Synthesis and evaluation of substrate analogues as mechanism-based inhibitors of type II isopentenyl diphosphate isomerase

Article abstract of DOI:10.1021/jo702061d

(Chemical Equation Presented) Type 2 isopentenyl diphosphate isomerase (IDI-2), which catalyzes the interconversion of isopentenyl diphosphate and dimethylallyl diphosphate, contains a tightly bound molecule of FMN. To probe the mechanism of the reaction,

Full text of DOI:10.1021/jo702061d

metalloprotein and requires a second divalent metal, typcially
Mg2+, for activity.7 Isomerization occurs by a protonation/
deprotonation mechanism that proceeds through a tertiary
carbocation as shown in Scheme 1.8-10 A second isomerase,
IDI-2, was discovered in 2001.11 It is clear from comparisons
of amino acid sequences12,13 and X-ray structures14,15 that IDI-1
and IDI-2 evolved independently. IDI-2, like IDI-1, requires
Mg2+ for activity. In addition, IDI-2 contains a tightly bound
molecule of FMN and requires a reductant, typically NADPH,
for activity.16 Two mechanisms have been proposed for IDI-2:
a protonation/deprotonation sequence similar to the mechanism
for IDI-1 and a hydrogen atom addition/abstraction (Scheme
1).16-20 While flavins normally facilitate oxidation/reduction
reactions, there are examples of the cofactors participating in
isomerizations with concomitant transient changes in oxidation
state.17
Synthesis and Evaluation of Substrate Analogues
as Mechanism-Based Inhibitors of Type II
Isopentenyl Diphosphate Isomerase
Joel R. Walker,† Steven C. Rothman,‡ and C. Dale Poulter*
Department of Chemistry, UniVersity of Utah, 315 South 1400
East RM 2020, Salt Lake City, Utah 84112
poulter@chemistry.utah.edu
ReceiVed September 19, 2007
Most bacteria synthesize isoprenoid compounds by the MEP
pathway, and although not required for their survival, most have
either IDI-1 or IDI-2 activity.21 However, a few pathogenic
bacteria, including Streptococcus pneumoniae and Staphylo-
coccus aureus, utilize the MVA pathway for isoprenoid bio-
synthesis and rely on IDI-2 for isomerization of IPP to DMAPP.
For these bacteria, IDI-2 presents an attractive target for the
development of antibacterial drugs.22
We were drawn to cyclopropyl or epoxy analogues of IPP,
where the methyl group has been replaced by a substituent, as
potential substrate analogues or mechanism-based inhibitors that
might be useful for distinguishing between the proton and
hydrogen atom mechanisms. Protonation of the double bond in
a cyclopropyl analogue, cIPP, would generate a cyclopropyl-
carbinyl cation, which could isomerize to cDMAPP or irrevers-
ibly inhibit the enzyme by reacting with an active site
nucleophile.23 Protonation of the oxirane ring in epoxy analogue,
Type 2 isopentenyl diphosphate isomerase (IDI-2), which
catalyzes the interconversion of isopentenyl diphosphate and
dimethylallyl diphosphate, contains a tightly bound molecule
of FMN. To probe the mechanism of the reaction, cyclo-
propyl and epoxy substrate analogues, designed to be
mechanism-based irreversible inhibitors, were synthesized
and evaluated with IDI-2 from Thermus thermophilus. The
cyclopropyl analogues were alternative substrates. The epoxy
analogue was an irreversible inhibitor, with kI ) 0.37 ( 0.07
min-1 and KI ) 1.4 ( 0.3 µM. LC-MS studies revealed
formation of an epoxide-FMN adduct.
(6) Cornforth, J. W.; Popjak, G. Methods Enzymol. 1969, 359-371.
(7) Lee, S.; Poulter, C. D. J. Am. Chem. Soc. 2006, 128, 11545-11550.
(8) Reardon, J. E.; Abeles, R. H. Biochemistry 1986, 25, 5609-5616.
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9955-9960.
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7328.
(11) Kaneda, K.; Kuzuyama, T.; Takagi, M.; Hayakawa, Y.; Seto, H.
Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 932-937.
(12) Barkley, S. J.; Desai, S. B.; Poulter, C. D. J. Bacteriol. 2004, 186,
8156-8158.
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186, 1811-1817.
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Biophys. Res. Commun. 2005, 338, 1515-1518.
