Synthesis and analysis of a fluorinated product analogue as an inhibitor for 1-deoxy-d-xylulose 5-phosphate reductoisomerase

Article abstract of DOI:10.1016/j.bmcl.2007.11.062

1-Deoxy-d-xylulose 5-phosphate (DXP) reductoisomerase (DXR) is an NADPH-dependent enzyme catalyzing the rearrangement and reduction of DXP to methyl-d-erythritol 4-phosphate (MEP). Two mechanisms for this enzymatic reaction have been proposed, involving either an α-ketol rearrangement or a retroaldol/aldol rearrangement. In this study, a fluorinated product analogue, FCH₂-MEP, was synthesized as a possible mechanism-based inactivator for DXR if the retroaldol/aldol mechanism is operative. FCH₂-MEP was found to be a weak competitive inhibitor, and thus was unable to discriminate between the mechanisms. This result is due to the inability of the targeted enzyme, DXR, to oxidize FCH₂-MEP to the aldehyde intermediate that is common to both mechanisms. While FCH₂-MEP failed to act as a mechanism-based inactivator, the insight gained from this study will assist in the future design of inhibitors of DXR.

Full text of DOI:10.1016/j.bmcl.2007.11.062

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 3090–3094
Synthesis and analysis of a fluorinated product analogue as
an inhibitor for 1-deoxy-D-xylulose 5-phosphate reductoisomerase
Jeffrey W. Munos, Xiaotao Pu and Hung-wen Liu*
Division of Medicinal Chemistry, College of Pharmacy, and Department of Chemistry and Biochemistry,
University of Texas, Austin, TX 78712, USA
Received 16 October 2007; revised 12 November 2007; accepted 15 November 2007
Available online 21 November 2007
Abstract—1-Deoxy-D-xylulose 5-phosphate (DXP) reductoisomerase (DXR) is an NADPH-dependent enzyme catalyzing the rear-
rangement and reduction of DXP to methyl-D-erythritol 4-phosphate (MEP). Two mechanisms for this enzymatic reaction have
been proposed, involving either an a-ketol rearrangement or a retroaldol/aldol rearrangement. In this study, a fluorinated product
analogue, FCH
operative. FCH
2
-MEP, was synthesized as a possible mechanism-based inactivator for DXR if the retroaldol/aldol mechanism is
2
-MEP was found to be a weak competitive inhibitor, and thus was unable to discriminate between the mechanisms.
This result is due to the inability of the targeted enzyme, DXR, to oxidize FCH -MEP to the aldehyde intermediate that is common
2
to both mechanisms. While FCH -MEP failed to act as a mechanism-based inactivator, the insight gained from this study will assist
2
in the future design of inhibitors of DXR.
Ó 2007 Elsevier Ltd. All rights reserved.
Isoprenoids are an important class of compounds that
1
Since the MEP pathway is absent in mammals but is
essential for many pathogens, including Plasmodium fal-
ciparum and Mycobacterium tuberculosis, all enzymes
8
in this pathway are potential drug targets.
are ubiquitous in nature. Included in this class of natu-
ral products are steroids, terpenoids, carotenoids, and
ubiquinones, many of which play important roles in liv-
6
7
2
ing organisms. The basic building blocks for assem-
bling these compounds are two 5-C precursors,
isopentenyl diphosphate (IPP, 1) and dimethylallyl
diphosphate (DMAPP, 2). For many years, it was be-
lieved that all organisms use the mevalonate (3) pathway
for the biosynthesis of IPP, where DMAPP is derived
from IPP by the action of IPP isomerase (Scheme 1,
pathway A). It was only recently that a new mevalo-
nate-independent pathway (generally referred to as the
MEP pathway, see Scheme 1, pathway B) was discov-
ered in eubacteria, archeabacteria, algae, and in the plas-
Much effort has been devoted to exploit this possibility
6
,9
and several leads have been identified. A notable exam-
ple is fosmidomycin (9), which has been demonstrated to
6
be an effective treatment for mice infected with malaria.
