Synthesis of (2E)-4-hydroxy-3-methylbut-2-enyl diphosphate, a key intermediate in the biosynthesis of isoprenoids

Article abstract of DOI:10.1039/b200712f

The synthesis of (2E)-4-hydroxy-3-methylbut-2-enyl diphosphate (HMBPP) was described by regioselective hydroxylation and diphosphorylation of dimethylallyl alcohol. The synthesis started from commercially available 3-methylbut-2-en-1-ol. The 1-alcohol function of starting material was converted to the acetate by treatment with pyridine acetic anhydride. Selective allylic hydroxylation was achieved using selenium dioxide and tert-butyl hydro-peroxide.

Full text of DOI:10.1039/b200712f

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Synthesis of (2E )-4-hydroxy-3-methylbut-2-enyl diphosphate,
a key intermediate in the biosynthesis of isoprenoids
Jane L. Ward and Michael H. Beale*
1
IACR-Long Ashton Research Station, Department of Agricultural Sciences, University of
Bristol, Long Ashton, Bristol, UK BS41 9AF. E-mail: mike.beale@bbsrc.ac.uk
Received (in Cambridge, UK) 21st January 2002, Accepted 19th February 2002
First published as an Advance Article on the web 26th February 2002
(2E)-4-Hydroxy-3-methylbut-2-enyl diphosphate, (HMB-
PP) has been synthesised by regioselective hydroxylation
and diphosphorylation of dimethylallyl alcohol.
studies with transgenic Arabidopsis plants expressing the ipt
gene from Agrobacterium tumefacians have suggested that 1
may be an alternate substrate for the ipt protein leading directly
to the zeatin-type cytokinins (Scheme 2).9
(2E)-4-Hydroxy-3-methylbut-2-enyl diphosphate (HMBPP),
1,1 is a natural product with potent immunomodulatory
activity, that has been recently identified in Escherichia coli.2,3
This compound is produced by the enzyme encoded by the
GcpE (IspG) gene3 and is a key intermediate in the 2-C-methyl-
-erythritol-4-phosphate (MEP) pathway (Scheme 1) leading to
isopentenyl diphosphate (IPP) and dimethylallyl diphosphate
(DMAPP), the universal isoprenoid precursors.4–6 In plants,
this pathway operates in chloroplasts, providing precursors
for the biosynthesis of monoterpenoids, diterpenoids and
carotenoids. The biosynthesis of cytokinins, the important
regulators of plant growth and development, has traditionally
been considered to proceed via a condensation of adenosine-5Ј-
monophosphate and DMAPP to form isopentenyladenosine-
5Ј-monophosphate (iPMP) catalysed by the enzyme encoded by
the ipt gene.7,8 The zeatin type cytokinins (e.g. ZMP, Scheme 2)
were thought to arise from subsequent hydroxylation of the
dimethyallyl moiety of iPMP. However, recent isotope-labelling
Scheme 2
There is an obvious need for a source of 1 for biochemical
studies of the MEP pathway and of zeatin biosynthesis and
for further study of its antigenicity. In this paper we describe
the first synthesis of HMBPP 1. The synthesis starts from
commercially available 3-methylbut-2-en-1-ol (dimethylallyl
alcohol) and is shown in Scheme 3. Although the key step in the
synthesis is the regiospecific hydroxylation of the 4-carbon,
an important factor in the route adopted was to fulfil the
requirement for an effective protecting group strategy that
differentiated between the C-1 and C-4 hydroxy groups.
The 1-alcohol function of starting material was converted to
the acetate by treatment with pyridine–acetic anhydride to
afford 2 in 99% yield. Selective allylic hydroxylation at C-4 of 2
was achieved using selenium dioxide and tert-butyl hydro-
peroxide. This reaction yielded a product with two components
(TLC) that were separable by flash chromatography. The less
polar product (24% yield) contained a mixture of compounds,
amongst which the 4-aldehyde was observed, by 1H NMR. The
more polar fraction was identified as the desired 4-hydroxy
compound 3 (25% yield). The regiochemistry of the reaction
was determined by a 1D-gradient NOE NMR experiment (1D-
GOESY),10 which demonstrated that the 3-methyl group
and 2-H atom were trans-orientated (no NOE), while the 4-
hydrogens and 2-H were cis-orientated (strong NOE). The
oxidation reaction was regiospecific and there was no indi-
cation (1H- and 13C-NMR) of the formation of any of the
corresponding cis-orientated alcohol.