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Bacher, A.; Rohdich, F. J. Mol. Biol. 2003, 329, 973-982.
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46, 5437-5445.
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Biochem. Biophys. Res. Commun. 2004, 322, 905-910.
(19) Johnston, J. B.; Walker, J. R.; Rothman, S. C.; Poulter, C. D. J.
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Isopentenyl diphosphate isomerase (IDI) catalyzes the inter-
conversion of isopentenyl diphosphate (IPP) and dimethylallyl
diphosphate (DMAPP), the two fundamental building blocks
in the isoprenoid biosynthetic pathway. IPP is synthesized from
acetyl CoA in eukarya, archaea, and some bacteria by the
mevalonate pathway (MVA),1 and isomerization of IPP to
DMAPP is an essential step required for survival of the
organisms. In plant chloroplasts and most bacteria, IPP and
DMAPP are synthesized from pyruvate and D-glyceraldehyde
phosphate by the methylerythritol phosphate (MEP) pathway.2
IDI activity, while typically found, is not required for survival
of these organisms.
IDI was discovered in the 1950s during studies of cholesterol
metabolism in yeast and rats.3-6 The enzyme is a zinc
† AMRI, 26 Corporate Circle, P.O. Box 15098, Albany, NY 12212.
‡ DuPont, Route 141 and Henry Clay, Wilmington, DE 19880.
(1) Kuzuyama, T.; Seto, H. Nat. Prod. Rep. 2003, 20, 171-183.
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G.; Schramek, N.; Steinbacher, S.; Huber, R.; Arigoni, D.; Bacher, A.;
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10.1021/jo702061d CCC: $40.75 © 2008 American Chemical Society
Published on Web 12/19/2007
726
J. Org. Chem. 2008, 73, 726-729

SCHEME 1. Protonation/Deprotonation and Hydrogen
Atom Addition/Abstraction Mechanisms
SCHEME 3. Synthesis of (E)-9-OH, (Z)-9-OH, and
(E,Z)-cDMAPP
SCHEME 2. Synthesis of cIPP
oIPP, could also result in inactivation of IDI by activating the
epoxide to nucleophilic capture.24 Addition of a hydrogen atom
to cIPP or oIPP would generate cyclopropylcarbinyl25 or epoxy
carbinyl26 radicals, which could then isomerize to cDMAPP or
oDMAPP upon abstraction or isomerize to ring-opened ho-
moallylic radicals that could inactivate the enzyme by hydrogen
atom abstraction or recombination reactions. We now report
the synthesis of cIPP, cDMAPP, and oIPP and an evaluation
of these compounds as alternative substrates and inhibitors of
Thermus thermophilus IDI-2.16
Synthesis of cIPP. The synthesis of cIPP is outlined in
Scheme 2. Cyclopropane carboxylic acid (1) was treated with
carbonyl diimidazole (CDI), and the resulting imidazolide was
condensed with the magnesium salt of monomethyl malonate
(MMM)27 to give a good yield of the Claisen product (2).28
Reduction of ketoester 2 with sodium borohydride to diol
3-OH, followed by protection of the primary hydroxyl group
and Dess-Martin oxidation29 of the secondary hydroxyl group,
gave cyclopropyl ketone 4. Treatment of 4 with Tebbe’s
reagent30 and removal of the silyl-protecting group gave
vinylcyclopropane alcohol 5-OH. Conversion of 5-OH to the
corresponding tosylate, followed by treatment with tetrabutyl
ammonium pyrophosphate, gave cyclopropyl analogue cIPP.
Synthesis of cDMAPP. We originally planned to prepare
the E and Z isomers of cDMAPP from the corresponding
alcohols (E)- and (Z)-9-OH. As shown in Scheme 3, Micheal
addition of thiophenol to ethyl 2-butynoate (6) gave a mixture
of (E)- and (Z)-7,31,32 which were then separated by column
chromatography. Each double bond isomer was treated with
cyclopropyl cuprate to give stereoisomeric cyclopropyl esters
(E)- and (Z)-8, and the esters were reduced with DIBAL to yield
cyclopropyl allylic alcohols (E)- and (Z)-9-OH.