Further analysis showed that fosmidomycin is a slow,
tight-binding inhibitor of DXP reductoisomerase
1
0,11
(DXR),
which catalyzes the conversion of 1-deoxy-
D-xylulose 5-phosphate (DXP, 4) to MEP (5), the first
committed step in the MEP pathway. The design of more
specific inhibitors for this enzyme depends on a detailed
understanding of its mechanism. Hence, we have initiated
3
–5
tids of plants.
In this pathway, both IPP (1) and
1
2
DMAPP (2) are produced directly from 1-hydroxy-2-
methyl-2-(E)-butenyl 4-diphosphate (8), which is derived
from 2C-methyl-D-erythritol 4-phosphate (MEP, 5).
mechanistic studies of the DXR catalyzed reaction.
DXR catalyzes the isomerization of DXP (4) to meth-
ylerythrose phosphate (10), followed by the reduction
of the aldehyde of methylerythrose phosphate using
NADPH to yield MEP (5). As shown in Scheme 2, there
are two plausible mechanisms for the rearrangement
step catalyzed by DXR. The first mechanism involves
an a-ketol rearrangement (route A), which is similar to
the mechanism catalyzed by ketol acid reductoisomer-
ase, a key enzyme in the biosynthesis of branched chain
Keywords: 1-deoxy-D-xylulose 5-phosphate reductoisomerase; Methyl-
D-erythritol 4-phosphate; Mechanism-based inactivator; Inhibitor.
*
Corresponding author. Tel./fax: +1 512 471 2746; e-mail:
h.w.liu@mail.utexas.edu
Present address: The University of Texas Southwestern Medical
Center at Dallas, 5323 Harry Hines Boulevard, Dallas, TX 75390,
USA.
0
960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2007.11.062

J. W. Munos et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3090–3094
3091
A. MVA Pathway
O
_
Me
CO2
O
O
O
O
O Me OHO
_
_
_
+
SCoA
O
SCoA
Me
CO2
Me
CO2
Me
Me
O Me OH
Me
_
O P2O63-
Me
O P2O63-
O
OH
MVA
3
1
2
B. MEP Pathway
DXR (IspC)
NADPH
O
OH
O
OH
HO Me
OH OH
_
O PO32-
O PO32-
+
O PO32-
Me
CO2
Me
OH
NADP+
O
DXP
MEP
4
5
O
=
HO
O
HO Me
O PO Me
3
P
IspD
IspE
O
Me
O
OCDP
OCDP
Me
P
O
OH
OH OH
OH OH
OH OH
6
7
O P2O63-
Me
1
O P2O63-
Me
Me
OH
8
3-
O P O
2
6
2
Scheme 1.
A. α-Ketol Rearrangement
NADPH
O
OH
HO Me
HO Me
OH OH
O PO 2-
3
1
3
O PO32-
PO32-
O
4
Me
2
5
O
O
OH
NADP+
10
H
4
5
B. Retroaldol/aldol Rearrangement
_
O
H
O
O
O
O PO32-
12
OH
N
PO32-
O PO32-
O
Me
O
Me
O
H
9
OH
4
11
Scheme 2.
1
3
amino acids. This mechanism is initiated by the depro-
tonation of the C-3 hydroxyl group followed by a 1,2-
migration to yield the aldehyde intermediate (10). The
second mechanism proceeds with a retroaldol/aldol rear-
rangement to produce the same intermediate, meth-
ylerythrose phosphate (10, Scheme 2, route B). Here
the enzyme first catalyzes the cleavage of the C3–C4
bond through a retroaldol mechanism to yield a three-
carbon (11) and a two-carbon phosphate (12) intermedi-
ate. These intermediates then condense through an aldol
reaction to form a new C–C bond in 10. Reduction of 10
by NADPH drives the equilibrium toward 5.

3092
J. W. Munos et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3090–3094
We envisioned that a fluoromethyl analogue of MEP, a
phosphate mono-((2S,3S)-3-fluoromethyl-2,4-dihy-
droxy-3-methyl-butyl) ester (FCH -MEP, 13), may be
uct(s). Clearly, the outcome of incubation of 13 with
DXR could be mechanistically informative.
2
a DXR inactivator whose mode of action could shed
light on the catalytic mechanism of DXR. Thus far, all
of the inhibitors that have been tested for DXR are
The synthesis of compound 13 was carried out by the
reactions shown in Scheme 4 starting with the commer-
cially available diisopropylidene protected glucose (20).
