(3R,4S)-3,4,5-Trihydroxy-4-methylpentylphosphonic acid, an isosteric phosphonate analogue of 2-C-methyl-D-erythritol 4-phosphate, a key intermediate in the new pathway for isoprenoid biosynthesis

Article abstract of DOI:10.1016/j.tetlet.2003.11.001

2-C-Methyl-D-erythritol 4-phosphate (MEP) is the first intermediate in the mevalonate-independent pathway for isoprenoid biosynthesis presenting the branched C₅ isoprene skeleton. Enantiopure (3R,4S)-3,4,5-trihydroxy- 4-methylpentylphosphonic a

Full text of DOI:10.1016/j.tetlet.2003.11.001

Tetrahedron
Letters
Tetrahedron Letters 45 (2004) 519–521
(3R,4S)-3,4,5-Trihydroxy-4-methylpentylphosphonic acid, an
isosteric phosphonate analogue of 2-C-methyl- -erythritol
D
4-phosphate, a key intermediate in the new pathway for
isoprenoid biosynthesis
Guillaume Hirsch, Catherine Grosdemange-Billiard, Denis Tritsch and Michel Rohmer*
CNRS UMR 7123, Institut Le Bel, Universiteꢀ Louis Pasteur, 4 rue Blaise Pascal, 67070 Strasbourg Cedex, France
Received 17 September 2003; revised 30 October 2003; accepted 3 November 2003
Abstract—2-C-Methyl-D-erythritol 4-phosphate (MEP) is the first intermediate in the mevalonate-independent pathway for iso-
prenoid biosynthesis presenting the branched C5 isoprene skeleton. Enantiopure (3R,4S)-3,4,5-trihydroxy-4-methylpentylphos-
phonic acid (MEPN), an isosteric phosphonate analogue of MEP was synthesized from 1,2-O-isopropylidene-a-
D-xylofuranose.
ꢀ 2003 Elsevier Ltd. All rights reserved.
Recently an alternative metabolic route, the methyl-
erythritol phosphate (MEP, 1) pathway (Scheme 1), was
discovered for the formation of isoprenoids in many
bacteria, green algae and plastids from plants and other
algal phyla.1 The presumed first committed step in the
cytidine triphosphate to produce 4-diphosphocytidyl
2-C-methyl-D
-erythritol 4-phosphate 3,3 which is sub-
sequently phosphorylated,4 cyclized and converted into
(E)-4-hydroxy-3-methylbut-2-enyl diphosphate 4, which
is the last intermediate of the pathway.5
MEP pathway is the conversion of 1-deoxy-
5-phosphate (DXP, 2) to 2-C-methyl-
D
-xylulose
-erythritol
D
Animals and humans synthesize their isoprenoids only
via the MVA pathway, with no evidence for the presence
of the MEP pathway. Each enzyme of the MEP path-
way may thus represent an attractive target for the
development of new biocides, which are of major
interest in the present situation of bacterial resistance
towards antibiotics. A first application is pointed out by
the DXP reducto-isomerase (DXR, Scheme 1) inhibitor,
the antibiotic fosmidomycin, which is effective against
bacteria and Plasmodium spp., the parasite responsible
for malaria, utilizing the MEP pathway.6 We decided
to synthesize (3R,4S)-3,4,5-trihydroxy-4-methylpentyl-
phosphonic acid (MEPN, 18, Scheme 2), an analogue of
MEP 1 that may act as an inhibitor of some of the MEP
pathway enzymes. In this analogue the phosphate group
of MEP is replaced by an isosteric phosphonate group.7
In addition, the chirality of the carbon chain and its
functionalization are the same in MEPN 18 as those in
MEP, the natural intermediate in the terpenoid bio-
synthetic pathway.
4-phosphate 1.2 In later steps, MEP is coupled with
Scheme 1. Methylerythritol phosphate pathway for isoprenoid bio-
synthesis.
Keywords: Biosynthesis; Isoprenoids; 2-C-Methyl-
phate; Phosphonate; 1-Deoxy- -xylulose reducto-isomerase; 2-C-
Methyl- -erythritol 4-phosphate cytidyltransferase.