Ready supply of 3 allowed us to develop a strategy of pro-
tection of the new C-4 hydroxy function, to allow deprotection
and diphosphorylation at the C-1-position. The 4-hydroxy
Scheme 1
710
J. Chem. Soc., Perkin Trans. 1, 2002, 710–712
This journal is © The Royal Society of Chemistry 2002
DOI: 10.1039/b200712f

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Scheme 3 Reagents and conditions; i, Ac2O–pyridine; ii, SeO2, t-BuOOH, CH2Cl2; iii, dihydropyran, CH2Cl2, cat. PPTS; iv, K2CO3, MeOH–H2
(3 : 5); v, N-chlorosuccinimide, Me2S, CH2Cl2, 0 ЊC; vi, tris(tetrabutylammonium)hydrogen diphosphate, (NH4)2CO3, MeCN; vii, CH3COOH,
(NH4)2CO3.
group was protected as the tetrahydropyranyl(THP) ether 4,
prepared in the standard way using dihydropyran in 84%
yield. Removal of the 1-acetate group from 4 by treatment
with potassium carbonate in methanol–water (3 : 5) afforded
the 1-hydroxy analogue 5 in 72% yield. Diphosphorylation
and isolation of the protected diphosphate 6 was carried out
according to the methods of Davisson et al.11,12 via the 1-
chloride. The purified product yield was low (14%) but this is
not atypical of this two step conversion and lengthy purification
procedure.11,12 The THP group was removed by treatment with
acetic acid. The target (2E)-4-hydroxy-3-methylbut-2-enyl
diphosphate 1 was obtained as the ammonium salt after
purification by cellulose chromatography.11 NMR spectroscopy
(D2O–NH4OD) confirmed loss of the –OTHP related proton
signals, accompanied by coalescence of the 4-H2 signal to a 2H
singlet at δ 4.03. 31P NMR (D2O–NH4OD) showed two peaks
corresponding to the diphosphate at δ Ϫ6.85 and δ Ϫ9.80. The
stereochemistry around the double bond in 1 was reconfirmed
at this final stage by performing a second 1D-gradient NOE
NMR experiment. Reciprocal nuclear Overhauser enhance-
ments were observed from 2-H (multiplet at δ 5.66) to both the
4-H2 singlet at δ 4.03 and the 1-H2 triplet at δ 4.56. Similarly,
reciprocal enhancements were observed from the 3-Me signal
(δ 1.72) to both 4-H2 at δ 4.03 and 1-H2 at δ 4.56. Significantly,
no NOE effect was observed between the 3-Me (δ 1.72) and 2-H
(δ 5.66) signals. This confirms that the 3-Me and 2-H reside in a
trans arrangement across the double bond. Absence of NOE
between 1-H2 and 4-H2 supported this assignment and therefore
1 is confirmed as having the E stereochemistry. Comparison of
the 1H and 13C NMR data with that reported2,3 for the natural
product, showed slight chemical shift differences, attributable
to use of different solvents (D2O–NH4OD versus D2O2 or
CD3OD–D2O3) but were otherwise in agreement. Negative
ion ESI MS-MS analysis of the neutralised ammonium salt
was in total agreement with data published2 for the natural
compound. Ions of note are the deprotonated molecular ion at
m/z 261 with a daughter ion at 243 (ϪH2O), and those at 177
with tetramethylsilane as an internal reference. For diphosphate
analogues NMR analysis was carried out in D2O, adjusted
to pH 8 by the addition of NH4OD. H- chemical shifts are
1
reported as δ values using sodium 3-(trimethylsilyl)propane-1-
sulfonate as an internal reference. 31P-NMR spectroscopy was
carried out in D2O–NH4OD and was externally referenced to
phosphoric acid (δ 0.0). HR-MS analyses were performed on a
Kratos MS80RFA mass spectrometer by direct insertion probe.
