769 R.J. Linderman et al. Insect Biochemistry and Molecular Biology 30 2000 767–774
regression analysis from at least four different inhibitor concentrations which bracketed the I
50
and fell within the linear range immediate to the I
50
. At each inhibitor concentration, the assays were run in triplicate. Inhibi-
tors were dissolved in ethanol, and the concentration of ethanol due to the addition of inhibitor never exceeded
1 of the reaction volume. Inhibition assays were always compared with ethanol controls.
2.4. Chemical synthesis Chemical reagents were purchased from Aldrich
Chemical Co Milwaukee and purified prior to use by either distillation or chromatography. Alkyllithium
reagents were titrated in toluene using 1,10-phenanthro- line as an indicator. Reactions were typically carried out
under an inert atmosphere of argon using solvents that were distilled immediately prior to use.
1
H,
13
C, and
19
F- NMR spectra were recorded on a 300 MHz spectrometer
in CDCl
3
. Chromatography was performed on silica gel 60, 230–400 mesh ASTM, obtained from EM Science.
Elemental analyses were carried out by Atlantic
Fig. 1. A: Stereospecific synthesis of trisubstituted and trans-disubstituted allylic alcohols, precursors for Sharpless asymmetric epoxidation reac-
tion. B: Stereospecific synthesis of cis-disubstituted allylic alcohol precursors for asymmetric epoxidation.
Microlab Inc. Norcross, GA. High resolution and rou- tine mass spectra were obtained at the Mass Spec-
troscopy Laboratory for Biotechnology at NC State Uni- versity. All new compounds were fully characterized by
spectroscopic methods and combustion or mass spectro- scopic analysis. A description of the chemical synthesis
of the inhibitors is given in Section 3.
3. Results
We chose a synthetic approach that provided MEMD, and the glycidol-ester and epoxy-ester MEMD analogs
in enantiomerically pure forms. Introduction of the absolute stereochemistry was accomplished by Sharpless
asymmetric epoxidation Katsuki and Sharpless, 1980 of allylic alcohols. The allylic alcohol precursors for the
trisubstituted epoxides were prepared stereospecifically by the Schlosser modification of the Witting reaction
Corey et al., 1970. The trans allylic alcohols, leading to the trans epoxide analogs, were obtained stereospec-
ifically by Horner Emmons Wadsworth homologation
770 R.J. Linderman et al. Insect Biochemistry and Molecular Biology 30 2000 767–774
and chemoselective reduction of the unsaturated ester to the alcohol. The cis allylic alcohols, leading to the cis
epoxide analogs, were obtained by stereospecific partial reduction of the corresponding alkyne. The synthetic
routes are detailed in Fig. 1. Asymmetric epoxidation of each alkene was then carried out using
+ and
2 tar- trate esters as the chiral auxiliary to independently pro-
vide each enantiomer of the epoxide. The enantiomeric excess was determined to be greater than or equal to
Fig. 2. A: Asymmetric epoxidation of trisubstituted allylic alcohols using the Sharpless methodology to provide glycidol-ester inhibitors of JH-
EH, and conversion of the glycidol-esters to epoxy-esters by chemoselective reduction. B: Asymmetric epoxidation of the cis-disubstituted allylic alcohols and conversion to the epoxy-ester inhibitors. The trans-disubstituted allylic alcohols were converted to the corresponding epoxides by the
same synthetic sequence. All asymmetric epoxidation reactions occurred in 95ee.
95 by
19
F-NMR analysis of the Mosher ester deriva- tives Dale et al., 1969 in all cases. The epoxy-ester
inhibitors 7–12 were finally obtained by chemoselective deoxygenation of the glycidols 1–6 by initial conversion
to the iodide and reduction with sodium cyanoborohyd- ride Hutchins et al., 1977 see Fig. 2. The results of
the inhibition assays of the glycidol-esters 1–6 and the epoxy-ester inhibitors 7–12 are given in Table 1.
The I
50
s for the MEMD analogs were determined
771 R.J. Linderman et al. Insect Biochemistry and Molecular Biology 30 2000 767–774
Table 1 I
50
data for enantiopure MEMD and MEMD analogs
a
a
I
50
is the molar inhibitor concentration that reduces recombinant T. ni JH-EH activity by one-half. The I
50
values were determined as described in Section 2. The r
2
was 0.98.
using crude cell homogenates prepared from TmEH-1 infected Sf9 cells as the enzyme source, [
3
H]-JH III as the substrate, with OTFP added as an inhibitor of JH
esterase as described in Section 2. All of the assays were completed within the linear range of enzyme activity
with respect to time and protein concentration. Complete JH esterase inhibition was confirmed by TLC analysis of
the assay products. Several general trends were observed from the data in Table 1. In terms of the epoxide substi-
tution pattern, as expected, the trisubstituted epoxides 1, 2, 11 and 12 were the most effective inhibitors of T. ni
JH-EH. The I
50
values for these compounds were gener- ally an order of magnitude less than the I
50
values for
the corresponding disubstituted epoxides, 3, 4, 5–10. Of
the disubstituted epoxides, the trans-disubstituted epox- ides 3, 4, 8 and 7 were more effective inhibitors than
the corresponding cis-epoxides. Most significantly, the R enantiomer of MEMD 11, I
50
= 9.4
× 10
28
M, proved to
be a more effective inhibitor than the S enantiomer 12, I
50
= 2.2
× 10
26
M. The trend of the C-10 R-configuration affording the highest degree of inhibitory efficacy is
noted throughout this series of compounds. All of the glycidol-esters 1–6 examined were also effective inhibi-
tors of T. ni JH-EH and tended to be more potent inhibi- tors than the corresponding epoxy-esters 7–12, by an
order of magnitude in most cases. Compound 1, the gly- cidol analog of R-MEMD, was the overall most effective
inhibitor determined in this study, I
50
= 1.2
× 10
28
M. As with the epoxy-esters, the R absolute configuration at C-
10 was a more potent inhibitor than the C-10 S com- pounds for the entire series of compounds.
4. Discussion
