Biosynthesis of terpenes. Preparation of (E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate, an intermediate of the deoxyxylulose phosphate pathway

Article abstract of DOI:10.1021/jo025705t

(E)-1-Hydroxy-2-methyl-but-2-enyl 4-diphosphate (E-6) was synthesized in six reaction steps from hydroxyacetone (9) and (ethoxycarbonylmethenyl)-triphenylphosphorane (11) with an overall yield of 38%. The compound was shown to be identical with the product of IspG protein, which serves as an intermediate in the nonmevalonate terpene biosynthetic pathway.

Full text of DOI:10.1021/jo025705t

4590
J . Org. Chem. 2002, 67, 4590-4594
Biosyn th esis of Ter p en es. P r ep a r a tion of
(E)-1-Hyd r oxy-2-m eth yl-bu t-2-en yl 4-Dip h osp h a te, a n In ter m ed ia te
of th e Deoxyxylu lose P h osp h a te P a th w a y†
Sabine Amslinger,‡,§ Klaus Kis,‡,§ Stefan Hecht,§ Petra Adam,§ Felix Rohdich,§ Duilio Arigoni,|
Adelbert Bacher,§ and Wolfgang Eisenreich*,§
Lehrstuhl fu¨r Organische Chemie und Biochemie, Technische Universita¨t Mu¨nchen, Lichtenbergstrasse 4,
D-85747 Garching, Federal Republic of Germany, and Laboratorium fu¨r Organische Chemie,
Eidgeno¨ssische Technische Hochschule Ho¨nggerberg HCI, CH-8093 Zu¨rich, Switzerland
wolfgang.eisenreich@ch.tum.de
Received March 11, 2002
(E)-1-Hydroxy-2-methyl-but-2-enyl 4-diphosphate (E-6) was synthesized in six reaction steps from
hydroxyacetone (9) and (ethoxycarbonylmethenyl)-triphenylphosphorane (11) with an overall yield
of 38%. The compound was shown to be identical with the product of IspG protein, which serves as
an intermediate in the nonmevalonate terpene biosynthetic pathway.
In tr od u ction
methyl-D-erythritol 4-phosphate (4), by isomerization and
reduction of the resulting branched chain aldose; both
reaction steps are catalyzed by the NADPH-dependent
2C-methyl-D-erythritol 4-phosphate synthase (IspC).15
The polyol phosphate 4 is converted into the cyclic
diphosphate 5 by the sequential action of three enzymes
specified by the ispDEF genes.16-21
More than 30 000 representatives make terpenoids one
of the largest groups of natural products that are all
assembled from the universal precursors, isopentenyl
diphosphate (IPP) (7) and dimethylallyl diphosphate
(DMAPP) (8).1
The formation of IPP and DMAPP via mevalonate has
been studied in considerable detail over a period of five
decades.2-5 More recently, a second pathway operating
in plants6 and certain bacteria7,8 has been uncovered by
independent studies in the research groups of Rohmer
and Arigoni (for review, see also refs 9-12).
The nonmevalonate pathway (Scheme 1) starts with a
thiamine diphosphate dependent condensation of pyru-
vate (1) and D-glyceraldehyde 3-phosphate (2) affording
1-deoxy-D-xylulose 5-phosphate (3).13,14 This product is
then converted into the branched polyol derivative, 2C-
Gene targeting analysis using Escherichia coli and
Synechocystis sp. had previously indicated an essential
role in the new paythway for the genes ispG (formerly
gcpE)22,23 and ispH (formerly lytB).24-26 In recent work
with E. coli cells engineered for the overexpression of the
xylB gene (specifying 1-deoxy-D-xylulose kinase), as well
as of the ispDEFG genes, we observed the formation of a
new intermediate, (E)-1-hydroxy-2-methyl-but-2-enyl
4-diphosphate (E-6),27 upon feeding of 13C-labeled 1-deoxy-
D-xylulose. The additional expression of the ispH gene
led to the in vivo formation of both IPP and DMAPP.28
(13) Sprenger, G. A.; Scho¨rken, U.; Wiegert, T.; Grolle, S.; deGraaf,
A. A.; Taylor, S. V.; Begley, T. P.; Bringer-Meyer, S.; Sahm, H. Proc.
Natl. Acad. Sci. U.S.A. 1997, 94, 12857.
(14) Lois, L. M.; Campos, N.; Putra, S. R.; Danielsen, K.; Rohmer,
M.; Boronat, A. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 2105.
(15) Takahashi, S.; Kuzujama, T.; Watanabe, H.; Seto, H. Proc. Natl.
Acad. Sci. U.S.A. 1998, 95, 9879.
(16) Rohdich, F.; Wungsintaweekul, J .; Fellermeier, M.; Sagner, S.;
Herz, S.; Kis, K.; Eisenreich, W.; Bacher, A.; Zenk, M. H. Proc. Natl.