D-erythritol 4-phos-
D
An efficient chemical synthesis of MEP from 1,2-O-
isopropylidene-a-
D
D-xylofuranose 7 was developed in our
laboratory.8 The synthesis of the MEP analogue, MEPN
* Corresponding author. Tel.: +33-3-90-24-13-49; fax: +33-3-90-24-13-
45; e-mail: mirohmer@chimie.u-strasbg.fr
0040-4039/$ - see front matter ꢀ 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.tetlet.2003.11.001

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G. Hirsch et al. / Tetrahedron Letters 45 (2004) 519–521
Scheme 2. Synthesis of (3R,4S)-3,4,5-trihydroxy-4-methylphosphonic acid MEPN 18: (i) (Bu3Sn)2O, Dean–Stark, BnBr, TBAB, toluene, 90 ꢁC, 3 d
(70%); (ii) (COCl)2, DMSO, TEA; (iii) NaBH4, MeOH, 0 ꢁC (42% over two steps); (iv) TBDMSOTf, 2,6-lutidine, CH2Cl2, 0 ꢁC to rt (90%); (v) H2,
Pd/C, AcOEt, 6 d (98%); (vi) Tf2O, TEA, CH2Cl2, )40 ꢁC; (vii) CH3P(O)(OBn)2, n-BuLi, HMPA, THF, )78 ꢁC; (viii) TBAF, THF, rt (67% over
three steps); (ix) (COCl)2, DMSO, TEA, then MeMgCl, THF (68%); (x) CCl3C(NH)OBn, CF3SO3H (cat.), CH2Cl2/cyclohexane (1/2), rt (73%); (xi)
TFA 90%, )15 ꢁC (65%); (xii) NaIO4, MeOH/H2O (1/1), rt (97%); (xiii) NaBH4, MeOH, )15 ꢁC, (69%); (xiv) H2, Pd/C, EtOH (92%).
18, was therefore performed, partly using the strategy
based on these previous results.
this approach was the introduction of the phosphonate
on ribofuranose derivative 10 using BerkowitzÕs meth-
odology.9 After activation of the primary hydroxyl by
conversion into a triflate and displacement by the dibenz-
ylmethylphosphonate anion,12 the 1,2-O-isopropylidene-
3-O-t-butyldimethylsilyl-5,6-deoxy-6-dibenzylphosphono-
Our first attempts to introduce the phosphonate group
on an already 3-C-methylated 1,2-isopropylidene-a-D-
ribofuranose or a xylofuranose derivative were unsuc-
cessful, most probably due to steric hindrance.9 The
strategy was therefore changed, and the phosphonate
a-D-allofuranose 11 was generated in satisfactory yield.
The silyl protecting group was removed in the presence
of tetrabutylammonium fluoride. Oxidation of the sec-
ondary alcohol 12 was performed in THF using the
Swern oxidation as modified by Ireland.13 Under these
conditions, the methyl group was directly introduced by
addition of methylmagnesium chloride without isolation
of the intermediate ketone to yield the tertiary alcohol
13. Methylation occurred on the less hindered b-face,
and the stereochemistry was verified by NOESY NMR
correlations. The NOE between H-1 and H-2 confirmed
the anomeric a-configuration and the position of the
two protons on the b-face of the five-membered ring.
The position of the methyl group at C-3 on the b-face of
the furanose ring was supported by a strong NOE
between H-1, H-2 and the protons of the C-3 methyl
group. Benzylation with benzyl-2,2,2-trichloroacetimi-
date14 yielded the protected tertiary alcohol 1,2-O-iso-
propylidene-3-C-methyl-O-benzyl-5,6-deoxy-6-dibenzyl-
group was introduced to a 1,2-isopropylidene-a-D-
ribofuranose derivative with an unhindered b-face and
the 3-C position was methylated afterwards. The
required 1,2-O-isopropylidene-a-
tive was not commercially available and was synthesized
from the 1,2-O-isopropylidene-a- -xylofuranose 7.
D-ribofuranose deriva-
D
The first step of the synthetic route is a regioselective
protection of the primary hydroxyl group with bistri-
butyltin oxide and benzyl bromide10 to give the 5-O
benzylated species 8 in 70% yield. Swern oxidation of
the free alcohol gave the corresponding ketone, which
was stereoselectively reduced by sodium borohydride10
to the corresponding ribose derivative 9. The reduction
selectively occurred on the furanose b-face, which is
more accessible to the reagent than the a-face, which is
hindered by the isopropylidene protecting group. NO-
ESY experiments showed a correlation between H-1 and
phosphono-a-
D
-allofuranose
14.
