Synthesis of 4-diphosphocytidyl-2-C-methyl-D-erythritol and 2-C-methyl-D-erythritol-4-phosphate.

Article abstract of DOI:10.1021/jo025736o

2-C-Methyl-D-erythritol 4-phosphate (MEP, 2) and 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDPME, 3) are metabolites in the MEP pathway for biosynthesis of isoprenoid compounds in bacteria, plant chloroplasts, and algae. The free phosphoacid of 2 was prepared from benzyloxyacetone in five steps with an overall yield of 27% and an enantiomeric ratio (er) of 75:25. Following titration to the corresponding tributylammonium salt, 2 was coupled to cytidine 5'-monophosphate using a protocol originally developed for synthesis of base-sensitive nucleoside diphosphate sugars to give 3 in 40% yield, following purification by size exclusion chromatography.

Full text of DOI:10.1021/jo025736o

SCHEME 1. Meth yler yth r itol P h osp h a te P a th w a y
to IP P a n d DMAP P
Syn th esis of
4-Dip h osp h ocytid yl-2-C-m eth yl-D-er yth r itol
a n d 2-C-Meth yl-D-er yth r itol-4-p h osp h a te
Andrew T. Koppisch and C. Dale Poulter*
Department of Chemistry, University of Utah,
Salt Lake City, Utah 84112
poulter@chem.utah.edu
Received March 19, 2002
Abst r a ct : 2-C-Methyl-D-erythritol 4-phosphate (MEP, 2)
and 4-diphosphocytidyl-2-C-methyl-D-erythritol (CDPME, 3)
are metabolites in the MEP pathway for biosynthesis of
isoprenoid compounds in bacteria, plant chloroplasts, and
algae. The free phosphoacid of 2 was prepared from benzy-
loxyacetone in five steps with an overall yield of 27% and
an enantiomeric ratio (er) of 75:25. Following titration to
the corresponding tributylammonium salt, 2 was coupled to
cytidine 5′-monophosphate using a protocol originally de-
veloped for synthesis of base-sensitive nucleoside diphos-
phate sugars to give 3 in 40% yield, following purification
by size exclusion chromatography.
After the discovery that isoprenoids were synthesized
from mevalonate in fungi and mammals in the late 1950s,
it was assumed this was the major route to the funda-
mental isoprenoid building blocks isopentenyl diphos-
phate (IPP1, 7) and dimethylallyl diphosphate (DMAPP,
8) in all organisms.2 More recently, labeling studies
conducted independently in the laboratories of Rohmer3
and Arigoni4 uncovered a novel pathway for the synthesis
of IPP and DMAPP in bacteria, plant chloroplasts, and
algae. Since then most of the transformations in the
“mevalonate-independent” or methylerythritol phosphate
5
(MEP) pathway have been established (Scheme 1).
The first committed step in the MEP pathway is the
conversion of 1-D-deoxyxylulose-5-phosphate (DXP, 1) to
2-C-methyl-D-erythritol-4-phosphate (MEP, 2).6 MEP is
then coupled with cytidine triphosphate (CTP) to produce
4-diphosphocytidyl-2-C-methyl-D-erythritol (CDPME, 3),7,8
which is subsequently phosphorylated and cyclized to
give 2-C-methyl-D-erythritol 2,4-cyclodiphosphate (cME-
PP, 5).9,10 The cyclic diphosphate is converted to 1-hy-
droxy-2-methyl-2-(E)-butenyl 4-diphosphate (HMBPP, 6)
by the enzyme encoded by gcpE,11,12 and recent evidence
indicates the lytB gene product is involved in the conver-
sion of 6 into both IPP and DMAPP.13
* To whom correspondence should be addressed. Phone: (801) 581-
6685. Fax: (801) 581-4391.
(1) Abbreviations used: CDPME, 4-diphosphocytidyl-2-C-methyl-
D-erythritol; CDPMEP, 4-diphosphocytidyl-2-C-methyl-D-erythritol-2-
phosphate; cMEPP, 2-C-methyl-D-erythritol-2,4-cyclodiphosphate; CMP,
cytidine monophosphate; CTP, cytidine triphosphate; DMA, dimethy-
laniline; DMAPP, dimethylallyl diphosphate; DXP, 1-D-deoxyxylulose-
5-phosphate; equiv, equivalents; er, enantiomeric ratio; FPLC, fast
protein liquid chromatography; HMBPP, 1-hydroxy-2-methyl-2-(E)-
butenyl 4-diphosphate; MeIm, methylimidazole; MEP, 2-C-methyl-D-
erythritol-4-phosphate; MSA, methane sulfonamide; MS(EI), electron
impact mass spectrometry; TBA, tributylamine; TEA, triethylamine;
TFAA, trifluoroacetic anhydride.