Acad. Sci. U.S.A. 1999, 96, 11758.
† Dedicated to Professor Helmut Simon on the occasion of his 75th
birthday.
* Corresponding author. Phone: +49-89-289-13043. Fax: +49-89-
289-13363.
‡ These authors contributed equally to this work.
§ Technische Universita¨t Mu¨nchen.
| Eidgeno¨ssische Technische Hochschule Ho¨nggerberg HCI.
(1) Sacchettini, J . C.; Poulter, C. D. Science 1997, 277, 1788.
(2) Bloch, K. Steroids 1992, 57, 378.
(3) Qureshi, N.; Porter, J . W. In Biosynthesis of Isoprenoid Com-
pounds; Porter, J . W., Spurgeon, S. L., Eds.; J ohn Wiley: New York,
1981; Vol. 1, pp 47-94.
(17) Kuzuyama, T.; Takagi, M.; Kaneda, K.; Dairi, T.; Seto, H.
Tetrahedron Lett. 2000, 41, 703.
(18) Lu¨ttgen, H.; Rohdich, F.; Herz, S.; Wungsintaweekul, J .; Hecht,
S.; Schuhr, C. A.; Fellermeier, M.; Sagner, S.; Zenk, M. H.; Bacher,
A.; Eisenreich, W. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 1062.
(19) Kuzuyama, T.; Takagi, M.; Kaneda, K.; Watanabe, H.; Dairi,
T.; Seto, H. Tetrahedron Lett. 2000, 41, 2925.
(20) Herz, S.; Wungsintaweekul, J .; Schuhr, C. A.; Hecht, S.;
Lu¨ttgen, H.; Sagner, S.; Fellermeier, M.; Eisenreich, W.; Zenk, M. H.;
Bacher, A.; Rohdich, F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2486.
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T.; Seto, H. Tetrahedron Lett. 2000, 41, 3395.
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M.; Boronat, A. FEBS Lett. 2001, 488, 170.
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M.; Beck, E.; J omaa, H. J . Bacteriol. 2001, 183, 2411.
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182, 5841.
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Bacteriol. 2001, 183, 7403.
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In Comprehensive Natural Product Chemistry; Cane, D., Ed.; Perga-
mon: Oxford, 1999; Vol. 2, pp 15-44.
(6) Schwarz, M. K. Ph.D. Thesis No. 10951, ETH Zu¨rich, Switzer-
land, 1994.
(7) Rohmer, M.; Knani, M.; Simonin, P.; Sutter, B.; Sahm, H.
Biochem. J . 1993, 295, 517.
(8) Broers, S. T. J . Ph.D. Thesis No. 10978, ETH Zu¨rich, Switzer-
land, 1994.
(9) Rohdich, F.; Kis, K.; Bacher, A.; Eisenreich, W. Curr. Opin.
Chem. Biol. 2001, 5, 535.
(10) Rohmer, M. In Comprehensive Natural Product Chemistry;
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6, 78.
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brand, S.; Hintz, M.; Beck, E.; J omaa, H. FEBS Lett. 2001, 499, 37.
10.1021/jo025705t CCC: $22.00 © 2002 American Chemical Society
Published on Web 05/22/2002

(E)-1-Hydroxy-2-methyl-but-2-enyl 4-Diphosphate
J . Org. Chem., Vol. 67, No. 13, 2002 4591
Sch em e 1. Biosyn th esis of IP P (7) a n d DMAP P
(8) via 1-Deoxy-D-xylu lose 5-P h osp h a te (3)a
Resu lts a n d Discu ssion
Treatment of hydroxyacetone (9) with an excess of 3,4-
dihydro-2H-pyran under catalysis by pyridinium toluene
4-sulfonate30 afforded the known tetrahydropyranyl (THP)
derivative 10 (Scheme 2). Wittig reaction of 10 with
(ethoxycarbonylmethylene)triphenylphosphorane (11)31
in toluene under reflux conditions gave a 6:1 mixture of
(E)- and (Z)-ethyl-2-methyl-1-tetrahydropyranyloxy-but-
2-enoate (E/Z-12), from which the pure E-isomer was
separated by preparative HPLC (for stereochemical as-
signment of the two isomers cf. below).
Reduction of E-12 by DIBALH32 in CH2Cl2 afforded (E)-
2-methyl-1-tetrahydropyranyloxy-but-2-ene-4-ol (E-13).
To avoid the formation of side products, it was important
to maintain the temperature at -78 °C. During workup,
it was essential to remove aluminum salts by extraction
with copious amounts of methanol; otherwise, the oily
product could not be purified by chromatography on silica
gel.
The allyl alcohol E-13 was transformed into the
chloride E-14 with DMAP and p-TsCl in CH2Cl2. The
chloride E-14 is unstable at room temperature.