The
acetonide
H-2, which are located on the b-face in a-
D
-xylose
protecting group was removed with 90% aqueous tri-
fluoroacetic acid solution. The resulting mixture of the
two anomers of the hemiketal 15 (a=b, 85:15, based on
1H NMR integration of the signals of the anomeric
proton H-1) underwent sodium metaperiodate oxidative
glycol cleavage into the (3R,4R)-dibenzyl-3-formyloxy-
4-methyl-O-benzyl-5-oxopentylphosphonate 16, which
was reduced using sodium borohydride to afford the
derivatives, and between H-3 and both H-1 and H-2,
indicating that H-3 is also located on the b-face and
confirming the stereochemistry. Protection of the sec-
ondary hydroxyl group using t-butyldimethylsilyl tri-
fluoromethanesulfonate11 followed by hydrogenolysis of
the benzyl group yielded 1,2-O-isopropylidene-3-O-t-
butyldimethylsilyl-a-D-ribofuranose 10. The key step of

G. Hirsch et al. / Tetrahedron Letters 45 (2004) 519–521
521
benzylated phosphonate 17.15 Hydrogenolysis of the
benzyl protecting groups yielded free MEPN, (3R,4S)-
3,4,5-trihydroxy-4-methylpentylphosphonic acid 18.16
€
4. Luttgen, H.; Rohdich, F.; Herz, S.; Wungsintaweekul, J.;
Hecht, S.; Schuhr, C. A.; Fellermeister, M.; Sagner, S.;
Zenk, M.; Bacher, A.; Eisenreich, W. Proc. Natl. Acad.
Sci. U.S.A. 2000, 97, 1062–1067.
5. Wolff, M.; Seemann, M.; Grosdemange-Billiard, C.;
Enantiopure MEPN 18, an isosteric phosphonate ana-
logue of MEP, was synthesized in 12 steps in an overall
yield of 6%. The influence of MEPN 18 on the growth of
Escherichia coli was tested by the agar diffusion method
on LB agar plates (5 cm diameter) inoculated with a
bacteria suspension (100 lL, 5 · 107 cells, exponential
growth phase). No growth inhibition zone was observed
around 6 mm Whatman No. 1 paper disks in the pres-
ence of MEPN (50 or 100 lg), whereas the presence of
fosmidomycin (10 lg), a strong inhibitor of the DXP
reducto-isomerase, induced the formation of a clear
growth inhibition zone. MEPN, like DXPN, also had no
effect on the activity of the DXP isomero-reductase from
E. coli. The DXP reducto-isomerase converted, how-
ever, DXPN into MEPN, which was identified by com-
parison of 31P NMR spectra with the synthetic sample.17
The reaction catalyzed by this enzyme is reversible.18
Using the same enzyme test and 31P NMR spectroscopy
for the characterization of the products,17 we showed
that MEPN is converted in DXPN by the DXP reducto-
isomerase. Additional tests with other MEP pathway
ꢀ
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7. Steinbacher, S.; Kaiser, J.; Eisenreich, W.; Huber, R.;
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18407.
8. Hoeffler, J.-F.; Pale-Grosdemange, C.; Rohmer, M. Tet-
rahedron 2000, 56, 1485–1489.
9. Berkowitz, D. B.; Bhuniya, D.; Peris, G. Tetrahedron Lett.
1999, 40, 1869–1872.
10. Alper, P. B.; Hendrix, M.; Sears, P.; Wong, C. H. J. Am.
Chem. Soc. 1998, 120, 1965–1978.
11. Emery, F.; Vogel, P. J. Org. Chem. 1995, 60, 5843–5854.
12. Stamm, H.; Gerster, G.; Baumann, T. Chem. Ber. 1983,
116, 2936–2957.
13. Ireland, R. E.; Norbeck, D. W. J. Org. Chem. 1985, 50,
2198–2200.
14. Iversen, T.; Bundle, D. R. J. Chem. Soc., Chem. Commun.
1981, 1240–1241.
enzymes, such as 2-C-methyl-D-erythritol 4-phosphate
cytidyltransferase, will follow to investigate if MEPN
can act as an inhibitor of this biosynthetic pathway.