Attempts to phosphorylate the alcohols using a two-step
chlorination/phosphorylation procedure we developed for allylic
diphosphates33 were unsuccessful. Introduction of the cyclo-
propane substituent was apparently too activating, and the
intermediate allylic chlorides decomposed. We then turned to
the direct phosphorylation of the alcohols using the Danilov
modification of the Cramer procedure.34,35 Because of the low
yields typically associated with Cramer phosphorylations, we
decided to use a 75:25 mixture of (E)- and (Z)-9-OH from
reduction of (E)- and (Z)-8 obtained by a Wittig condensation
of methylcyclopropyl ketone and triethyl phosphonoacetate36
to optimize the phosphorylation step. We discovered that both
isomers of cDMAPP were unstable at room temperature when
dissolved in water, and the diphosphates decomposed with a
half-life of ∼5 h at pH 8 and ∼10 h at pH 11. As a result, a
substantial amount of material was lost during purification by
ion exchange and column chromatography, and we did not
pursue synthesis of the individual double bond isomers of
cDMAPP.
Inorganic pyrophosphate is an excellent leaving group whose
reactivity depends on its total negative charge.37 The pH
dependence of the half-life of cDMAPP in water suggested that
(24) Xiang, J. L.; Christensen, D. J.; Poulter, C. D. Biochemistry 1992,
31, 9955-9960.
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Wiley & Sons: New York, 1993; Collect. Vol. 8, pp 610-616.
(34) Danilov, L. L.; Druzhinina, T. N.; Kalinchuk, N. A.; Maltsev, S.
D.; Shibaev, V. N. Chem. Phys. Lipids 1989, 51, 191-203.
(27) Hutchinson, C. R.; Nakane, M.; Gollman, H.; Knutson, P. L. Organic
Syntheses; Wiley & Sons: New York, 1990; Collect. Vol. 7, pp 323-326.
(28) Durham, T. B.; Miller, M. J. J. Org. Chem. 2003, 68, 27-34.
(29) Dess, D. B.; Martin, J. C. J. Org. Chem. 1983, 48, 4155-4156.
(30) Pine, S. H.; Shen, G. S.; Hoang, H. Synthesis 1991, 165-167.
(35) Keller, R. K.; Thompson, R. J. Chromatogr. 1993, 645, 161-167.
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J. Org. Chem, Vol. 73, No. 2, 2008 727

SCHEME 4. Synthesis of oIPP
FIGURE 1. Double reciprocal plot of kinact versus [oIPP].
TABLE 1. Inhibition of IDI-2 with Substrate Analoguesa
compound
IC50 (µΜ)
in the peaks for cDMAPP as the intermediate solvolyzed to
give tertiary alcohol 11-OH.19 A similar experiment starting
with cDMAPP resulted in the transient production of cIPP along
with the competing solvolysis reaction. These diphosphates did
not appear to irreversibly inhibit IDI-2.
Incubation of IDI-2, which had been reduced with NADPH,
with oIPP resulted in a rapid first-order irreversible loss of
activity of the enzyme. Kinetic parameters for the time-
dependent inactivation were calculated from a double reciprocal
plot of the first-order rate constants (kinact) at different inhibitor
concentrations (Figure 1); kI ) 0.37 ( 0.07 min-1 and KI )
1.4 ( 0.3 µΜ. oIPP was not an irreversible inhibitor in the
absence of NADPH.
In a preparative-scale experiment, reduced IDI-2 was incu-
bated with oIPP. Negative ion LC-MS analysis of the enzyme‚
inhibitor complex gave a peak at m/z 731 for a FMN‚oIPP
adduct and fragment ions at m/z 456 and 274 for the flavin and
oIPP components of the adduct.19 A UV spectrum of the
inhibited enzyme was characteristic of an alkylated flavin. A
transient absorption seen at 600 nm when the enzyme‚inhibitor
complex was denatured with guanidinium hydrochloride was
similar to spectra reported for N5 flavin adducts.16,40-43 The
FMN‚oIPP adduct from inactivation of IDI-2 in D2O followed
by workup in H2O did not contain deuterium.19 This result shows
that the epoxide ring was activated for inactivation of IDI-2 by
protonation at oxygen rather than by hydrogen atom addition
to the double bond of oIPP, followed by trapping of a radical
intermediate by flavin semiquinone. This mechanism for ir-
reversible inhibition by oIPP is similar to that for inhibition of
IDI-1 by eIPP, the epoxy derivative of IPP.9 Recently, Hoshino
and co-workers20 reported that eIPP also inhibits IDI-2 with
concomitant formation of a covalent adduct with the reduced
flavin cofactor. These observations are consistent with the recent
report by Kittleman and co-workers44 that IDI-2‚1-deazaFMN
is active when incubated with IPP and NADPH, while the
5-deazaFMN complex is not, suggesting that the proton at N5
is an integral part of the catalytic machinery.