The diphosphate group was introduced by reaction of
E-14 with 1.2 equiv of tris(tetra-n-butylammonium)
hydrogen diphosphate in MeCN,33 and the resulting E-15
could be purified by two chromatographic steps using
columns of DOWEX 50 WX8 and cellulose. Alternatively,
the mixture was subjected to deprotection without prior
purification.
The THP group of E-15 was removed by acid hydrolysis
(pH 1.3) at ambient temperature. Prolonged incubation
of the reaction mixture resulted in progressive hydrolysis
of the diphosphate motif of E-6. (E)-1-Hydroxy-2-methyl-
but-2-enyl 4-diphosphate (E-6) was purified by cation
exchange chromatography on DOWEX 50 WX8 followed
by chromatography on cellulose. The overall yield for the
last two steps was 66%.
A 6:1 mixture of (E)- and (Z)-1-hydroxy-2-methyl-but-
2-enyl 4-diphosphate (E/Z-6) was prepared by the same
route from the original unresolved mixture of E/Z-12 and
used for the spectroscopic characterization of both iso-
mers. NMR data are summarized in Table 1. E/Z assign-
ments are based on NOESY experiments. Specifically,
the geometry of the double bond was established by
strong NOE interactions between H-3/H-1 and H-4/H-2-
methyl for E-6 and H-3/H-2-methyl and H-4/H-1 for Z-6,
respectively.
For comparison with the synthetic specimen of E-6, a
sample of the biosynthetic material was produced fol-
lowing the published procedure27 by incubating [U-13C5]1-
deoxy-D-xylulose with an E. coli strain engineered for
expression of the xylB and ispCDEFG genes. An extract
prepared by ultrasonic treatment of the cells was used
without purification for NMR analysis of the biosynthetic
product. The relevant 13C NMR signals are shown in
Figure 1A; in agreement with published data,27 they form
complex multiplets due to 13C-13C coupling.
a
Dashed arrows under ipi are meant to indicate that an
isomerization step is not essential.25
In agreement with these findings, accumulation of a
compound identified spectroscopically as E-6 was inde-
pendently observed in an ispH-deficient mutant of E.
coli.29 The possible cooperation of additional but yet
unidentified proteins in channeling the electron flow
required by the reductive nature of the last two steps of
Scheme 1 remains to be investigated.
In our original work on the role of the IspG protein,
identification of the reaction product was greatly helped
by the availability of an authentic specimen of E-6 of
synthetic origin. This paper describes the synthesis of
E-6 and its spectroscopic comparison with the biosyn-
thetic material. The synthetic route outlined in this paper
could stimulate future work on the synthesis of isotope-
labeled E-6 as a substrate for biosynthetic studies.
(27) Hecht, S.; Eisenreich, W.; Adam, P.; Amslinger, S.; Kis, K.;
Bacher, A.; Arigoni, D.; Rohdich, F. Proc. Natl. Acad. Sci. U.S.A. 2001,
98, 14837.
(28) Rohdich, F.; Hecht, S.; Ga¨rtner, K.; Adam, P.; Krieger, C.;
Amslinger, S.; Arigoni, D.; Bacher, A.; Eisenreich, W. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 1158.
(29) Hintz, M.; Reichenberg, A.; Altincicek, B.; Bahr, U.; Gschwind,
R. M.; Kollas, A.-K.; Beck, E.; Wiesner, J .; Eberl, M.; J omaa, H. FEBS
Lett. 2001, 509, 317.
(30) Hagiwara, H.; Uda, H. J . Chem. Soc., Perkin Trans. 1 1984,
91.
(31) Miyaoka, H.; Isaji, Y.; Kajiwara, Y.; Kunimune, I.; Yamada, Y.
Tetrahedron Lett. 1998, 39, 6503.
(32) Watanabe, H.; Hatakeyama, S.; Tazumi, K.; Takano, S.; Ma-
suda, S.; Okano, T.; Kobayashi, T.; Kubodera, N. Chem. Pharm. Bull.
1996, 44, 2280.
(33) Davisson, V. J .; Woodside, A. B.; Neal, T. R.; Stremler, K. E.;
Muehlbacher, M.; Poulter, C. D. J . Org. Chem. 1986, 51, 4768.

4592 J . Org. Chem., Vol. 67, No. 13, 2002
Amslinger et al.
Sch em e 2a
a
Reagents and conditions: (a) DHP, PPTS, 25 °C (2.5 h); (b) toluene, reflux (23 h); (c) (1) DIBALH, CH2Cl2, -78 °C (2.5 h), (2) 1 M
NaOH/H2O; (d) p-TsCl, DMAP, CH2Cl2, 25 °C (3 h); (e) tris(tetra n-butylammonium) hydrogendiphosphate, MeCN, 25 °C (3 h); (f), HCl/
H2O pH 1.3, 25 °C (20 min).
