DXR from E. coli19 as well as a complex of DXR with
NADPH have been determined.20 Despite the advances made
so far in our understanding of the catalytic properties of
DXR, it remains elusive whether catalysis is better described
by route A or route B. In an attempt to gain more insight
into the mechanism of this intriguing enzyme, we prepared
three fluorinated substrate analogues 5-7 and examined their
competence as substrates or inhibitors upon incubation with
DXR.
Scheme 1
for the development of new antibiotics.4-6 In fact, fosmido-
mycin, which inhibits the conversion of 1 to 2,10 has been
shown to be effective for the treatment of malaria.11,12
The enzyme catalyzing the conversion of DXP (1) to MEP
(2), 1-deoxy-D-xylulose-5-phosphate reducto-isomerase (DXR,
also known as MEP synthase), has been isolated from several
sources. It is a NADPH-dependent catalyst, which also
requires a divalent metal ion for activity.9,13-15 Two possible
mechanisms for the conversion of DXP (1) to MEP (2) have
been proposed. As shown in Scheme 1, the first mechanism
involves an R-ketol rearrangement to yield methylerythrose
phosphate (3) as an intermediate, followed by the reduction
of the aldehyde group in 3 by NADPH to drive the
equilibrium to completion (route A). A mechanism involving
a retroaldolization/aldolization rearrangement is another
alternative (route B). This route yields first a bimolecular
intermediate (4), which is condensed to form 3. Subsequently,
3 is reduced by NADPH to generate the product 2.
The catalysis by the Escherichia coli enzyme has been
shown to proceed via an ordered mechanism in which
NADPH binds before the substrate, DXP, and NADP+ is
released after the discharge of MEP.14,15 Interestingly, a
random mechanism has recently been determined for the
Mycobacterium tuberculosis enzyme.16 However, due to the
significant differences in Km of the substrates in the forward
and reverse reaction, a preferred order of binding exists in
the latter case rendering its mechanism effectively similar
to that of the E. coli enzyme. Early studies have also
established that the C-1 pro-S hydrogen in MEP (2) is derived
from H-3 of DXP (1) and the hydride transfer from NADPH
is pro-S specific.17,18 In addition, the crystal structures of
If the hydroxyl substituent(s) of DXP indeed play(s) a
crucial role in DXR catalysis, its replacement with a
chemically inert fluorine will effect the catalytic turnover.
We therefore replaced each of these hydroxyl groups with
an inert fluorine atom and examined the effect of the
substitution on the catalytic turnover. In route A, deproto-
nation of the C-3 hydroxyl group followed by a 1,2-migration
to yield an aldehyde intermediate is the key step in the
mechanism. Since the C-4 hydroxyl group is not directly
involved in catalysis, compound 7 is likely to be a substrate,
while compound 6 would not be a substrate if the reaction
proceeds via an R-ketol rearrangement. The catalytic rate
for 7 might be retarded, however, due to the increased
electronegativity of fluorine. In the case of a retroaldolization
mechanism, while the C-4 hydroxyl group is involved in the
first step of the reaction, the C-3 hydroxyl group participates
in the aldolization step such that a new bond forms between
carbons derived from C-2 and C-4 of DXP to yield the
aldehyde intermediate. Thus, it is expected that if the reaction
proceeds via route B, both compounds 6 and 7 will not be
turned over by DXR. In contrast, compound 5 should always
be recognized by DXR as a substrate, since C-1 is not directly
involved in catalysis. However, the electron-withdrawing
nature of the fluorine substituent at C-1 will render the 2-keto
group more electrophilic and thus facilitate the R-ketol
rearrangement. Although a similar argument holds for the
retroaldol bond cleavage step in route B, stabilization of the
resulting enolate anion by the fluorine substituent at C-1 may
compromise its capability as a nucleophile and slow the
second half of the reaction. The effect on the overall catalysis
will depend on which step is more rate limiting.
(10) Kuzuyama, T.; Shimizu, T.; Takahashi, S.; Seto, H. Tetrahedron
Lett. 1998, 39, 7913-7916.
(11) Jomaa, H.; Wiesner, J.; Sanderbrand, S.; Altincicek, B.; Weidemeyer,
C.; Hintz, M.; Tu¨rbachova, I.; Eberl, M.; Zeidler, J.; Lichtenthaler, H. K.;
Soldati, D.; Beck, E. Science 1999, 285, 1573-1576.
(12) Lell, B.; Ruangweerayut, R.; Wiesner, J.; Missinou, M. A.;
Schindler, A.; Baranek, T.; Hintz, M.; Hutchinson, D.; Jomaa, H.; Kremsner,
P. G. Antimicrob. Agents Chemother. 2003, 47, 735-738.
(13) Kuzuyama, T.; Takahashi, S.; Takagi, M.; Seto, H. J. Biol. Chem.
2000, 275, 19928-19932.
To test these predictions, we developed methodology to
prepare this series of molecules.21 The reaction sequence used
to synthesize the 1-fluoro analogue 5 from 822 is shown in
Scheme 2. Fluorination at C-5 followed by hydrolysis,
benzylation, and reductive ring opening of 10 led to the key
intermediate 11, which upon benzoylation and Swern oxida-
tion gave 13 as the product. Subsequent ketalization and de-
(14) Koppisch, A. T.; Fox, D. T.; Blagg, B. S. J.; Poulter, C. D.
Biochemistry 2002, 41, 236-243.
(15) Hoeffler, J.-F.; Tritsch, D.; Grosdemange-Billiard, C.; Rohmer, M.
Eur. J. Biochem. 2002, 269, 4446-4457.
(19) Reuter, K.; Sanderbrand, S.; Jomaa, H.; Wiesner, J.; Steinbrecher,
I.; Beck, E.; Hintz, M.; Klebe, G.; Stubbs, M. T. J. Biol. Chem. 2002, 277,
5378-5384.
(16) Argyrou, A.; Blanchard, J. S. Biochemistry 2004, 43, 4375-4384.
(17) Proteau, P. J.; Woo, Y.-H.; Williamson, T.; Phaosiri, R. C. Org.
Lett. 1999, 1, 921-923.
(20) Yajima, S.; Nonaka, T.; Kuzuyama, T.; Seto, H.; Ohsawa, K. J.
Biochem. 2002, 131, 313-317.
(18) Radykewicz, T.; Rohdich, F.; Wungsintaweekul, J.; Herz, S.; Kis,
K.; Eisenreich, W.; Bacher, A.; Zenk, M. H.; Arigoni, D. FEBS Lett. 2000,
465, 157-160.
(21) For experimental details, see the Supporting Information.
(22) Pakulski, Z.; Zamojski, A. Tetrahedron 1995, 51, 871-908.
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Org. Lett., Vol. 6, No. 20, 2004