Because enzymes in the MEP pathway are not found
in animals,14 they are attractive targets for the develop-
(7) 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, 11758-11763.
(8) Kuzuyama, T.; Takagi, M.; Kaneda, K.; Dairi, T.; Seto, H.
Tetrahedron Lett. 2000, 41, 703-706.
(2) Qureshi, N.; Porter, J . W. In Biosynthesis of Isoprenoid Com-
pounds; Porter, J . W., Spurgeon, S. L., Eds.; Wiley: New York, 1981;
Vol. 1, pp 47-94.
(9) Herz, S.; Wungsintaweekul, J .; Schuhr, C. A.; Hecht, S.; Luttgen,
H.; Sagner, S.; Fellermeier, M.; Eisenreich, W.; Zenk, M. H.; Bacher,
A.; Rohdich, F. Proc. Natl. Acad. Sci. U.S.A. 2000, 97, 2426-2490.
(10) Takagi, M.; Kuzuyama, T.; Kaneda, K.; Watanabe, H.; Dairi,
T.; Seto, H. Tetrahedron Lett. 2000, 41, 3395-3398.
(11) 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, 14837-14842.
(3) Rohmer, M.; Knani, M.; Simonin, P.; Sutter, B.; Sahm, H.
Biochem. J . 1993, 295, 517-524.
(4) Eisenreich, W.; Schwarz, M.; Cartayrade, A.; Arigoni, D.; Zenk,
M. H.; Bacher, A. Chem. Biol 1998, 5, R221-R233
(5) Rohdich, F.; Kis, K.; Bacher, A.; Eisenreich, W. Curr. Opin.
Chem. Biol. 2001, 5, 535-540.
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E.; Duvold, T.; Tritsch, D.; Boronat, A.; Rohmer, M. Tetrahedron Lett.
2002, 43, 1413-1415.
(6) Takahashi, S.; Kuzuyama, T.; Watanabe, H.; Seto, H. Proc. Natl.
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10.1021/jo025736o CCC: $22.00 © 2002 American Chemical Society
Published on Web 06/22/2002
5416
J . Org. Chem. 2002, 67, 5416-5418

SCHEME 2. Syn th esis of F r ee Acid of MEP (2)a
ment of herbicides and antimicrobial agents. The MEP
synthase inhibitor fosmidomycin is effective as a treat-
ment for malaria in an animal model.15 At this time only
a few of the enzymes in the MEP pathway have been
studied in detail,5 and a major limitation to this research
has been the unavailability of substrates. Although large-
scale enzymatic preparations of several MEP pathway
intermediates have been reported,16-19 there are no
efficient organic syntheses available for many of the
advanced intermediates. Reliable syntheses of DXP20 and
MEP21 were important in our studies of DXP synthase22
and MEP synthase.23 We now report a chemical synthesis
CDPME (3), the next intermediate in the MEP pathway,
and an improved procedure for the synthesis of MEP (2).
Syn th esis of 2-C-Meth yl-D-er yth r itol-4-p h osp h a te
(2). We recently reported the synthesis of the monoso-
dium salt of 2 in seven steps from 1,2-propane diol.21
Although the synthesis provided ample quantities of 2
for our enzymatic studies, the limited solubility of the
sodium salt in organic solvents compromised its use as
a precursor for the synthesis of 3 by coupling with CMP.
The nucleophilicity of unprotected phosphates is ex-
tremely dependent on the solvent used in coupling
reactions24 and the nature of the counterion. Alkylam-
monium salts of the phosphoacids are often employed to
increase their solubility in organic solvents.25 Since
replacement of alkali metal cations with alkylammonium
ions is an unfavorable process for most commercial ion-
exchange resins, we chose to modify our synthesis of 2
to obtain the free acid as shown in Scheme 2.
a
(a) KH, (2,2,2-trifluoroethyl) methoxycarbonylmethyl-phos-
phonate, THF, -20 °C (67%, 7:3 c:t); (b) LiAlH4, CH2Cl2, -40 °C
(85%); (c) tribenzyl phosphite/iodine, CH2Cl2 0 °C (88%); (d) AD-
mix â, t-BuOH/H2O, 0 °C; (55%); (e) Pd/C, MeOH/H2O (95%).