Ta ble 1. NMR Da ta of (E)-1-Hyd r oxy-2-m eth yl-bu t-2-en yl 4-Dip h osp h a te (E-6) a n d (Z)-1-Hyd r oxy-2-m eth yl-bu t-2-en yl
4-Dip h osp h a te (Z-6)e in D2O a t p H 7
position
chemical shifts [ppm]
coupling constants [Hz]
J HH J PC J PP
13C isotope shiftsc [ppb]
NMR correlation pattern
NOESY (rel NOE)d
HMBC
1Ha
13Ca
31Pb
(E)-1-Hydroxy-2-methyl-but-2-enyl 4-Diphosphate (E-6)
1
2
3.88
68.6
141.6
15.1
-8
-67
-15
-100
-30
2-methyl (53), 3 (100) 2, 2-methyl, 3
2-methyl 1.57
3
4
Pâ
PR
4 (79), 1 (53)
1 (100)
1, 2, 3
2, 3
5.52 122.7
4.39
6.89, 1.2 8.0
7.2 5.1
64.3
2-methyl (79)
-3.49 (-4.48)e
-7.17 (-7.06)e
22.3 (20.8)e
22.0 (20.8)e
(Z)-1-Hydroxy-2-methyl-but-2-enyl 4-Diphosphate (Z-6)e
1
2
4.03
62.0
141.8
22.7
2-methyl (34), 4 (100) 2, 2-methyl, 3
2-methyl 1.70
3
4
1 (34), 3 (88)
2-methyl (88)
1 (100)
1, 2, 3
2, 3
5.49 125.6
4.41 63.8
6.8
7.3
7.7
5.1
Pâ
PR
-4.48
-7.06
20.8
20.8
a
Referenced to external trimethylsilylpropane sulfonate. b Referenced to external 85% orthophosphoric acid. c Determined with [U-13C5]1-
hydroxy-2-methyl-but-2-enyl 4-diphosphate. Intensities of cross-signals are given as relative units. e Determined from E/Z-6.
d
Figure 1C displays the 13C NMR signals of synthetic
(E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (E-6),
and Figure 1B shows the signals of the sample of Figure
1A after addition of the synthetic compound. The singlets
representing the synthetic compound are slightly down-
field-shifted from the center of the multiplets represent-
ing the uniformly 13C-labeled biosynthetic compound as
a consequence of heavy isotope chemical shift effects in
the latter (cf. Table 1). It should also be noted that the
line widths of the 13C-labeled biosynthetic material
exceed those of the unlabeled synthetic material as a
consequence of long-range 13C-13C couplings, which are
too small to be fully resolved but can be gleaned from
the slight line broadening. It could also be shown that
displayed the full biological activity described by other
authors29 for the novel compound, i.e., the stimulation
of the proliferation of γδ T cells (data not shown).
Exp er im en ta l Section
Gen er a l. Commercially available reagents were used with-
out further purification. Chromatography was performed on
silica gel 60 (230-400 mesh), DOWEX 50 WX8 (200-400
mesh), and cellulose. TLC plates were stained with a 1:2:100
v/v mixture of anisaldehyde/H2SO4/HOAc. NMR spectra were
recorded at room temperature.
Aceton yl Tetr a h yd r op yr a n yl Eth er (10). A mixture of
339 mg (1.35 mmol) of pyridinium toluene-4-sulfonate, 9.35
mL (10.0 g, 0.135 mol) of hydroxyacetone, and 24.7 mL (22.7
g, 0.270 mol) of 3,4-dihydro-2H-pyran was stirred at room
temperature for 2.5 h. Residual 3,4-dihydro-2H-pyran was
removed by evaporation under reduced pressure. The crude
mixture was purified by flash chromatography on a column of
silicagel (6 × 20 cm; 4:1 hexane/acetone) to yield 18.7 g (0.118
mol, 88%) of 10 as a colorless liquid: 1H NMR (CDCl3, 500
MHz) δ 4.62 (t, J ) 3.6 Hz, 1H), 4.22 (d, J ) 17.3 Hz, 1H),
1
the H NMR signals of synthetic E-6 coincide with those
of the 13C-decoupled 1H NMR spectrum of the biosyn-
thetic product (Figure 2). These findings provide conclu-
sive proof for the correctness of the structure assigned
to the new intermediate of the nonmevalonate terpene
pathway. Not surprisingly, the synthetic species has also

(E)-1-Hydroxy-2-methyl-but-2-enyl 4-Diphosphate
J . Org. Chem., Vol. 67, No. 13, 2002 4593
F igu r e 2. 13C-decoupled 1H NMR signals of 1-hydroxy-2-
methyl-but-2-enyl 4-diphosphate: A, a crude cell extract of an
E. coli strain engineered for expression of the xylB and
ispCDEFG genes and proffered with [U-13C5]1-deoxy-D-xylu-
lose; B, the sample in A after addition of a 6:1 mixture of (E)-
and (Z)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate; C, the
sample in B after addition of a second equivalent of the 6:1
mixture of (E)- and (Z)-1-hydroxy-2-methyl-but-2-enyl 4-diphos-
phate. An * indicates signals of an impurity.
