Synthesis of Enantiopure 2-C-Methyl-D-erythritol 4-Phosphate and 2,4-Cyclodiphosphate from D-Arabitol

Article abstract of DOI:10.1021/ol0362562

(Equation presented) Two key intermediates of the newly discovered mevalonate-independent pathway for isoprenoid biosynthesis were prepared. Optically pure 2-C-methyl-D-erythritol 4-phosphate and 2,4-cyclodiphosphate were chemically synthesized from D-arabitol using a convenient benzylidene and tert-butyldimethylsilyl protection of polyhydroxylated intermediates. The new scheme offers a straightforward route to analogues and labeled forms.

Full text of DOI:10.1021/ol0362562

ORGANIC
LETTERS
2004
Vol. 6, No. 1
135-138
Synthesis of Enantiopure
2-C-Methyl-D-erythritol 4-Phosphate and
2,4-Cyclodiphosphate from D-Arabitol
Marek Urbansky, Chad E. Davis, Jacob D. Surjan, and Robert M. Coates*
Department of Chemistry, UniVersity of Illinois at Urbana-Champaign,
600 South Mathews AVenue, Urbana, Illinois 61801
coates@scs.uiuc.edu
Received November 18, 2003
ABSTRACT
Two key intermediates of the newly discovered mevalonate-independent pathway for isoprenoid biosynthesis were prepared. Optically pure
2-C-methyl-D-erythritol 4-phosphate and 2,4-cyclodiphosphate were chemically synthesized from D-arabitol using a convenient benzylidene
and tert-butyldimethylsilyl protection of polyhydroxylated intermediates. The new scheme offers a straightforward route to analogues and
labeled forms.
Terpenes, one of the largest groups of natural products, are
assembled from two precursors, dimethylallyl diphosphate
(DMAPP) and isopentenyl diphosphate (IPP) by electrophilic
elongations, cyclizations, and rearrangements. Since the first
identification of the acetate-replacement factor in 1956, the
mevalonate pathway was considered to be the universal
source of these biosynthetic isoprene units.1 However, the
recent discovery of the mevalonate-independent (methyl-
erythritol phosphate, MEP) pathway for isoprenoid biosyn-
thesis in bacteria, plant chloroplasts, and algae has triggered
an intense burst of research activity.2 Since the MEP pathway
is absent in animal cells, new opportunities have become
available to develop herbicides and drugs against pathogenic
bacteria and the malaria parasite.3 Although many details of
this novel route are unknown, some intermediates, mecha-
nisms, enzymes, and genes have been identified (Scheme 1,
Cy ) cytidyl).2,4
erythritol 4-phosphate (MEP, 4), the first pathway-specific
intermediate. MEP is then coupled with cytidine triphosphate
(CTP) to produce 4-diphosphocytidyl-2-C-methyl-D-erythritol
(CDP-ME, 5), which is subsequently phosphorylated to give
4-diphosphocytidyl-2-C-methyl-D-erythritol 2-phosphate (6).
Cyclization of 6 leads to 2-C-methyl-D-erythritol 2,4-
cyclodiphosphate (MECDP, 7), previously identified as an
oxidative stress metabolite of gram-negative bacterial para-
(1) Bochar, D. A.; Friesen, J. A.; Stauffacher, C. V.; Rodwell, V. W. In
ComprehensiVe Natural Products Chemistry. Isoprenoids Including Caro-
tenoids and Steroids; Cane, D. E., Ed.; Elsevier: Amsterdam, 1999; Vol.
2, pp 15-44.
(2) (a) Kuzuyama, T.; Seto, H. Nat. Prod. Rep. 2003, 20, 171-183. (b)
Dewick, P. M. Nat. Prod. Rep. 2002, 19, 181-222. (c) Sponsel, V. M. J.
Plant Growth Regul. 2001, 20, 332-345. (d) Rohdich, F.; Kis, K.; Bacher,
A.; Eisenreich, W. Curr. Opin. Chem. Biol. 2001, 5, 535-540. (e)
Eisenreich, W.; Rohdich, F.; Bacher, A. Trends Plant Sci. 2001, 6, 78-84.
(f) Lichtenthaler, H. K. Biochem. Soc. Trans. 2000, 28, 785-789. (g)
Rohmer, M. Pure Appl. Chem. 1999, 71, 2279-2284. (h) Lichtenthaler,
H. K. Annu. ReV. Plant Physiol. Plant Mol. Biol. 1999, 50, 47-65. (i)
Rohmer, M. In ComprehensiVe Natural Products Chemistry. Isoprenoids
Including Carotenoids and Steroids; Cane, D. E., Ed.; Elsevier: Amsterdam,
1999; Vol. 2, pp 45-67. (j) Rohmer, M. Nat. Prod. Rep. 1999, 16, 565-
574. (k) Eisenreich, W.; Schwarz, M.; Cartayrade, A.; Arigoni, D.; Zenk,
M. H.; Bacher, A. Chem. Biol. 1998, 5, R221-R233. (l) Lichtenthaler, H.
K. Fett/Lipid 1998, 100, 128-138.
The initial transketolase-like condensation between D-
glyceraldehyde 3-phosphate (1) and pyruvate (2) forming
1-deoxy-D-xylulose 5-phosphate (3) is followed by semi-
benzilic rearrangement and reduction to 2-C-methyl-D-
10.1021/ol0362562 CCC: $27.50 © 2004 American Chemical Society
Published on Web 12/09/2003