The asymmetric dihydroxylation of 12 to form 13 is
an adaptation of a similar procedure first reported for
the synthesis of methylerythritol.29 We had previously
observed that standard AD-mix conditions for dihydroxy-
lation of allylic phosphates frequently gave low yields,
accompanied by substantial degradation. For example,
in the previously reported synthesis of 2, dihydroxylation
of an analogue of 12, where the primary alcohol was
protected as a TBS ether and the phosphate was a
dimethyl ester,21 required a substantial increase in the
concentrations of (DHQD)2PHAL and OsO4 and addition
of NaHCO3 to buffer the reaction.30 In addition, the usual
rate enhancement of dihydroxylation associated with
using (methanesulfonamide, NMO) as a co-oxidant was
minimal for the TBS containing allylic phosphate. Com-
pound 12 was more robust to the standard AD-mix
conditions than the TBS derivative. Both modified condi-
tions and buffering had little effect on yield of the
dihydroxylation. By using standard conditions, 12 was
converted to 13 in 55% yield. The er of the dihydroxyla-
tion was estimated to be 75:25 by integration of the
diastereomeric 19F resonances in the Mosher’s ester of
13.31 The benzyl groups in 13 were removed by hydro-
genation over Pd/C in water/methanol to give 2 as the
free acid in five steps and an overall yield of 27%.
Fontana32 recently described a synthesis of MEP from
dimethyl fumarate where dihydroxylation was accom-
plished by asymmetric epoxidation of the trans-isomer
of allylic alcohol 10, followed by opening of the epoxide
with perchloric acid. The E-isomer from our olefination
step could be converted to 2 using this strategy.
The Still modification of the Horner-Emmons reac-
tion26 was used to convert commercially available ben-
zyloxyacetone 9 to the protected olefin 10, with increased
selectivity for the Z-isomer. The isomers were separated
by flash column chromatography, and the Z-ester was
reduced with LiAlH4 to give allylic alcohol 11. The 1H
and 13C NMR spectra of 11 were identical to those
reported by Sato and co-workers.27 Phosphorylation of 11
with iodine and tribenzyl phosphite28 provided the fully
benzylated olefin phosphate 12 in good yield.
(13) Rohdich, F.; Hecht, S.; Gartner, K.; Adam, P.; Krieger, C.;
Amslinger, S.; Arigoni, D.; Bacher, A.; Eisenreich, W. Proc. Natl. Acad.
Sci. U.S.A. 2002, 99, 1158-1163.
(14) Lange, B. M.; Rujan, T.; Martin, W.; Croteau, R. Proc. Natl.
Acad. Sci. U.S.A. 2000, 97, 13172-13177.
(15) J omaa, H.; Wiesner, J .; Sanderbrand, S.; Altincicek, B.; Weide-
meyer, C.; Hintz, M.; Turbachova, I.; Eberl, M.; Zeidler, J .; Lichtentha-
ler, H. K.; Soldati, D.; Beck, E. Science 1999, 285, 1573-1576.
(16) Rohdich, F.; Schuhr, C. A.; Hecht, S.; Herz, S.; Wungsin-
taweekul, J ,; Eisenreich, W.; Zenk, M. H.; Bacher, A. J . Am. Chem.
Soc. 2000, 122, 9571-9574.
(17) Hecht, S.; Wungsintaweekul, J.; Rohdich, F.; Kis, K.; Radykewicz,
T.; Schuhr, C. A.; Eisenreich, W.; Richter, G.; Bacher, A. J . Org. Chem.
2001, 66, 7770-7775.
(18) Hecht, S.; Kis, K.; Eisenreich, W.; Amslinger, S.; Wungsin-
taweekul, J .; Herz, S.; Rohdich, F.; Bacher, A. J . Org. Chem. 2001,
66, 3948-3952.
(19) Hoeffler, J .-F.; Pale-Grosdemange, C.; Rohmer, M. Tetrahedron
2000, 56, 1485-1489.
(20) Blagg, B. S. J .; Poulter, C. D. J . Org. Chem. 1999, 64, 1508-
1511.