pressure to yield 9 g of an orange-colored oil that was purified
by FC on a column of silica gel (6.5 × 28 cm, 9:1 hexane/
acetone) to afford 7.03 g (30.8 mmol, 89%) of a mixture of (E)/
(Z) stereoisomers of 12 in a ratio of 86:14. The isomers were
separated by preparative reversed-phase HPLC (column,
250 × 40.0 mm; eluent, 33:22:45 methanol/2-propanol/water;
flow rate, 76 mL/min). The retention time of E-12 was 13 min.
Fractions were combined, and the volume was reduced to about
one-half by evaporation under reduced pressure. The solution
was extracted with CH2Cl2. The organic phase was dried over
Na2SO4, filtered, and evaporated to dryness under reduced
pressure to yield 5.64 g (24.7 mmol, 71%) of isomerically pure
E-12: 1H NMR (CDCl3, 500 MHz) δ 5.93 (q, J ) 1.4 Hz, 1H),
4.59 (t, J ) 3.4 Hz, 1H), 4.17 (dd, J ) 15.6 Hz, 1.4 Hz, 1H),
4.12 (q, J ) 7.1 Hz, 2H), 3.90 (dd, J ) 15.5, 1.4 Hz, 1H), 3.81-
3.76 (m, 1H), 3.49-3.45 (m, 1H), 2.06 (s, 3H), 1.87-1.78 (m,
2H), 1.72-1.50 (m, 4H), 1.23 (t, J ) 7.1 Hz, 3H); 13C NMR
(CDCl3, 126 MHz) δ 166.8, 154.7, 114.5, 98.0, 70.6, 62.0, 59.7,
30.3, 25.3, 19.1, 15.9, 14.3; MS (CI, isobutane) m/z 229 [M +
1]+. Anal. Calcd for C12H20O4: C, 63.14; H, 8.83. Found: C,
63.13; H, 8.44.
(E)-2-Meth yl-1-tetr ah ydr opyr an yloxy-bu t-2-en e-4-ol (E-
13). A solution of E-12 (3.00 g, 13.1 mmol) in 30 mL of dry
CH2Cl2 was cooled to -78 °C. A 1.0 M solution of DIBALH
(31.5 mL, 31.5 mmol) in hexane was added over a period of 2
h under an atmosphere of nitrogen. The solution was stirred
for 30 min at -78 °C. The reaction was terminated by the
addition of 0.5 mL of 1 M NaOH. The solvent was removed by
evaporation under reduced pressure. Methanol (200 mL) was
added, and the mixture was passed through a column of Na2-
SO4/silica gel (3.5 × 5; 21 cm). The column was developed with
600 mL of methanol. The effluent was concentrated under
reduced pressure. The resulting oil was dissolved in 5 mL of
CH2Cl2. The solution was loaded on a column of silica gel
(3.5 × 12 cm), which was developed with 450 mL of methanol.
F igu r e 1. 13C NMR signals of 1-hydroxy-2-methyl-but-2-enyl
4-diphosphate: A, obtained with a crude cell extract of an E.
coli strain engineered for expression of the xylB and ispCDEFG
genes and proffered with [U-13C5]1-deoxy-D-xylulose; B, ob-
tained with the sample in A after addition of synthetic (E)-1-
hydroxy-2-methyl-but-2-enyl 4-diphosphate (E-6); C, synthetic
(E)-1-hydroxy-2-methyl-but-2-enyl 4-diphosphate (E-6). An *
indicates signals of residual [U-13C5]2C-methyl-D-erythritol 2,4-
cyclodiphosphate (5) present in the crude E. coli extract.
Couplings with coupling constants below 5 Hz are not indi-
cated. The concentration of biosynthetic [U-13C5]-E-6 was 0.1
mM. The acquisition time was approximately 1 h for each of
the 13C NMR experiments.
4.09 (d, J ) 17.3 Hz, 1H), 3.83-3.79 (m, 1H), 3.51-3.47 (m,
1H), 2.15 (s, 3H), 1.87-1.49 (m, 6H); 13C NMR (CDCl3, 126
MHz) δ 206.7, 98.7, 72.3, 62.3, 30.2, 26.5, 25.2, 19.2; MS (CI,
isobutane) m/z 159 [M + 1]+. Anal. Calcd for C8H14O3: C, 60.74;
H, 8.92. Found: C, 60.39; H 8.65.