cIPP
(E/Z)-cDMAPP
oIPP
100 ( 15 at 25 °C, pH 8.6
278 ( 185 at 25 °C, pH 8.6
1.14 ( 0.27 at 37 °C, pH 7.0
a Incubations were for 10 min in 50 µL of 200 mM Tris buffer containing
8 nM IDI-2, 5 µM IPP (59 µCi/µmol), 20 µM FMN, 5 mM NADPH, and
10 mM MgCl2. The higher pH and lower temperature for (E/Z)-cDMAPP
were used to minimize decomposition and for cIPP to permit a direct
comparison between the allylic isomers.
the diphosphate had solvolyzed. 1H NMR analysis of the
reaction showed a first-order disappearance of cDMAPP to give
vinylcyclopropyl alcohol 11-OH38 as the sole product, suggest-
ing that vinyl-substituted cyclopropylcarbinyl cation 10 is an
intermediate in the reaction.
Synthesis of oIPP. The synthesis of oIPP is shown in Scheme
4. Ethyl ester 12, obtained by condensation of ethyl acetate with
acrolein,39 was reduced with sodium borohydride to give allylic
diol 13. The primary hydroxyl group was selectively protected
with TBSCl, followed by treatment with m-chloroperbenzoic
acid. The epoxide was then subjected to a Dess-Martin
oxidation to give epoxy ketone 16. Attempts to convert the
ketone to an olefin with Tebbe’s reagent as described for
conversion of 4 to 5-OH were unsuccessful. After exploring a
variety of Wittig conditions, we were able to obtain 17-OH in
a modest yield using methyltriphenylphosphonium bromide and
sodium hexamethyl disilazide. The alcohol was converted to
oIPP using the phosphorylation procedure described for cIPP.
Studies with IDI-2. cIPP, E/Z-cDMAPP, and oIPP were
examined as inhibitors of recombinant T. thermophilus IDI-
2.16 Preliminary experiments to determine IC50 values gave
substantial errors (Table 1) due to a combination of chemical
instability of cDMAPP and irreversible inhibition by oIPP (see
the following paragraph). The isomerization of cIPP to cD-
MAPP was followed by 1H NMR spectroscopy. Initially, signals
for cIPP decrease with a concomitant increase in the intensity
of the signals for cDMAPP. This was followed by a decrease
In summary, cyclopropyl analogues of IPP and DMAPP and
an epoxy analogue of IPP were synthesized. cIPP and cDMAPP
(40) Kemal, C.; Bruice, T. C. J. Am. Chem. Soc. 1976, 98, 3955-3964.
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(43) Hemmerich, P.; Ghisla, S.; Hartmann, U.; Muller, F. In FlaVins and
FlaVoproteins; Kamin, H., Ed.; University Park Press: Baltimore, 1971.
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Biochemistry 2007, 46, 8401-8413.
(38) Babler, J. H.; Coghlan, M. J.; Giacherio, D. J. J. Org. Chem. 1977,
42, 2172-2175.
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728 J. Org. Chem., Vol. 73, No. 2, 2008

Hz), 3.35-3.37 (m, 1H), 3.66-3.74 (m, 2H), 5.01-5.02 (m, 1H),
5.21 (s, 1H); 13C NMR (CDCl3): δ -5.3, 18.3, 25.9, 34.1, 47.8,
53.8, 62.6, 114.3, 142.8; HRMS (CI) calcd for C12H24O2Si (M +
H) 229.1624, found 229.1617.
oIPP. To a solution of p-toluenesulfonyl chloride (175 mg, 0.92
mmol) and DMAP (150 mg, 1.23 mmol) in methylene chloride
was added by cannula a solution of 70 mg (0.61 mmol) of alcohol
17-OH in 2 mL of methylene chloride. The mixture was stirred at
rt for 16 h. Solvent was removed at reduced pressure, and the
residue was chromatographed on silica (7:3 hexanes/ethyl acetate)
to give 118 mg (72%) of an oil. The tosylate was immediately used
in the next step without further characterization.
were substrates for IDI-2, and no evidence was found for
irreversible inhibition by either compound. In contrast, oIPP
was a potent irreversible inhibitor that formed a covalent adduct
with the reduced FMN cofactor in the enzyme. At this point, a
protonation/deprotonation mechanism provides the simplest
explanation for our observations.
