCl
35.6 (4ϫ CH), 134.9 (C ), 134.0 (2ϫ C ), 129.5 (2ϫ CH), 127.6
), 39.8 (CH ), 31.1 (CH ), 26.8
), 6.1 (quint., JC,D = 22.8 Hz,
2
), 2.05 (t, JH,H = 7.1 Hz,
), 1.42 (s, 3 H, CH ),
): δ =
–
1
–1
–1
2
2
3
2 2
m) cm . UV/Vis (CH Cl ): λmax (ε, Lmol cm ) = 271 (502), 265
1
1
3
3
714), 260 (704), 230 (4609) nm.
q
q
2
(
(
(
E)-[1,1- H
2
]-6-[(tert-Butyldiphenylsilyl)oxy]-4-methylhex-4-en-1-ol
(3.6 g, 86 mmol, 1.2 equiv.) in THF
500 mL) was cooled to 0 °C, and methyl ester 14 (57.0 g,
44 mmol, 1.0 equiv.) was added dropwise. The reaction mixture
was stirred for 2 h at 0 °C and overnight at room temperature. For
workup, H O was added carefully, and the precipitate was dissolved
by addition of 2 m HCl. The aqueous layer was extracted three
(4ϫ CH), 125.4 (CH), 61.0 (CH
(3 ϫ CH ), 19.1 (C ), 16.1 (CH
) ppm. MS (EI, 70 eV): m/z (%) = 480 (Ͻ1) [M] , 423 (40),
2
2
2
1
15): A suspension of LiAlD
4
3
q
3
2
+
C H
2
1
309 (18), 249 (4), 199 (100), 181 (9), 135 (4), 97 (12). IR (ATR): ν˜
= 3070 (w), 2931 (w), 2891 (w), 2856 (w), 1427 (w), 1109 (s), 1062
–
1
2
(m), 822 (m), 739 (m), 700 (s), 610 (m) cm . UV/Vis (CH
λ
2
Cl
2
):
–
1
–1
max (ε, Lmol cm ) = 265 (1078), 259 (1157), 229 (5598) nm.
Eur. J. Org. Chem. 2014, 7684–7691
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7689

J. S. Dickschat et al.
FULL PAPER
2
(
E)-[1,1- H
2
]-{6-[(tert-Butyldiphenylsilyl)oxy]-4-methylhex-4-en-1-
59.3 (CH
2 2 2 3 3
), 39.5 (CH ), 26.2 (CH ), 25.5 (CH ), 17.6 (CH ), 16.2
+
yl}triphenylphosphonium Iodide (18): Triphenylphosphine (38.0 g,
45 mmol, 1.2 equiv.) was dissolved in toluene (400 mL) and
(CH ) ppm. MS (EI, 70 eV, MSTFA): m/z (%) = 227 (10) [M] , 212
3
1
(15), 183 (8), 169 (23), 157 (27), 143 (55), 122 (62), 108 (11), 93
2
treated with [6,6- H
2
]-17 (57.9 g, 121 mmol, 1.0 equiv.). The reac-
(34), 73 (100), 41 (47). IR (ATR): ν˜ = 3323 (br), 2967 (w), 2912
–
1
tion was refluxed for 3 h, cooled to room temperature, and filtered.
The resulting solid was washed with toluene and dried under re-
duced pressure to give the Wittig salt [1,1- H
(m), 2857 (w), 1445 (m), 1375 (m), 1100 (w), 997 (s) cm . UV/Vis
–
1
–1
(CH
2
Cl
2
): λmax (ε, Lmol cm ) = 227 (262) nm.