Eth yl (E)-2-Meth yl-1-tetr a h yd r op yr a n yloxy-bu t-2-en -
oa te (E-12). Acetonyl tetrahydropyranyl ether (10, 3.88 g, 24.5
mmol) was dissolved in 50 mL of dry toluene under a nitrogen
atmosphere at room temperature. A suspension of (ethoxycar-
bonylmethylene)triphenylphosphorane (11, 12.8 g, 36.8 mmol)
in 150 mL of dry toluene was added, and the mixture was
heated under reflux for 23 h. The solvent was evaporated
under reduced pressure to yield an orange oil. Triphenylphos-
phinoxide was precipitated by the addition of 100 mL of 9:1
hexane/acetone. The mixture was filtered, and the filtrate was
concentrated to a volume of about 1 mL. A 9:1 mixture of
hexane/acetone (100 mL) was added. The precipitate was
filtered off, and the solvent was evaporated under reduced

4594 J . Org. Chem., Vol. 67, No. 13, 2002
Amslinger et al.
Evaporation of the solvent gave 2.39 g (12.8 mmol, 98%) of
E-13 as a colorless liquid: 1H NMR (CDCl3, 500 MHz) δ 5.66
(tq, J ) 6.8, 1.3 Hz, 1H), 4.59 (t, J ) 3.6 Hz, 1H), 4.17 (t, J )
5.0 Hz, 2H), 4.10 (d, J ) 12.5 Hz, 1H), 3.86-3.81 (m, 1H), 3.84
(d, J ) 12.5 Hz, 1H), 3.50-3.46 (m, 1H), 1.86-1.66 (m, 2H),
1.67 (s, 3H), 1.61-1.48 (m, 4H); 13C NMR (CDCl3, 126 MHz) δ
135.5, 125.6, 97.8, 71.9, 62.1, 59.0, 30.5, 25.4, 19.3, 14.1; MS
(CI, isobutane) m/z 169 [M - H2O + 1]+.
made to isolate the Z compounds in pure form, the pertinent
NMR data could be easily collected from the spectra of the
mixtures and are summarized below.
Eth yl (Z)-2-Meth yl-1-tetr a h yd r op yr a n yloxy-bu t-2-en -
oa te (Z-12): 1H NMR (CDCl3, 500 MHz) δ 5.71 (q, J ) 1.4 Hz,
1H), 4.60 (t, J ) 3.6 Hz, 1H), 4.20 (dd, J ) 15.5 Hz, 1.3 Hz,
1H), 4.11 (q, J ) 7.1 Hz, 2H), 3.93 (dd, J ) 15.6, 1.3 Hz, 1H),
3.84-3.79 (m, 1H), 3.52-3.48 (m, 1H), 1.97 (d, J ) 1.4 Hz,
3H), 1.88-1.50 (m, 6H), 1.24 (t, J ) 7.1 Hz, 3H); 13C NMR
(CDCl3, 126 MHz) δ 165.9, 156.8, 116.9, 98.7, 66.5, 62.3, 59.8,
30.6, 25.3, 21.9, 19.5, 14.3; MS (CI, isobutane) m/z 229 [M +
1]+; Anal. Calcd for C12H20O4: C, 63.14; H, 8.83. Found: C,
63.13; H, 8.44.
(Z)-2-Meth yl-1-tetr ah ydr opyr an yloxy-bu t-2-en e-4-ol (Z-
13): 1H NMR (CDCl3, 500 MHz) δ 5.64 (tq, J ) 6.6, 1.3 Hz,
1H), 4.63 (t, J ) 3.3 Hz, 1H), 4.20 (d, J ) 6.8 Hz, 2H), 4.15 (d,
J ) 11.8 Hz, 1H), 3.87-3.82 (m, 1H), 3.83 (d, J ) 11.3 Hz,
1H), 3.52-3.48 (m, 1H), 1.86-1.48 (m, 6H), 1.79 (s, 3H); 13C
NMR (CDCl3, 126 MHz) δ 136.2, 128.6, 96.6, 65.1, 61.8, 58.1,
30.3, 25.3, 21.9, 19.0; MS (CI, isobutane) m/z 169 [M - H2O +
1]+.
(Z)-4-Ch lor o-2-m eth yl-1-tetr a h yd r op yr a n yloxy-bu t-2-
en (Z-14): 1H NMR (CDCl3, 500 MHz) δ 5.65 (t, J ) 8.1 Hz,
1H), 4.61 (t, J ) 3.6 Hz, 1H), 4.18 (d, J ) 12.8 Hz, 1H), 4.15
(d, J ) 8.0 Hz, 2H), 3.92 (d, J ) 12.8 Hz, 1H), 3.90-3.86 (m,
1H), 3.59-3.52 (m, 1H), 1.92-1.52 (m, 6H), 1.86 (s, 3H); 13C
NMR (CDCl3, 126 MHz) δ 138.3, 124.6, 97.5, 64.7, 62.2, 40.1,
30.5, 25.4, 21.8, 19.4; MS (CI, isobutane) m/z 205 [M + 1]+.