merically pure MEP and MECDP, which appears to be read-
ily adaptable to access the cytidine intermediates, to prepara-
tion of various analogues, and to stereoselective labeling.17
Literature procedures18,19 were used to prepare 1,3-ben-
zylidene-D-threitol (12) from commercially available D-arabi-
tol (Scheme 2). The primary hydroxyl group of the diol
Scheme 1
Scheme 2
was protected with a tert-butyldimethylsilyl group (TBSCl,
DMAP, Et3N, DMF) to give monoether 13. TPAP/NMO
oxidation20 afforded the key intermediate 14. Alternatively,
phase-transfer-promoted RuO4 oxidation (RuO2/NaIO4, CCl4,
H2O, BnEt3NCl)21 proved to be of similar efficiency with
the advantage of lower cost for the oxidant. The re-
quired 2S configuration for 2-C-methyl-D-erythritol was
expected to be obtained by nucleophilic additions to the
protected D-erythrulose 14 since highly selective axial attack
in the reactions of complex metal hydrides22 and various
sites.5 The last reductive steps leading to IPP (9) and DMAPP
(10) via (E)-4-hydroxy-DMAPP (8) involving the gcpE and
lytB genes are still a matter of investigation.
Efficient synthesis of MEP pathway intermediates, their
labeled forms, and their derivatives is important in the mech-
anistic study of these unusual enzymes and for discovery of
specific inhibitors and therapeutic agents. Enzymatic prepa-
rations of phosphate 46,7 and cyclodiphosphate 78-10 have
been reported. Known chemical syntheses are either based
on asymmetric oxidation methods of suitably functionalized
3-methylbut-2-enyl-1,4-diols11-14 or utilize carbohydrate pre-
cursors.15,16 We report a new, versatile approach to enantio-
(7) Hecht, S.; Wungsintaweekul, J.; Rohdich, F.; Kis, K.; Radykewicz,
T.; Schuhr, C. A.; Eisenreich, W.; Richter, G.; Bacher, A. J. Org. Chem.
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2000, 56, 1485-1489.
(17) A number of singly and multiply labeled forms of MEP and MECDP
(4 and 7) bearing deuterium, tritium, carbon-13, or carbon-14 have been
prepared, mostly by enzymatic methods. See refs 5e, 7, and 10 and the
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Zenk, M. H. Tetrahedron Lett. 1999, 40, 2743-2746. (b) Rohdich, F.;
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136
Org. Lett., Vol. 6, No. 1, 2004