2
2
]-18 (69.5 g, 94 mmol,
2
2
Isolation of (R)-[3- H]-Tuberculosinol, and (S)-[3- H]-Tuberculosin-
ol: The purified Rv3377c protein was prepared as described pre-
1
7
8%) as a colorless solid. R
NMR (400 MHz, CDCl , TMS): δ = 7.85–7.62 (m, 19 H, 19ϫ
CH), 7.41–7.30 (m, 6 H, 6ϫ CH), 5.38–5.34 (m, 1 H, CH), 4.17
f 2 2
= 0.65 [CH Cl /MeOH (10:1)]. H
3
[3]
viously. To isolate the product from the incubation of Rv3377c
2
with [14- H]-GGPP, the reaction mixture (300 mL) containing Tris-
3
3
(
d, JH,H = 6.3 Hz, 2 H, CH
2
), 2.32 (t, JH,H = 7.1 Hz, 2 H, CH
), 0.99 (s, 9 H, 3ϫ CH
): δ = 135.3 (4ϫ CH), 135.0 (d,
2
),
2
HCl buffer (pH 7.5, 50 mm), MgCl
(
3
2
(0.1 mm), [14- H]-GGPP
1
.86–1.72 (m, 2 H, CH
2
), 1.34 (s, 3 H, CH
3
3
)
6 mg), and purified Rv3377c protein (4 mg), was incubated at
0 °C for 20 h. To hydrolyze the diphosphate moiety of the product,
the phosphatase reaction mixture (900 mL) containing Rv3377c re-
ppm. 3C NMR (100 MHz, CDCl
1
3
4
3
J
P,C = 3.0 Hz, 3ϫ CH), 134.9 (C
q
), 133.6 (2ϫ C
q
), 133.5 (d, JP,C
2
=
10.0 Hz, 6ϫ CH), 130.4 (d, JP,C = 12.5 Hz, 6ϫ CH), 129.4 (2ϫ
action mixture (300 mL), acetate buffer (0.2 m, pH 5.6), 2-PrOH
1
CH), 127.4 (4ϫ CH), 125.9 (CH), 117.8 (d, JP,C = 86.1 Hz, 3ϫ
–1
(
20%, v/v), and acid phosphatase (0.25 mgmL , Sigma) was then
2
C
1
q
), 60.6 (CH
2
), 38.9 (d, JP,C = 15.7 Hz, CH
9.9 (d, JP,C = 3.9 Hz, CH ), 18.9 (C ), 16.0 (CH
122 MHz, CDCl ): δ = 24.9 ppm. IR (ATR): ν˜ = 3052 (w), 3014
w), 2930 (w), 2856 (w), 2183 (w), 1437 (m), 1110 (s), 1064 (m), 917
2
), 26.6 (3ϫ CH
3
),
further incubated at 37 °C for 12 h. After addition of MeOH
3
31
2
q
3
) ppm. P NMR
(
(
400 mL), the reaction mixture was extracted with n-hexane
300 mLϫ 3). The n-hexane layer was dried with Na SO and the
(
(
(
(
3
2
4
solvents were evaporated to dryness. The crude materials were dis-
solved in a small amount of hexane. The enzymatic product was
purified by SiO column chromatography with hexane/ethyl acetate
2
–
1
2 2
m), 731 (s), 703 (s), 687 (s), 610 (m) cm . UV/Vis (CH Cl ): λmax
ε, Lmol–1 cm ) = 229 (33630) nm.
–1
2
(
E)-[6- H]-tert-Butyl[(3,7-dimethylocta-2,6-dien-1-yl)oxy]diphenyl- (100:0 to 100:5) followed by reverse-phase HPLC [CAPCELL PAK
2
silane (11): Wittig salt [1,1- H
2
]-18 (20.0 g, 27 mmol, 1.0 equiv.) was
C18 MG S5 column (250ϫ15 mm), SHISEIDO, Tokyo, Japan,
mobile phase CH CN, flow rate 3 mLmin , detection at 210 nm],
3
yielding 1.0 mg of (R)-[3- H]-tuberculosinol (deuterium atom con-
tent: 93%). To isolate the product from the incubation of Rv3377c
with non-labeled GGPP in deuterium oxide, the reaction mixture
–
1
suspended in THF (250 mL) and cooled to 0 °C. A solution of
nBuLi (1.6 m, 18.6 mL, 29.7 mmol, 1.1 equiv.) in hexane was added,
and stirring was continued for 90 min at 0 °C. After cooling to
2
–
78 °C, acetone (1.7 g, 29.7 mmol, 1.1 equiv.) was added dropwise,
and the mixture was stirred overnight at room temperature. The
reaction was quenched by the addition of H O and extracted three
times with EtOAc. The combined organic layers were dried with
MgSO and concentrated under reduced pressure. Column
chromatography with hexane/EtOAc (10:1) yielded [6- H]-11 (8.1 g,
0.6 mmol, 76%) as a colorless oil (deuterium atom content: 90%).
(150 mL) containing Tris-HCl buffer (1 m, pH 7.5, 7.5 mL), MgCl
2
–
1
(0.1 m, 0.15 mL), GGPP (3 mgmL , 1 mL), purified Rv3377c pro-
tein (0.5 mgmL , 4 mL), and D O (99.9 atom-% D, Aldrich,
2
2
–
1
137.35 mL) was incubated at 30 °C for 20 h. After the incubation,
EDTA (0.5 m, pH 8.0, 3 mL) was added to terminate the reaction.
The phosphatase reaction mixture (total volume 300 mL, same
composition as described above) was incubated at 37 °C for 12 h.