Anal. Calcd for C10H17ClO2: C, 58.68; H, 8.37; Cl, 17.32.
Found: C, 58.56; H, 8.54; Cl, 16.96.
(Z)-2-Meth yl-1-tetr a h yd r op yr a n yloxy-bu t-2-en yl 4-Di-
p h osp h a te Tr ia m m on iu m Sa lt (Z-15): 1H NMR (D2O, 500
MHz) δ 5.52 (t, J ) 6.8, 1H), 4.65 (s, 1H), 4.31 (t, J ) 7.1 Hz,
2H), 3.98 (d, J ) 12.3 Hz, 1H), 3.84 (d, J ) 12.1 Hz, 1H), 3.74-
3.70 (m, 1H), 3.42-3.38 (m, 1H), 1.64 (s, 3H), 1.61-1.57 (m,
2H), 1.40-1.32 (m, 4H); 13C NMR (D2O, 126 MHz) δ 136.3,
125.8 (d, J ) 8.6 Hz), 98.6, 72.5, 63.2, 61.8 (d, J ) 5.1 Hz),
29.9, 24.5, 20.8, 19.0; 31P NMR (D2O, 101 MHz) δ -5.69 (d,
J ) 20.8 Hz), -7.68 (d, J ) 20.8 Hz).
(E)-4-Ch lor o-2-m eth yl-1-tetr a h yd r op yr a n yloxy-bu t-2-
en (E-14). To a solution of E-13 (1.10 g, 5.91 mmol) in 12 mL
of dry CH2Cl2, solutions of 1.01 g (8.27 mmol) of DMAP in 12
mL of dry CH2Cl2 and of 1.35 g (7.09 mmol) of p-TsCl in 12
mL of dry CH2Cl2 were added. The resulting solution was
stirred at room temperature for 3 h. After evaporation of the
solvent under reduced pressure, the residue was purified by
FC on silica gel (3.5 × 12.5 cm, CH2Cl2 100%) to obtain 1.13 g
(5.52 mmol, 93%) of E-14 as a colorless liquid: 1H NMR (CDCl3,
500 MHz) δ 5.72 (tq, J ) 8.0, 1.5 Hz, 1H), 4.59 (t, J ) 3.6 Hz,
1H), 4.18 (d, J ) 12.8 Hz, 1H), 4.15 (d, J ) 8.0 Hz, 2H), 3.92
(d, J ) 12.8 Hz, 1H), 3.90-3.86 (m, 1H), 3.59-3.52 (m, 1H),
1.92-1.52 (m, 6H), 1.77 (s, 3H); 13C NMR (CDCl3, 126 MHz) δ
138.7, 121.8, 97.9, 71.3, 62.1, 40.2, 30.5, 25.4, 19.4, 13.8; MS
(CI, isobutane) m/z 205 [M + 1]+. Anal. Calcd for C10H17ClO2:
C, 58.68; H, 8.37; Cl, 17.32. Found: C, 58.56; H, 8.54; Cl, 16.96.
(E)-2-Meth yl-1-tetr a h yd r op yr a n yloxy-bu t-2-en yl 4-Di-
p h osp h a te Tr ia m m on iu m Sa lt (E-15). To a solution of E-14
(475 mg, 2.32 mmol) in 2.5 mL of MeCN was slowly added a
solution of 2.51 g (2.78 mmol) of tris(tetra-n-butylammonium)
hydrogen pyrophosphate in 5.5 mL of MeCN at room temper-
ature. After 3 h, the orange-colored solution was concentrated
by evaporation under reduced pressure. The resulting orange-
colored oil was dissolved in 2.5 mL of water. The solution was
passed through a column of DOWEX 50 WX8 (2.5 × 11 cm,
+
NH4 form) which had been equilibrated with 100 mL of 25
mM NH4HCO3. The column was developed with 250 mL of 25
mM NH4HCO3. Fractions were combined and lyophilized. The
residue was dissolved in 5 mL of a 1:1 v/v mixture of
2-propanol/100 mM NH4HCO3. The solution was loaded on a
cellulose column (2 × 18 cm), which was developed with the
same mixture of 2-propanol/100 mM NH4HCO3. The effluent
was lyophilized affording 444 mg (1.12 mmol, 88%) of E-15 as
a white powder: 1H NMR (D2O, 500 MHz) δ 5.52 (tq, J ) 6.8
Hz, 1H), 4.65 (s, 1H), 4.34 (t, J ) 7.0 Hz, 2H), 3.98 (d, J )
12.3 Hz, 1H), 3.84 (d, J ) 12.1 Hz, 1H), 3.74-3.70 (m, 1H),
3.42-3.38 (m, 1H), 1.61-1.57 (m, 2H), 1.54 (s, 3H), 1.40-1.32
(m, 4H); 13C NMR (D2O, 126 MHz) δ 136.4, 123.9 (dd, J ) 8.0,
2.3 Hz), 98.5, 72.5, 63.2, 62.2 (d, J ) 5.3 Hz), 29.9, 24.5, 19.0,
13.4; 31P NMR (D2O, 101 MHz) δ -5.62 (d, J ) 20.9 Hz), -7.57
(d, J ) 20.8 Hz).
