erythritol phosphate originates from C3 of DXP and which
derives from NADPH? (2) Delivery of hydride from NADPH
occurs on which face of the intermediate aldehydesre or
si? (3) Which hydride from NADPH is delivered in the
reduction (class A or class B dehydrogenase)?
of all five backbone protons, and this solvent was used for
further analysis. HSQC and HMBC experiments were used
1
to assign the H and 13C resonances.9 GNOESY and 1D
DPFGSE10 NOE experiments were utilized to assign the
diastereotopic groups in the molecule. The C1 hydrogens
appeared as a pair of doublets, one centered at 3.71 ppm
and the other at 3.95 ppm. Strong NOE correlations between
the 3.71 ppm proton, the C2 methyl group, and one of the
C5 isopropylidene methyls11 allowed us to assign all three
of these groups to the same face of the dioxolane ring. On
the basis of NOE experiments, the pro-S proton at C1 has a
shift of 3.95 ppm and the pro-R proton resonates at 3.71
ppm.
To provide a monodeuterated compound for the stereo-
chemical analysis, it was necessary to synthesize deoxy-
xylulose phosphate specifically deuterated at C3. This was
accomplished by modifying the synthesis of 3-2H-deoxy-
xylulose12 to allow for synthesis of the 5-phosphate from
the known intermediate 6 (Scheme 2). The labeled substrate
Our earlier work examining the MEP pathway in the
cyanobacterium Synechocystis6 prompted us to examine the
cyanobacterial isomeroreductase. The Synechocystis protein
sequence has 42% identity to the Escherichia coli DXR
sequence. A cosmid clone containing the dxr gene was
obtained from the Kazusa DNA Research Institute. The
desired gene was subcloned into pUC18, and unique site-
elimination mutagenesis was used to introduce BamH I and
Hind III sites at the 5′ and 3′ ends of the gene, respectively.
The gene was then ligated into a pBAD/His expression
vector, and expression upon arabinose induction in E. coli
provided the recombinant isomeroreductase with a N-terminal
6xHis sequence. Partial purification using Ni-NTA spin
columns (Qiagen) provided recombinant enzyme for stere-
ochemical studies. The enzyme was assayed by monitoring
the decrease in absorbance at 340 nm.4 The product of the
enzymatic reaction was converted to 2-C-methylerythritol
triacetate (3) by treating the crude mixture with alkaline
phosphatase, followed by acetylation with acetic anhydride
and pyridine (Scheme 1). The purified derivative was
Scheme 2a
1
analyzed by comparing GC/MS and H NMR data with an
authentic standard.
Scheme 1a
a
(a) CH3C(OCH3)2CH3, pTsOH, THF; (b) H2, Pd/C, EtOH; (c)
tetrazole, (BnO)2PNiPr2; (d) tBuOOH, CH2Cl2; (e) H2, Pd(OH)2/
C; (f) Dowex 50, H2O.
7 was incubated with recombinant isomeroreductase, provid-
ing monodeuterated MEP which was dephosphorylated with
alkaline phosphatase. The proteins from the mixture were
precipitated with ethanol, and the supernatant was concen-
trated and then treated with acetic anhydride and pyridine.
The conversion of methylerythritol to the triacetate provided
a derivative that was readily purified by flash chromatog-
raphy (40% EtOAc/hexanes). The triacetate was deprotected
with a basic ion-exchange resin and then converted to the
a (a) DXR; (b) alkaline phosphatase; (c) Ac2O, pyr; (d) Amberlite
IRA400 (OH-); (e) CH3C(OCH3)2CH3, pTsOH, THF.
1
bisacetonide (Scheme 1). The H NMR spectrum of this
To distinguish the pro-R and pro-S protons at C1 of MEP,
a derivative was required in which the two protons could be
unambiguously assigned. A derivative that potentially could
fulfill this requirement was the bisacetonide derivative.7
Unlabeled 2-C-methylerythritol (4) was prepared8 and con-
verted to the bisacetonide 5 with 2,2-dimethoxypropane and
toluenesulfonic acid. A survey of NMR solvents revealed
that the 1H NMR spectrum in acetone-d6 provided resolution
derivative showed a slightly broadened singlet at 3.69 ppm
(9) 1H NMR (acetone-d6): δ 4.09 (dd, J ) 7.0, 5.8 Hz, H3), 4.00 (dd,
J ) 8.7, 7.0 Hz, H4S), 3.95 (d, J ) 8.7 Hz, H1S), 3.84 (dd, J ) 8.7, 5.8
Hz, H4R), 3.71 (d, J ) 8.7 Hz, H1R), 1.35 (s, C6-CH3R), 1.310 (s, C5-
CH3S), 1.306 (s, C5-CH3R), 1.27 (s, C6-CH3S), 1.21 (s, C2-CH3). 13C NMR
(acetone-d6): δ 110.11 (C5), 110.00 (C6), 81.83 (C2), 79.44 (C3), 73.49
(C1), 66.00 (C4), 27.58 (C5-CH3S), 27.02 (C5-CH3R), 26.60 (C6-CH3R),
25.04 (C6-CH3S), 19.80 (C2-CH3).
(10) GNOESY ) Gradient NOESY. Wagner, R.; Berger, S. J. Magn.
Reson. 1996, 123A, 229-232. DPFGSE ) double pulsed field gradient
spin-echo. Stott, K.; Keeler, J.; Van, Q. N.; Shaka, A. J. J. Magn. Reson.
1997, 125, 302-324.
(6) Proteau, P. J. Tetrahedron Lett. 1998, 39, 9373-9376.
(7) Anthonsen, T.; Hagen, S.; Sallam, M. A. E. Phytochemistry 1980,
19, 2375-2377.
(8) Duvold, T.; Cali, P.; Bravo, J. M.; Rohmer, M. Tetrahedron Lett.
1997, 38, 6181-6184.
(11) The selective excitation and high resolution possible with the
DPFGSE experiment allowed us to assign the correlations from the C2
methyl and the 3.71 ppm signal specifically to the methyl at 1.306 ppm.
(12) Giner, J.-L. Tetrahedron Lett. 1998, 39, 2479-2482.
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Org. Lett., Vol. 1, No. 6, 1999