11,12,14–20
24
either DXP (4) or fosmidomycin (9) analogues.
Interestingly, while the DXR reaction is reversible,
Based on a literature procedure, the preparation was
1
1
initiated by the oxidation of 20 using PDC to generate
the 3-keto product (21) in 70% yield. Condensation of
21 with nitromethane gave 22, which underwent dehydra-
tion upon treatment with acetic anhydride in DMSO to
generate a nitroalkene intermediate. Stereoselective
nucleophilic addition of lithium dimethylcuprate on the
si face of the nitroalkene group afforded 23, having the
desired configuration at the quaternary C-3 center. Oxi-
there are no reports where MEP (5) analogues are
exploited as inhibitors for this enzyme. There are also
no known examples of compounds that irreversibly inac-
tivate DXR in a mechanistically relevant manner. In view
of this void, compound 13 was designed as a possible
MEP-based suicide inhibitor. If DXR proceeds via the
a-ketol rearrangement mechanism (Scheme 2, route A),
compound 13 may act as a competitive inhibitor against
MEP (5) when the DXR reaction is carried out in the re-
verse direction (5 ! 4). In contrast, if a retroaldol/aldol
mechanism (Scheme 2, route B) is operative, compound
Ò
dation of the nitronate 23 using Oxone , followed by lith-
ium aluminum hydride reduction, yielded 24, which was
2
4
then converted to 25. Selective hydrolysis of the 5,6-O-
isopropylidene group, treatment of the resulting diol with
sodium metaperiodate, followed by sodium borohydride
reduction, produced alcohol 26. After investigating vari-
ous phosphorylation reagents, trimethyl phosphine and
13 may covalently modify DXR and irreversibly inacti-
vate the enzyme.
The possible scenarios for inactivation of DXR by 13
are depicted in Scheme 3. When the reaction is run in
the direction of DXP (4) formation, FCH -MEP (13)
2
TeCl were found to efficiently phosphorylate the pri-
4
1
2
mary alcohol of 26 in high yield (90%) to provide 27.
The 1,2-O-isopropylidene protecting group was then re-
moved with aqueous trifluoroacetic acid, and the result-
ing diol was reacted with NaIO /NaBH to shorten the
will be oxidized to the corresponding aldehyde (14),
which then proceeds through the same retroaldol mech-
anism, as MEP would, to cleave the C2–C3 bond. Be-
cause the subsequent aldol condensation cannot occur
in this case, the resulting 4-C fragment 15 may instead
undergo fluoride elimination to yield the Michael accep-
4
4
chain by one carbon unit to give 28. Upon treatment with
trimethylsilyl bromide followed by aqueous hydrolysis,
the phosphotriester 28 was converted to 13. The crude
product was purified by cellulose chromatography under
2
1
25
tor 16. As shown in path A, this intermediate could
trap a nearby active site nucleophile resulting in the
basic conditions (6:3:1 i-propanol/H O/NH OH).
2
4
2
2
inactive, covalently modified enzyme, 17. Alterna-
tively, 1,4-reduction of 16 by NADPH (generated
in situ) to give 18 followed by aldol condensation with
To investigate whether FCH -MEP (13) could inactivate
2
DXR, FCH -MEP (1.8 mM) was incubated with DXR
2
+
(500 nM), NADP (300 lM), BSA (1 mg/mL), and
2
3
12 could generate 19(path B), which, in equilibrium
with 18 and 12, may be released as the turnover prod-
MgCl (2 mM) in 100 mM tris(hydroxymethyl)amino-
methane (Tris)HCl buffer (pH 7.6) at room temperature
2
NADP+
F
O
F
Me
F
Me
O PO32-
O PO 2-
O PO32-
3
Me
12
OH OH
O
O
O
_
NADPH
O
H
1
5
13
14
_
F
Enz Nu
Enz Nu
A
_
H
O
O PO32-
O PO32-
12
Me
Me
1
2
O
O
16
17
NADPH
Inactivation
B
NADP+
O
Me Me
Me
Me
O PO32-
O PO32-
12
O
OH
O
H
19
18
turnover
Scheme 3.