(Z)-1-Hyd r oxy-2-m eth yl-bu t-2-en yl 4-Dip h osp h a te Tr i-
a m m on iu m Sa lt (Z-6): 1H NMR (D2O, 500 MHz) δ 5.49 (tm,
J ) 6.8 Hz, 1H), 4.41 (t, J ) 7.3 Hz, 2H), 4.03 (s, 2H), 1.70 (s,
3H); 13C NMR (D2O, 126 MHz) δ 141.8, 125.6 (d, J ) 7.7 Hz),
63.8 (d, J ) 5.1 Hz), 62.0, 22.7; 31P NMR (D2O, 101 MHz) δ
-4.48 (d, J ) 20.8 Hz), -7.06 (d, J ) 20.8 Hz).
[U-13C5]1-Hyd r oxy-3-m eth yl-bu t-2-en yl 4-Dip h osp h a te.
Biosynthetic [U-13C5]-6 was obtained as described earlier using
the E. coli strain XL1-pBSxispC-G27 engineered for expression
of the xylB and ispCDEFG genes. About 300 µL of cells were
suspended in 440 µL of 20 mM NaF in D2O and 210 µL of
MeOD-d3. The suspension was ultrasonically treated at 0 °C
and centrifuged. The supernatant was used directly for NMR
characterization.
(E)-1-Hyd r oxy-2-m eth yl-bu t-2-en yl 4-Dip h osp h a te Tr i-
a m m on iu m Sa lt (E-6). For the following reaction step, crude
E-15 could be used with omission of the chromatographic
purification described above. A solution of orange-colored,
crude E-15 (2.32 mmol) in 8 mL of MeCN was diluted by the
addition of 8 mL of water. The pH was adjusted to pH 1.3 by
addition of 400 µL of 37% HCl. The reaction was monitored
by NMR and terminated by addition of 350 µL of 40% NaOH.
The solution was passed through a column of DOWEX 50 WX8
Note Ad d ed in P r oof. In a paper published after submis-
sion of this manuscript, M. Wolff et al. have reported an
independent synthesis of (E)-1-hydroxy-2-methyl-but-2-enyl
4-diphosphate.35
+
Ack n ow led gm en t. This work was supported by the
Deutsche Forschungsgemeinschaft, the Fonds der Che-
mischen Industrie, and the Hans-Fischer-Gesellschaft.
We thank Silverio Ruggieri (Universita` di Ancona) for
support. Financial support by Novartis International
AG, Basel, is gratefully acknowledged (D.A.).
(2.5 × 6 cm; NH4 form). The column was developed with 90
mL of 25 mM NH4HCO3. Fractions were combined and
lyophilized. The residue was dissolved in 3 mL of a 1:1 v/v
mixture of 2-propanol/100 mM NH4HCO3. The solution was
loaded on a cellulose column (2.5 × 19 cm), which was
developed with the same mixture of 2-propanol/100 mM NH4-
HCO3. The effluent was lyophilized affording 516 mg (1.65
mmol, 66% based on E-14) of E-6 as a white powder: 1H NMR
(D2O, 500 MHz) δ 5.52 (t, J ) 6.9 Hz, 1H), 4.39 (t, J ) 7.2 Hz,
2H), 3.88 (s, 2H), 1.57 (s, 3H); 13C NMR (D2O, 126 MHz) δ
141.6, 122.7 (d, J ) 8.0 Hz), 68.6, 64.3 (d, J ) 5.1 Hz), 15.1;
31P NMR (D2O, 101 MHz) δ -3.49 (d, J ) 22.3 Hz), -7.17 (d,
J ) 22.0 Hz).
Su p p or tin g In for m a tion Ava ila ble: NMR spectra of
compounds E-6, E/Z-6, and E-12 through E-15. This material
is available free of charge via the Internet at http://pubs.acs.org.
J O025705T
(34) Hahn, F. M.; Hurlburt, A. P.; Poulter, C. D. J . Bacteriol. 1999,
181, 4499.
(35) Wolff, M.; Seemann, M.; Grosdemange-Billiard, C.; Tritsch, D.;
Campos, N.; Rodriguez-Concepcion, M.; Boronat, A.; Rohmer, M.
Tetrahedron Lett. 2002, 43, 2555.
(E/Z)-1-Hydr oxy-2-m eth yl-bu t-2-en yl 4-Diph osph ate Tr i-
a m m on iu m Sa lt (E/Z-6). An E/Z mixture of 6 was prepared
by the reaction sequence described above using the E/Z
mixture of 12 as a starting material. Whereas no attempt was