ESI-MS-MS was carried out on an LCQ mass spectrometer
(Thermoquest) using negative ion mode and HR-ESI-TOF MS
analysis was carried out on a Micromass Q-TOF spectrometer.
3-Methylbut-2-enyl acetate 2
3-Methylbut-2-en-1-ol (20 g) in pyridine (50 mL) and acetic
anhydride (100 mL) was stirred at room temperature for 2.5 h.
The solution was then poured into water (250 mL), acidified to
pH 3 with c.HCl and extracted into diethyl ether (2 × 200 mL)
to afford the crude acetate 2 (29.5 g, 99%) as a colourless oil,
which was used without further purification. 1H NMR (CDCl3)
δ 1.71(s, 3-CH3), 1.77(s, 4-H3), 2.06(s, –OCOCH3), 4.58(d, J = 7
Hz, 1-H2), 5.37(m, 2-H). HR-MS (EI) m/z 128.0851(2%, Mϩ,
C7H12O2 requires 128.0837), 86(12%), 71(18), 68(100), 60(6),
53(39).
(2E )-4-Hydroxy-3-methylbut-2-enyl acetate 3
3-Methylbut-2-enyl acetate 2 (29.5 g) in dichloromethane
(200 mL) was treated with selenium dioxide (19.4 g) and tert-
butyl hydroperoxide (74 mL). The solution was stirred under
nitrogen at room temperature overnight. Dilute HCl (pH 3)
was added and the organic layer was collected and washed
with water, dried (MgSO4) and evaporated to afford the crude
oxidised product. Flash chromatography using hexane–EtOAc
mixtures afforded a mixture containing the 4-aldehyde [δ 9.46-
(s, 4-CHO), 6.52(m, 2-H), 4.92(dd, J = 6 and 1 Hz, 1-H2)] con-
taminated with tert-butyl hydroperoxide containing residues
(8.01 g, 24%) followed by the desired (2E)-4-hydroxy-3-methyl-
but-2-enyl acetate 3 (8.51 g, 25%) as a colourless oil. 1H NMR
(CDCl3) δ 1.71(s, 3-CH3), 2.06(s, –OCOCH3), 4.02(s, 4-H2),
4.65(d, J = 7 Hz, 1-H2), 5.60(m, 2-H); 13C NMR (CDCl3, 100
MHz) δ 14.0(q, 3-CH3), 21.1(q, –OCOCH3), 60.9(t, 1-C),
67.3(t, 4-C), 118.4(d, 2-C), 141.3(s, 3-C), 171.6(s, –OCOCH3).
HR MS (EI) m/z 144.0793(2%, Mϩ, C7H12O3 requires
144.0786), 129(37%), 119(12), 103(30), 101(100), 86(25), 71(15),
59(89).
Ϫ
Ϫ
(H3P2O7 ) and 159 (base peak, H2P2O6 ).
In conclusion, the establishment of this synthesis of HMBPP
now provides a means to investigate further the pivotal role
of this compound in the MEP pathway, and in particular the
function of the lytB gene, its role in cytokinin biosynthesis and
its pharmacological properties.
Experimental
General techniques were as previously described.13 Tris(tetra-n-
butylammonium) hydrogen pyrophosphate was prepared by the
method of Davisson et al.10 1H- and 13C-NMR spectra were
recorded on a Bruker Avance 400 MHz spectrometer, in CDCl3,
(2E )-4-Tetrahydropyranyloxy-3-methylbut-2-enyl acetate 4
(2E)-4-Hydroxy-3-methylbut-2-enyl acetate
3 (4.5 g) in
dichloromethane (100 mL) was treated with dihydropyran
J. Chem. Soc., Perkin Trans. 1, 2002, 710–712 711

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(5.7 mL) and PPTS (15 mg). The resultant solution was stirred
overnight under nitrogen. Removal of the solvent under
reduced pressure afforded the crude product which upon flash
chromatography with hexane–ethyl acetate mixtures gave the
clean THP derivative 4 (6.01 g, 84%). 1H NMR (CDCl3) δ 1.50–
1.70(5H, m, 2Ј-H, 3Ј-H2, 4Ј-H2), 1.77(s, 3-CH3), 1.88(m, 2Ј-H),
2.06(s, –OCOCH3), 3.50(m, 5Ј-H), 3.85(m, 5Ј-H), 3.91(d, J = 12
Hz, 4-H), 4.16(d, J = 12 Hz, 4-H), 4.63(m, 1Ј-H), 4.65(d, J = 7
Hz, 1-H2), 5.63(m, 2-H); 13C NMR (CDCl3, 100 MHz) δ 14.5(q,
3-CH3), 20.1(t), 21.3(q, –OCOCH3), 25.7(t), 30.9(t), 60.7(t,
1-C), 62.4(t, 5Ј-C), 71.8(t, 4-C), 98.1(d, 1Ј-C), 120.4(d, 2-C),
138.5(s, 3-C), 171.3(s, COCH3). EI-MS m/z 213(1%, M Ϫ 15),
117(12), 101(22), 85(100).