J. W. Munos et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3090–3094
3093
O
O
Me
Me
Me
O
CH3NO2, nBu4NI, NaOH
O
PDC, Ac2O, NaHCO3
CH Cl , reflux
Me
O
O
O
O
aq benzene, rt
80%
2
2
HO
O
Me
Me
Me
Me
70%
O
O
O
21
2
0
O
O
1. OXONE, Na HPO4
Me
Me
2
Me
Me
1
. Ac O, DMSO, rt
MeOH, rt
2
O
O
O
O
HO
O
O
2. LiAlH4, THF, 0 o
2. MeLi, CuI
THF, -78 o
Me
O N
C
Me
Me
Me
Me
C
O
2 steps 40%
O2N
2
steps 80%
2
23
22
O
O
Me
Me
Me
Me
1
. Tf O, pyr
2
1. HOAc, H O, rt
2
2. NaIO4, aq EtOH, rt
O
CH2Cl2, 0 o
O
O
C
O
O
O
Me
HO
Me
3. NaBH , aq EtOH, rt
Me 2. TBAF, THF, rt
steps 86%
4
Me
Me
O
O
3 steps 54%
2
Me
F
2
4
25
O
O
P
O
HO
Me
P(OMe) , TeCl4
(MeO)2
O
3
O
O
Me
2
,6-lutidine, CH Cl , rt
2 2
Me
Me
Me
Me
O
O
9
0%
F
F
26
27
O
O
P
1
2
. TFA
. NaIO , aq EtOH, rt
P
TMSBr
pyr
OH
OH
(HO)2
O
4
(MeO)2
O
3. NaBH , aq EtOH, rt
Me
Me
4
30%
3
steps 82%
OH
OH
F
F
28
13
Scheme 4.
for 16 h. An identical incubation mixture without 13 was
run as a control. An aliquot (10 lL) of each incubation
mixture was assayed for activity by adding it to a solu-
tion (190 lL) of NADPH (150 lM), DXP (285 lM),
The fact that FCH -MEP (13) is neither a suicide inhib-
2
itor nor a substrate for DXR makes it unsuitable to dis-
tinguish between the two proposed rearrangement
mechanisms for DXR (Scheme 2). The inability of
DXR to catalyze the initial oxidation of 13, which is a
prerequisite for both rearrangement mechanisms, may
be due to the increased steric bulk of the fluoromethyl
group, which may prevent the required preorganization
of 13 in the active site for the oxidation to occur. Inter-
estingly, incubation of 13 in the presence of MEP (5) re-
MgCl (2 mM), and BSA (1 mg/mL) in 100 mM Tris–
2
HCl buffer (pH 7.6). No loss in activity was observed
as compared to the control. Evidently, this compound
is not an irreversible inactivator for DXR. TLC analysis
of the incubation mixture also failed to detect any new
product formation. To determine if DXR could catalyze
the elimination of a fluoride ion from 13, 13 (10 mM)
2
7
vealed that it is a weak inhibitor against DXR. As
shown in Figure 1, when the reaction was run in the re-
verse direction with MEP (96 lM, which is roughly half
its Km value) as the substrate, the addition of excess
was mixed in a NMR tube with 2 mM MgCl and
2
+
0 mM NADP in 100 mM Tris–HCl buffer in D O
(
corded. DXR was then added to this mixture to a final
1
2
1
pD 7.8). The F NMR spectrum of this sample was re-
9
FCH -MEP (2.9 mM) led to 45% inhibition of the
2
concentration of 60 lM, and the solution was incubated
at room temperature for 15 h. The F NMR spectrum
of this sample was again recorded at the end of the incu-
reaction.
1
9
In summary, in this study, we report the synthesis and
analysis of FCH -MEP (13) as a potential inhibitor of
DXR. Although compound 13 was not able to inacti-
vate DXR as originally proposed, it was found to be a
weak competitive inhibitor of DXR. The cytidylated
1
9
bation period. No new peak in the F NMR spectrum
was detected. It was determined that our failure to ob-
serve fluoride elimination was due to its inability to oxi-
2
+
dize 13 to 14 using NADP , and not to the inability of
DXR to catalyze the retroaldol reaction on 14. This con-
clusion was made based on the absence of NADPH pro-
duction, monitored at 340 nm, upon mixing 13 with
form of FCH -MEP (equivalent of 6) could be made
2
synthetically or possibly by IspD (see Scheme 1) and
the resulting compound could be a viable inhibitor for
IspE, which is also a potential drug target. More impor-
+
26
DXR, NADP , and MgCl .