Experimental Section
Epoxy Ketone 16. Epoxy alcohol 16 (520 mg, 2.25 mmol) in
5 mL of CH2Cl2 was added via cannula to 9.5 g (15 wt %,
3.38 mmol) of Dess-Martin periodinane in 100 mL of CH2Cl2.
After being stirred for 18 h at rt, a saturated solution of NaHCO3
and solid Na2S2O3 were added. After 30 min, the mixture was
diluted with ether and washed with water and brine. The aqueous
layers were extracted with ether. The combined organic layers were
dried (MgSO4) and concentrated at reduced pressure. The residue
was chromatographed on silica (8:2 hexanes/ethyl acetate) to give
A solution of 17-OTs (118 mg, 0.44 mmol) in 2 mL of
acetonitrile was added dropwise to 595 mg (0.66 mmol) of tris-
(tetra-butylammonium) pyrophosphate in 20 mL of acetonitrile.
The mixture was allowed to stir for 2 h at rt and was concentrated
at reduced pressure. The residue was loaded onto an ion exchange
column (DOW-EX 50W NH4+ form) in 25 mM NH4HCO3/2% (v)
IPA. The resin was washed with two column volumes of 25 mM
NH4HCO3/2% (v) IPA. The eluent was concentrated by lyophiliza-
tion, and the residue was chromatographed on cellulose (8:2 IPA/
1
486 mg (94%) of an oil. H NMR (CDCl3): δ 0.03 (s, 6H), 0.86
(s, 9H), 2.42-2.65 (m, 2H), 2.92-3.00 (m, 2H), 3.43-3.45 (m,
1H), 3.80-3.97 (m, 2H); 13C NMR (CDCl3): δ -5.6, 18.1, 25.7,
39.2, 45.3, 53.6, 58.3, 206.7; HRMS (CI) calcd for C11H22O3Si (M
+ H) 231.1416, found 231.1421.
1
25 mM NH4HCO3) to give 22 mg (16%) of a white powder. H
NMR (DMSO-d6): δ 2.18 (br s, 2H), 2.65-2.67 (m, 1H), 2.83-
2.86 (m, 1H), 3.39 (br s, 1H), 3.84 (br s, 2H), 5.06 (s, 1H), 5.18 (s,
1H); 13C NMR (DMSO-d6): δ 31.2, 47.2, 53.2, 62.0, 114.1, 142.6;
31P NMR (DMSO-d6) δ -8.45 (br s), -9.90 (br s); HRMS
(MALDI) calcd for C6H12O8P2 (M-H) 272.9929, found 272.9943.
Silyl-Protected Alcohol 17-OTBS. A suspension of methylt-
riphenylphosphonium bromide (232 mg, 0.65 mmol) in 20 mL of
THF was cooled to 0 °C before sodium bis(trimethylsilyl)amide
(1.0 M, 0.52 mL, 0.52 mmol) was added by syringe. The reaction
was stirred for 30 min and then cooled to -78 °C before a solution
of 100 mg (0.43 mmol) of epoxy ketone 16 in 5 mL of THF was
added by cannula. The reaction was stirred for 1 h while warming
to rt and was then quenched with a saturated solution of NH4Cl.
Ether was added, and the resulting solution was washed with water
and brine. The aqueous layers were extracted with ether, and the
combined organic layers were dried (MgSO4) and concentrated at
reduced pressure. The residue was chromatographed on silica (95:5
Acknowledgment. This work was supported by NIH Post-
doctoral Fellowship GM 071114 to S.C.R. and NIH Grant GM
25521.
Supporting Information Available: General methods, proce-
dures for synthesis of all other compounds, enzymatic assays, and
1H and 13C NMR spectra. This material is available free of charge
via the Internet at http://pubs.acs.org.
1
hexanes/ethyl acetate) to give 42 mg (42%) of an oil. H NMR
(CDCl3): δ 0.05 (s, 6H), 0.88 (s, 9H), 2.17 (td, 2H, J ) 1.2, 6.9
Hz), 2.66 (dd, 1H, J ) 2.7, 5.4 Hz), 2.87 (dd, 1H, J ) 4.2, 5.4
JO702061D
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