organometallic reagents with 1,3-dioxan-5-ones is well
established.23-25 In agreement with this precedent, reduction
of 14 with NaBH4/MeOH yielded exclusively equatorial
alcohol 15. Cleavage of the primary silyl ether gave 1,3-
benzylidene-D-erythritol (16), the physical data of which
are similar to those reported for the known L enantio-
mer.26 In like manner, Grignard addition (MeMgBr, Et2O,
-78 °C) was accomplished in g20:1 selectivity, and
silica gel chromatography afforded pure tertiary alcohol 17
(Scheme 3).
diammonium salt of MEP (4), [R]D +11.3° (c 1.6, H2O),
with dry gaseous ammonia.
Double phosphorylation27 of 18 by the phosphoramidite
method led to the benzyl-protected diphosphate 21 (Scheme
5). Selective debenzylation (H2, Pd/C, MeOH, -10 °C) gave
Scheme 5
Scheme 3
Cleavage of the TBS protecting group (Bu4NF, THF)
afforded 1,3-benzylidene derivative 18, mp 99-100 °C, and
subsequent hydrogenolysis (H2, 200 psi, Pd(OH)2/C, EtOH,
HCOOH) gave the parent C5 tetrol, 2-C-methyl-D-erythritol
(19), that had spectroscopic properties in agreement with
those reported by Kis et al.15
Selective phosphorylation of the primary hydroxyl group
of benzylidene diol 18 was effected by lithiation with BuLi
(1.2 equiv, THF, -78 °C) and reaction with dibenzyl
chlorophosphoridate (Scheme 4). The resulting monophos-
bis-phosphomonoester 22, which was characterized as its
tetraammonium salt 23. 13C NMR spectra (126 MHz, D2O)
of 23 show three-bond 13C-31P coupling at the C-3 methine
(85.6 ppm, dd, JPC ) 10.5, JP′C ) 8.1 Hz). Additional
signals indicate the presence of one methyl (C-2′, 19.82
ppm), five benzylidene (104.68, 129.18, 131.47, 132.68,
139.04 ppm), and one quatermary and two methylene (65.60,
73.15, 78.35 ppm) carbons. The 2-C-methylerythritol part
of the molecule is represented in the 1H NMR spectrum (500
MHz, D2O) by a singlet (2′-CH3, 1.45 ppm), an AB pattern
3
3
for the endocyclic methylene (3.92 and 4.13 ppm, JAB
)
Scheme 4
10.8 Hz), and three clearly resolved signals (3.67, 4.00, 4.07
ppm) indicating the POCH2CH fragment.
We found that bisphosphate 22 is smoothly converted to
cyclic diphosphate 24 under the influence of 1,1′-carbonyl-
diimidazole (anhyd DMSO, room temperature, 4 h).28 The
crude imidazolium salt was exchanged to the ammonium
form (Dowex 50WX8-200) and purified by silica gel
chromatography. The 31P NMR spectrum of the product
displays signals at -15.80 and -11.09 ppm as doublets with
31P-31P coupling (J ) 25 Hz), confirming the presence of
the phosphoryl anhydride linkage. The 13C NMR spectrum
reflects changes of the P-O-C-C dihedral angles by
3
splitting of the C-2′ methyl (19.30 ppm, d, JPC ) 6.4 Hz)
phate 20, mp 86-87 °C, underwent simultaneous hydro-
genolysis of the benzyl and benzylidene protecting groups
to form the phosphomonoester that was converted to the
and by disappearance of the strong 13C-31P coupling at C-3
(80.50 ppm). Final hydrogenolytic removal of the ben-
(23) Kobayashi, Y. M.; Lambrecht, J.; Jochims, J. C.; Burkert, U. Chem.
Ber. 1978, 111, 3442-3459.
(24) Wu, Y.-D.; Houk, K. N. J. Am. Chem. Soc. 1987, 109, 908-910.
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R.; Marco, J. A. Tetrahedron: Asymmetry 1997, 8, 559-577.
(26) Ghavami, A.; Johnston, B. D.; Pinto, B. M. J. Org. Chem. 2001,
66, 2312-2317.
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(28) Schaller, H.; Staab, H. A.; Cramer, F. Chem. Ber. 1961, 94, 1621-
1633.
Org. Lett., Vol. 6, No. 1, 2004
137

zylidene group provided 2-C-methyl-D-erythritol 2,4-cyclo-
diphosphate (7) as the diammonium salt, mp 220-222 °C
(dec), [R]D + 1.8° (c 1.00).
tural analogues and labeled forms17 of 4, 7, and 19 can be
readily devised. These compounds and labeled variants, like
their mevalonate counterparts,29 are certain to find useful
applications in research on MEP pathway enzymes and
terpene biosynthesis.
25
Benzylidene acetals 23 and 24 were also evaluated under
1
conditions of acid-catalyzed hydrolysis. H NMR kinetic
experiments in HCOOH/D2O demonstrated that cleavage of
the benzylidene moiety of 23 is approximately two times
faster than that of 24. The diphosphate anhydride of 24
proved to be hydrolytically stable, and besides formation of
7, no other products were observed in 1H and 31P NMR spec-
tra. Thus, the hydrolytic lability of the benzylidene acetals
can be utilized as an alternative method of deprotection.
In conclusion, this straightforward synthesis affords opti-
cally pure phosphates 4 and 7 in substantial amounts and
satisfactory yields. The benzylidene protecting group proved
to be stable to nucleophilic and basic conditions; its rigid
structure favors cyclic diphosphate formation, and depro-
tections under neutral or weakly acidic conditions avoid facile
cyclic phosphate rearrangements.14 Schemes to access struc-
Acknowledgment. The authors thank R. B. Miles for
assistance in obtaining optical rotations and mass spectral
data and the National Institutes of Health (GM 13956) for
financial support.
Supporting Information Available: Details of experi-
mental procedures, characterization data, and reproductions
of NMR spectra. This material is available free of charge
via the Internet at http://pubs.acs.org.
OL0362562
(29) (a) Retey, J.; Robinson, J. A. Stereospecificity in Organic Chemistry
and Enzymology; Verlag Chemie: Weinheim, 1982. (b) Hanson, J. R. AdV.
Steroid Biochem. Pharmacol. 1970, 1, 51-72.
138
Org. Lett., Vol. 6, No. 1, 2004