(D2O–NH4OD) δ 1.50–1.70(5H, m, 2Ј-H, 3Ј-H2, 4Ј-H2), 1.73(s,
3-CH3), 1.77(m, 2Ј-H), 3.58(m, 5Ј-H), 3.92(m, 5Ј-H and 1Ј-H),
4.02(d, J = 12 Hz, 4-H), 4.16(d, J = 12 Hz, 4-H), 4.53(t, J = 7 Hz,
1-H2), 5.71(m, 2-H); 13C NMR (D2O–NH4OD) δ 13.9(q,
3-CH3), 19.5(t), 24.9(t), 30.4(t), 62.4(t, 1-C), 63.7(t, 5Ј-C),
73.0(t, 4-C), 98.9(d, 1Ј-C), 124.4(d, 2-C), 136.7(s, 3-C). 31P
NMR (D2O–NH4OD): δ = Ϫ6.10, Ϫ9.88.
(2E )-4-Hydroxy-3-methylbut-2-enyl diphosphate 1
(2E)-4-Tetrahydropyranyloxy-3-methylbut-2-enyl diphosphate
6 (100 mg) was treated with acetic acid and left at room
temperature overnight. The mixture was then neutralised by
the addition of sodium hydrogen carbonate solution. Cellulose
chromatography as above followed by lyophilization afforded
the pure ammonium salt of (2E)-4-hydroxy-3-methylbut-2-enyl
4-Tetrahydropyranyloxy-3-methylbut-2-en-1-ol 5
1
4-Tetrahydropyranyloxy-3-methylbut-2-enyl acetate 4 (6 g) in
methanol–water (3 : 5, 80 mL) was treated with potassium
carbonate (4 g). The reaction was stirred at room temperature
for 2 h whereupon a more polar product was observed by TLC.
The methanol was removed by evaporation and the resultant
residue partitioned between water and ethyl acetate. The
organic layer was separated, dried (MgSO4) and the solvent
removed under reduced pressure to afford the crude product
(4.5 g). Flash chromatography using hexane–ethyl acetate
diphosphate 1 (40 mg, 51%) as a white solid. H NMR (D2O–
NH4OD) δ 1.72(s, 3-CH3), 4.03(s, 4-H2), 4.56(t, J = 7 Hz, 1-H2),
5.66(m, 2-H); 13C NMR (D2O–NH4OD): δ = 13.4(q, 3-CH3),
62.9(t, 1-C), 66.9(t, 4-C), 121.0(d, 2-C), 140.4(s, 3-C); 31P NMR
(D2O–NH4OD): δ = Ϫ6.85, Ϫ9.80. ESI-TOF-MS (Ϫve) m/z
260.9913 (M Ϫ H), C5H12O8P2 requires M Ϫ H = 260.9929;
242.9844 (M Ϫ H Ϫ H2O), C5H12O8P2 Ϫ H Ϫ H2O requires
242.9824. ESI-MS-MS (Ϫve) 261(1%), 243(56), 177(3), 163(9),
159(100), 97(9), 79(6).