2

3094
J. W. Munos et al. / Bioorg. Med. Chem. Lett. 18 (2008) 3090–3094
4
5
6
. Rohmer, M. Nat. Prod. Rep. 1999, 16, 565.
2
2
2
2
1
1
1
1
1
6
4
2
0
8
6
4
2
0
. Kuzuyama, T.; Seto, H. Nat. Prod. Rep. 2003, 20, 171.
. Jomaa, H.; Wiesner, J.; Sanderbrand, S.; Altincicek, B.;
Weidemeyer, C.; Hintz, M.; Turbachova, I.; Eberl, M.;
Zeidler, J.; Lichtenthaler, H. K.; Soldati, D.; Beck, E.
Science 1999, 285, 1573.
7
. Argyrou, A.; Blanchard, J. S. Biochemistry 2004, 43, 4375.
u
v
8. Eisenreich, W.; Bacher, A.; Arigoni, D.; Rohdich, F. Cell.
Mol. Life Sci. 2004, 61, 1401.
9
. Hirsch, A. K.; Lauw, S.; Gersbach, P.; Schweizer, W. B.;
Rohdich, F.; Eisenreich, W.; Bacher, A.; Diederich, F.
Chem. Med. Chem. 2007, 2, 806.
1
1
1
1
1
0. Kuzuyama, T.; Shimizu, T.; Takahashi, S.; Seto, H.
Tetrahedron Lett. 1998, 39, 7913.
1. Koppisch, A. T.; Fox, D. T.; Blagg, B. S.; Poulter, C. D.
Biochemistry 2002, 41, 236.
2. Wong, A.; Munos, J. W.; Devasthali, V.; Johnson, K. A.;
Liu, H.-w. Org. Lett. 2004, 6, 3625.
3. Dumas, R.; Biou, V.; Halgand, F.; Douce, R.; Duggleby,
R. G. Acc. Chem. Res. 2001, 34, 399.
4. Kodai, Yajima S. H.; Sanders John, M.; Yin, Fenglin;
Ohsawa, Kanju; Wiesner, Jochen; Jomaa, Hassan; Old-
field, Eric J. Am. Chem. Soc. 2004, 126, 10824.
5. Phaosiri, C.; Proteau, P. J. Bioorg. Med. Chem. Lett. 2004,
0
1
2
3
4
[
FCH -MEP] (mM)
2
Figure 1. Dixon plot showing the inhibition by FCH
formation of DXP (4) catalyzed by DXR. The incubation mixture
2
-MEP (13) on the
+
contained 40 nM DXR, 400 lM NADP , 2 mM MgCl
MEP (5) in 100 mM Tris–HCl buffer, pH 7.6.
2
, and 96 lM
1
tantly, our results provide useful information to guide
the design of future inhibitors of DXR. The inability
of DXR to oxidize 13–14 and the weak inhibition of
1
4, 5309.
1
1
6. Fox, D. T.; Poulter, C. D. Biochemistry 2005, 44, 8360.
7. Walker, J. R.; Poulter, C. D. J. Org. Chem. 2005, 70,
955.
8. Hoeffler, J. F.; Tritsch, D.; Grosdemange-Billiard, C.;
1
3 toward DXR are most likely due to the steric hin-
9
drance caused by the substitution of a fluoromethyl
group for a hydroxyl group. This conclusion is consis-
tent with an early observation in which one carbon
extension of the backbone of DXP (4) rendered the
resulting analogue (Et-DXP, (2R,3S)-2,3-dihydroxy-4-
oxohexyl dihydrogen phosphate) a weak inhibitor in-
stead of a substrate for DXR. Thus, future inhibitor
design might consider the steric limitations of the ac-
tive-site of DXR. However, the possibility that replace-
ment of the hydroxyl group with –FCH2 disrupts
necessary hydrogen bonding interactions cannot be ru-
1
Rohmer, M. Eur. J. Biochem. 2002, 269, 4446.