(27) Sato, K.; Inoue, S.; Takagi, Y.; Morii, S. Bull. Chem. Soc. J pn.
1976, 49, 3351-3352.
(21) Koppisch, A. T.; Blagg, B. S. J .; Poulter, C. D. Org. Lett. 2000,
2, 315-317.
(28) Gefflaut, T.; Lemaire, M.; Valentin, M. L.; Bolte, J . J . Org.
Chem. 1997, 62, 5920-5922.
(22) Hahn, F. M.; Eubanks, L. M.; Testa, C. A.; Blagg, B. S. J .; Baker,
J . A.; Poulter, C. D. J . Bacteriol. 2001, 183, 1-11.
(23) Koppisch, A. T.; Fox, D. T.; Blagg, B. S. J .; Poulter, C. D.
Biochemistry 2002, 41, 236-243.
(29) Duvold, T.; Cali, P.; Bravo, J . M.; Rohmer, M. Tetrahedron Lett.
1997, 38, 6181-6184.
(30) Kolb, H. C.; VanNieuwenhze, M. S.; Sharpless, K. B. Chem.
Rev. 1994, 94, 2483-2547.
(24) Slotin, L. A. Synthesis 1977, 737-752.
(25) Davisson, V. J .; Woodside, A. B.; Neal, T. R.; Stremler, K. E.;
Muehlbacher, M.; Poulter, C. D. J . Org. Chem. 1986, 51, 4768-4779.
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(32) Fontana, A. J . Org. Chem. 2001, 66, 2506-2508.
J . Org. Chem, Vol. 67, No. 15, 2002 5417

moieties during the time required for the reaction.36 We
anticipated similar problems for the conversion of 2 to
3, which were confirmed in small-scale reactions.
SCHEME 3. Cou p lin g of CMP (14) a n d MEP (2) To
F or m CDP ME (3)a
Triethylammonium cytidine 5′-monophosphate (14)
was prepared by titration of the corresponding phospho-
acid with triethylamine. The nucleoside phosphate was
activated by treatment, in succession, with trifluoroacetic
anhydride and methylimidazole34,35 and then coupled
with the tributylammonium salt of 2. The reaction was
complete after 2 h as judged by 31P NMR and was then
quenched to minimize formation of side products. The
crude mixture was chromatographed over Biogel P2-fine35
with elution by 100 mM (NH4)2HCO3, pH 7.8. Fractions
were monitored both by UV and 31P NMR spectroscopy.
Those containing product were combined and lyophilized
to give a 40% yield of 3 free of buffer salts. Our yield is
comparable to that reported for UDP-Galf.33 1H, 13C, and
31P NMR spectra for 3 agreed with those reported for a
sample prepared enzymatically.6,7 Overall, 3 was ob-
tained in 11% yield in six steps from benzyloxyacetone.
a
(a) TFAA, TEA, CH3CN; (b) MeIm, DMA, TEA, CH3CN; (c)
tributylammonium-2, TEA, CH3CN, 2 Å mol sieves (40%).
Exp er im en ta l Section
Often preparations of 2 contained small quantities of
4-Dip h osp h ocytid yl-2-C-m eth yl-D-er yth r itol (3).6,7 To a
solution of triethylammonium cytidine monophosphate 14 (103
mg, 0.23 mmol) in CH3CN was added dimethylaniline (117 µL,
0.93 mmol) and triethylamine (32 µL, 0.23 mmol). The solution
was cooled to 0 °C before trifluoroacetic anhydride (164 µL, 1.16
mmol) in CH3CN was added over 30 s. The reaction was stirred
for 15 min before excess anhydride and trifluoroacetic acid were
removed at reduced pressure. A solution of methylimidazole (55
µL, 0.69 mmol) and triethylamine (164 µL, 1.16 mmol) in CH3-
CN was added over 3 min, and stirring was continued for 30
min before the mixture was transferred to a solution of tribu-
tylammonium 2 (125 mg, 0.20 mmol) and 50 mg of finely crushed
and activated 4 Å molecular sieves in CH3CN. The reaction was
stirred at 0 °C for 4 h before cold 1 M NH4HCO3, pH 8, was
added. The mixture was extracted with CHCl3, and residual
alkylammonium salts were exchanged with NH4+ by passing the
paramagnetic contaminants from the debenzylation step,
as indicated by broadening of the H NMR resonances.