1
mixtures afforded the pure alcohol 5 (3.5 g, 72%). H NMR
(CDCl3) δ 1.50–1.70(5H, m, 2Ј-H, 3Ј-H2, 4Ј-H2), 1.71(s, 3-CH3),
1.88(m, 2Ј-H), 3.50(m, 5Ј-H), 3.85(m, 5Ј-H), 3.90(d, J = 12 Hz,
4-H), 4.11(d, J = 12 Hz, 4-H), 4.19(d, J = 7 Hz, 1-H2), 4.65(t,
J = 3.5 Hz, 1Ј-H), 5.67(m, 2-H); 13C NMR (CDCl3, 100 MHz)
δ 14.3(q, 3-CH3), 19.4(t), 25.7(t), 30.8(t), 58.8(t, 1-C), 62.3(t,
5Ј-C), 72.4(t, 4-C), 97.9(d, 1Ј-C), 126.5(d, 2-C), 134.8(s, 3-C).
HR-MS (EI) m/z 168.1139[1%, Mϩ Ϫ 18, C10H16O2 (M Ϫ H2O)
requires 168.1150], 141(1%), 116(3), 101(27), 85(100), 67(18),
55(22).
Acknowledgements
IACR receives grant-aided support from the Biotechnology
and Biological Sciences Research Council of the United
Kingdom. We thank Mervyn Lewis and Nathan Hawkins for
MS analysis.
References
1 Synonyms also in use for 1 are (2E)-1-hydroxy-2-methylbut-2-enyl
4-diphosphate and 4-hydroxydimethylallyl diphosphate (HO-
DMAPP).
2 M. Hintz, A. Reichenberg, B. Altincicek, U. Bahr, R. M. Gschwind,
A.-K. Kollas, E. Beck, J. Wiesner, M. Eberl and H. Jomaa, FEBS
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Oxford, 1999, pp. 45–68.
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M. Muehlbacher and C. D. Poulter, J. Org. Chem., 1986, 51, 4768.
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(2E )-4-Tetrahydropyranyloxy-3-methylbut-2-enyl diphosphate 6
N-Chlorosuccinimide (2.67 g) in dry dichloromethane (100 mL)
was cooled to 0 ЊC. Dimethyl sulfide (2.24 mL) was added
followed by 4-tetrahydropyranyloxy-3-methylbut-2-en-1-ol 5
(3.4 g). After stirring for 1 h at 0 ЊC, the temperature was then
raised to room temperature and the mixture was stirred for a
further 15 min. The mixture was diluted with saturated brine.
The organic layer was separated, dried (MgSO4) and the solvent
removed under reduced pressure to afford the crude chloride,
which was used without further purification. 1H NMR (CDCl3)
δ 1.50–1.70(5H, m, 2Ј-H, 3Ј-H2, 4Ј-H2), 1.79(s, 3-CH3), 1.88(m,
2Ј-H), 3.50(m, 5Ј-H), 3.85(m, 5Ј-H), 3.90(d, J = 12 Hz, 4H),
4.11(d, J = 7 Hz, 1-H2), 4.14(d, J = 12 Hz, 4-H), 4.62(t, J =
3.5 Hz, 1Ј-H), 5.71(m, 2-H); 13C NMR (CDCl3, 100 MHz)
δ 14.3(q, 3-CH3), 19.7(t), 25.8(t), 30.9(t), 40.6(t, 1-C), 62.5(t,
5Ј-C), 71.7(t, 4-C), 98.2(d, 1Ј-C), 122.1(d, 2-C), 139.0(s,
3-C). The chloride was dissolved in acetonitrile (50 mL) and
treated with tris(tetrabutylammonium)hydrogen diphosphate
(34.7 g). The resultant solution was stirred under nitrogen
overnight. The solvent was removed under reduced pressure
to give the crude diphosphate. Purification by ion exchange
ϩ
chromatography (Dowex 50W-8X, NH4 form) eluted with
1 : 49 isopropanol–aqueous ammonium bicarbonate (25 mM)
and lyophilisation, followed by chromatography on cellulose
(Whatman CF11) eluted with 9 : 5 : 6 isopropanol–acetonitrile–
ammonium bicarbonate (100 mM) and final lyophilisation gave
1
the diphosphate 6 (1.02 g, 14%) as a white solid. H NMR
712
J. Chem. Soc., Perkin Trans. 1, 2002, 710–712