19. Fox, D. T.; Poulter, C. D. J. Org. Chem. 2005, 70, 1978.
20. Meyer, O.; Grosdemange-Billiard, C.; Tritsch, D.; Roh-
mer, M. Org. Biomol. Chem. 2003, 1, 4367.
1. Pongdee, R.; Liu, H.-w. Bioorg. Chem. 2004, 32, 393.
22. Chang, C.-w. T.; He, X.; Liu, H.-w. J. Am. Chem. Soc.
998, 120, 9698.
3. Parikh, S.; Moynihan, D. P.; Xiao, G.; Tonge, P. J.
Biochemistry 1999, 38, 13623.
24. Hart, D. J.; Patterson, S.; Unch, J. P. Synlett 2003, 1334.
2
1
9
1
2
1
2
2
2
5. Characterization of FCH -MEP (13):
2
H
NMR
led out. While FCH -MEP (13) fails to act as a mecha-
2
(300 MHz, D O) d 0.78 (s, 3H, J = 2.1 Hz), 3.46 (ddd,
2H, J = 1.5, 11.4, 12.3 Hz), 3.70 (dd, 2H, J = 5.4, 6.9 Hz),
3.85 (dd, 1H, J = 5.4, 6.6 Hz), 4.31 (d, 1H,
2
nism-based inactivator, MEP-based inhibitors remain
one option to modulate the function of DXR. Attempts
to resolve the mechanism of DXR-catalyzed reaction
and to design effective inhibitors against this enzyme
are in progress.
1
3
J
= 47.4 Hz), 4.32 (d, 1H,
J
= 47.4 Hz).
C
HꢀF
NMR (75 MHz, D
HꢀF
2
O) d 13.9, 14.0, 42.8, 43.0, 63.55,
19
6
3.62, 65.9, 72.63, 72.68, 72.72, 72.77, 85.1, 87.3.
= 49.2 Hz).
F
P
3
1
NMR (282 MHz, D O) d 2.45 (t, J
2
HꢀF
NMR (121 MHz, D O) d 3.24 (s). HRMS (CI) calcd for
2
Acknowledgment
6 6
C H13FO P 231.0434; Found: 231.0430.
+
6. To determine if NADP can carry out the initial oxidation
of the primary hydroxyl group in 13 to generate the
aldehyde intermediate 14, the formation of NADPH upon
, and DXR was investigated. A
This work was supported by the Welch Foundation
Grant F-1511.
+
mixing 13, NADP , MgCl
2
+
solution of 8.5 mM 13, 1 mM NADP , 2 mM MgCl
mg/mL BSA in 100 mM Tris–HCl buffer (pH 7.6) was
placed in a cuvette, and the absorbance of the solution at
40 nm was determined. DXR, with a final concentration
2
, and
1
Supplementary data
3
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/
j.bmcl.2007.11.062.
of 77 lM, was then added to the cuvette, and the solution
was monitored at 340 nm for the production of NADPH.
7. The assays were run at 25 °C in degassed and N
2
saturated
1
00 mM Tris–HCl buffer (pH 7.6) containing 2 mM
+
MgCl , 1 mg/mL BSA, 400 lM NADP , 96 lM MEP,
2
References and notes
2
and varying concentrations of FCH -MEP (0–2.9 mM).
The reactions were initiated by the addition of enzyme to a
final concentration of 50 nM. All reactions were moni-
tored by following the rate of production of NADPH at
1
. Poulter, C. D.; Rilling, H. C. Biosynthesis of Isoprenoid
Compounds; Wiley: New York, 1981.
. Sacchettini, J. C.; Poulter, C. D. Science 1997, 277, 1788.
. Eisenreich, W.; Schwarz, M.; Cartayrade, A.; Arigoni, D.;
Zenk, M. H.; Bacher, A. Chem. Biol. 1998, 5, R221.
P
ꢀ
1
ꢀ1
3
40 nm ð
¼ 6:22 mM cm Þ. The concentrations of
2
3
340
MEP and DXR were determined as previously described,
see Refs. 11 and 12.