1
Contaminated samples gave poorer yields of the corre-
sponding alkylammonium salt, and attempts to purify
the debenzylated product over cellulose33 did not remove
the contaminants. The metal contaminants were removed
by ion exchange over CHELEX. Typically the resin was
converted to the proton form by repeatedly washing with
2 M HCl, followed by exhaustive washing with deionized
water. However, the material obtained by titration of 2
following treatment with CHELEX typically contami-
nated by 0.8-1.2 equiv of an extraneous tri-n-butylam-
monium species. We had previously observed degradation
of 2 and migration of the phosphate moiety when the
protecting groups were removed with acid and the
resulting solution was neutralized with NH4HCO3.21 In
contrast, titration of an aqueous solution of the free acid
of 2 to pH 7 with tri-n-butylamine did not promote
intramolecular phosphate migration, and the tributy-
lammonium salt was obtained upon lyophilization.
Syn t h esis of 4-Dip h osp h ocyt id yl-2-C-m et h yl-D-
er yth r itol (3). Our synthesis of 3 (Scheme 3) follows a
recently reported procedure that produces the nucleoside/
carbohydrate diphosphate rapidly, with a minimum of
undesired side products.34
+
aqueous layer over a column of DOWEX 50WX8-200 resin (NH4
form), and eluting with 100 mM NH4HCO3, pH 7.8. After
lyophilization, the crude material was suspended in a minimum
volume of elution buffer (100 mM NH4HCO3, pH 7.8) and applied
to a FPLC column (1.5 cm × 100 cm, Bio-gel P2 fine) that had
been preequilibrated in elution buffer. The column was eluted
at a flow rate of 0.14 mL/min, and fractions were monitored by
UV spectroscopy and 31P NMR. Fractions containing product
1
were lyophilized to give 45 mg (40%) of a white solid. H NMR
(D2O): δ 1.10 (s, 3H), 3.46 (d, J ) 12.2 Hz, 1H), 3.55 (d, J )
12.2 Hz, 1H), 3.81 (d, J ) 6.8 Hz, 1H), 3.94-3.96 (m, 1H), 4.16-
4.33 (m, 6H), 5.94 (d, J ) 4 Hz, 1H), 6.14 (d, J ) 7.3 Hz, 1H),
8.02 (d, J ) 7.3 Hz, 1H). 13C NMR (D2O): δ 18.5, 64.9 (d, J CP
6.0 Hz), 66.4, 66.5, 67.3 (d, J CP ) 6.0 Hz), 69.5, 73.7 (d, J CP
)
)
The overall procedure involves transient activation of
the phosphoester moiety as a trifluoroacetyl anhydride,
followed by its conversion to the corresponding phos-
phoramide by treatment with methylimidazole. This
procedure was first developed for the synthesis of nucleo-
side triphosphates35 and was subsequently adapted for
the synthesis of sugar/nucleoside diphosphates by Mar-
low and Kiessling.34 The TFAA/methylimidazole protocol
provided a higher yield of coupled nucleotide in a much
shorter time than a previously reported synthesis based
on morpholidate/tetrazole activation, where significant
degradation can occur in base-sensitive carbohydrate
7.5 Hz), 74.1, 74.5, 78.0, 83.0 (d, J CP ) 9 Hz), 89.5, 96.4, 141.7,
158.0, 166.4. 31P NMR (D2O): δ -10.59 (d, J ) 20.6 Hz), -11.28
(d, J ) 21.1 Hz). IR (KBr pellet): 3515 (br), 3159 (br), 1642,
1619, 1580, 1401, 1160 cm-1. MS (EI, 70 eV): m/z 551 (M +
NH4, 1), 275 (M - MEP + PO4, 10), 184 (MEP - 2OH, 5). [R]20
D
(c 0.015) +10.2 °.
Ack n ow led gm en t. We would like to thank Dr.
Mark Brown of Echelon Inc. for helpful discussions. This
work was supported by NIH grant GM 25521.
Su p p or tin g In for m a tion Ava ila ble: General methods
and experimental procedures for the synthesis of 2 and 10-
1
13, as well as H, 13C, and 31P NMR spectra. This material is
available free of charge via the Internet at http://pubs.acs.org.
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(35) Bogachev, V. S. Russ. J . Bioorg. Chem. 1996, 22, 599-604.
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5418 J . Org. Chem., Vol. 67, No. 15, 2002