15. 1H NMR (300 MHz, CDCl3 + 1 drop D2O): d ¼ 1:11 (3H,
s, CH3), 1.64 (1H, m), 1.86 (2H, m), 2.05 (1H, m), 3.66 and
3.72 (2 · 1H, 2d, JHa;Hb ¼ 11:9 Hz, 4-C benzylic CH2),
2
3
3
3.73 (1H, dd, J3;H-4a ¼ 10:4 Hz, J3;H-4b ¼ 1:5 Hz, 3-H),
4.49 (2H, s, 5-H), 4.96 and 5.06 (2 · 1H, 2dd,
3
3
2JHa;Hb ¼ 11:8 Hz, JHa;P ¼ 8:2 Hz, JHb;P ¼ 9:1 Hz, phos-
phonate benzylic ester CH2), 4.96 and 5.07 (2 · 1H, 2dd,
2JHa;Hb ¼ 11:8 Hz, 3JHa;P ¼ 8:2 Hz, 3JHb;P ¼ 9 Hz, phospho-
nate benzylic ester CH2), 7.32 (15H, m, 3Ph). 13C NMR
(75 MHz, CDCl3): d ¼ 16:0 (CH3), 23.5 (CH2,
Acknowledgements
We thank Mr. J.-D. Sauer for the 2D NMR measure-
ments and Mr. R. Huber for the MS analyses. This
investigation was supported by a grant to M.R. from the
ÔInstitut Universitaire de FranceÕ. G.H. thanks the
2
1JC;P ¼ 137:5 Hz), 24.4 (CH2, JC;P ¼ 5:5 Hz), 64.2 (CH2),
2
65.2 (CH2), 67.4 (CH2, JC;P ¼ 6:2 Hz), 67.5 (CH2,
1
2JC;P ¼ 6:2 Hz), 75.4 (CH, JC;P ¼ 12:9 Hz), 79.1 (quater-
nary C), 127.7, 128.1, 128.6 and 128.7 (15 aromatic CH),
ꢁ
ÔMinistere de la Jeunesse, de lÕEducation Nationale et de
3
136.4 (2 aromatic quaternary C, JC;P ¼ 6:2 Hz), 138.9
la RechercheÕ for financial support.
(aromatic quaternary C). 31P NMR (121 MHz, CDCl3):
d ¼ 34:7 (s). HRMS (FABþ): calcd for C27H34O6P
(M+H)þ, m=z ¼ 485:2093; found, m=z ¼ 485:2099.
16. 1H NMR (300 MHz, D2O): d ¼ 1:10 (3H, s, CH3), 1.40
(2H, m), 1.68 (2H, m), 3.49 and 3.59 (2 · 1H, 2d,
References and Notes
3
2JHa;Hb ¼ 11:7 Hz, 5-H). 3.50 (1H, dd, J3;4-Ha ¼ 10:3 Hz,
3J3;4-Hb ¼ 1:6 Hz, 3-H), 13C NMR (75 MHz, D2O):
1. Rohmer, M. In Comprehensive Natural Product Chemistry,
Isoprenoids Including Steroids and Carotenoids; Cane, D.
E., Ed.; Pergamon, 1999; Vol. 2, Chapter 2, pp 45–68.
2. Takahashi, S.; Kuzuyama, T.; Watanabe, H.; Seto, H.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 9879–9884.
3. Rohdich, F.; Wungsintaweekul, J.; Fellermeister, M.;
Sagner, S.; Herz, S.; Kis, K.; Eisenreich, W.; Bacher, A.;
Zenk, M. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 11758–
11763.
1
d ¼ 17:7 (CH3), 24.5 (CH2), 25.3 (CH2, JC;P ¼ 131 Hz),
66.4 (CH2), 74.8 (quaternary C), 75.2 (CH, 3JC;P ¼ 16 Hz).
31P NMR (121 MHz, D2O): d ¼ 24:4 (s). Electrospray MS:
m=z ¼ 213 (M)Hþ, molecular monoanion of 18).
17. Meyer, O.; Grosdemange-Billiard, C.; Tritsch, D.; Roh-
mer, M. Org. Biomol. Chem., in press.
18. Hoeffler, J. F.; Tritsch, D.; Grosdemange-Billiard, C.;
Rohmer, M. Eur. J. Biochem. 2002, 269, 4446–4457.