The enzymatic product was extracted with n-hexane and then puri-
4
2
2
1
R
(
7
f
= 0.78 [hexane/EtOAc (10:1)]. GC (BPX-5): I = 2548. H NMR
400 MHz, CDCl , TMS): δ = 7.71–7.68 (m, 4 H, 4ϫ CH), 7.43–
.34 (m, 6 H, 6ϫ CH), 5.38 (tq, JH,H = 6.3, JH,H = 0.9 Hz, 1 H,
3
3
4
3
fied by reverse-phase HPLC in CH CN, yielding 1.1 mg of (S)-[3-
3
2
CH), 4.23 (d, JH,H = 6.3 Hz, 2 H, CH
2
), 2.08–2.04 (m, 2 H, CH
), 1.68 (s, 3 H, CH ), 1.60 (s, 3 H, CH
), 1.05 (s, 9 H, 3 ϫ CH
): δ = 137.0 (C ), 135.6 (4ϫ CH), 134.2 (2ϫ C
), 129.5 (2ϫ CH), 127.6 (4ϫ CH), 124.1 (CH), 123.8 (t,
2
),
H]-tuberculosinol (deuterium atom 91%).
1
1
.99–1.96 (m, 2 H, CH
.44 (s, 3 H, CH
2
3
3
),
Supporting Information (see footnote on the first page of this arti-
) ppm. 13C NMR
3
3
cle): Synthetic procedures and spectroscopic data for compounds
(100 MHz, CDCl
3
q
q
),
2
2
2
2
2
2
2
[
[
[
H
H
2
]-2, [ H
2
2
]-9, [ H
5
]-6, [ H
7
]-3a, [ H
9
]-4a, [ H12]-7a, [ H14]-1b,
1
J
31.4 (C
q
2
2
2
2
2
15]-3b, [9- H]-1c, [13- H]-1d, [10- H]-3c, [14- H]-3d, [10- H]-4c,
1
2
C,D = 23.0 Hz, C H), 61.2 (CH
6.3 (CH ), 25.6 (CH ), 19.2 (C ), 17.7 (CH
MS (EI, 70 eV): m/z (%) = 393 (3) [M] , 336 (66), 258 (11), 199
100), 181 (30), 135 (24), 121 (13), 70 (29), 41 (23). IR (ATR): ν˜ =
2
), 39.5 (CH
2
), 26.9 (3ϫ CH
3
),
2
2
2
2
1
13
14- H]-4d, [8- H]-7b, [12- H]-7c, and [14- H]-8d. H NMR,
C
2
2
3
q
3
), 16.3 (CH
3
) ppm.
NMR, and DEPT spectra of all synthesised compounds.
+
(
3
7
2
071 (w), 2960 (w), 2857 (w), 1428 (w), 1109 (s), 1060 (m), 822 (w), Acknowledgments
–1 –1 –1
2 2
38 (m), 700 (s) cm . UV/Vis (CH Cl ): λmax (ε, Lmol cm ) =
71 (426), 265 (628), 259 (703), 230 (3306) nm.
This work was funded by the Deutsche Forschungsgemeinschaft
DFG) with a Heisenberg fellowship (grant DI1536/4-1, to J. S. D.
and project “Fragrants from Actinomycetes”, grant DI1536/2-1)
and by the Beilstein Institut zur Förderung der Chemischen Wis-
senschaften with a Scholarship (to P. R.).
(
2
2
(
1
E)-[6- H]-3,7-Dimethylocta-2,6-dien-1-ol (4b): Compound [6- H]-
1 (29.0 g, 73.6 mmol, 1.0 equiv.) was dissolved in THF (360 mL)
and cooled to 0 °C. A solution of TBAF (1.0 m, 88.3 mL,
8
0
8.3 mmol, 1.2 equiv.) in THF was added and stirred for 3 h at
°C. The solution was diluted with Et O and washed with H O.
and concentrated under
reduced pressure. Column chromatography with hexane/EtOAc
2
2
The organic layer was dried with MgSO
4
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(
5:1) yielded [6- H]-4b (11.1 g, 71.4 mmol, 97%) as a colorless oil.
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R
f
= 0.26 [hexane/EtOAc (5:1)]. GC (BPX-5, MSTFA): I = 1363.
1
3
4
3
H NMR (400 MHz, CDCl , TMS): δ = 5.31 (tq, JH,H = 7.0, JH,H
3
=
1.3 Hz, 1 H, CH), 4.15 (d, JH,H = 7.0 Hz, 2 H, CH
m, 2 H, CH ), 2.05–2.01 (m, 2 H, CH ), 1.68 (s, 3 H, CH
s, 3 H, CH ), 1.60 (s, 3 H, CH
) ppm. 13C NMR (100 MHz,
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2
), 2.12–2.08
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1
2
CDCl
3
q
q
7690
www.eurjoc.org
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
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Isotopically Labelled Oligoprenyl Diphosphates
[
[
[
[
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[
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Received: July 28, 2014
Published Online: October 24, 2014
2011, 18, 32–37.
Eur. J. Org. Chem. 2014, 7684–7691
© 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.eurjoc.org
7691