Introduction and the most effective epoxy-ester inhibitor, compound 11, exhibited an

Insect Biochemistry and Molecular Biology 30 2000 767–774 www.elsevier.comlocateibmb Inhibition of insect juvenile hormone epoxide hydrolase: asymmetric synthesis and assay of glycidol-ester and epoxy-ester inhibitors of Trichoplusia ni epoxide hydrolase Russell J. Linderman a, , R. Michael Roe b , Shannon V. Harris a , Deborah M. Thompson b a Department of Chemistry, North Carolina State University, Raleigh, NC 27695, USA b Department of Entomology, North Carolina State University, Raleigh, NC 27695, USA Received 31 October 1999; received in revised form 31 December 1999; accepted 25 January 2000 Abstract Juvenile hormone JH undergoes metabolic degradation by two major pathways involving JH esterase and JH epoxide hydrolase EH. While considerable effort has been focussed on the study of JH esterase and the development of inhibitors for this enzyme, much less has been reported on the study of JH-EH. In this work, the asymmetric synthesis of two classes of inhibitors of recombi- nant JH-EH from Trichoplusia ni, a glycidol-ester series and an epoxy-ester series is reported. The most effective glycidol-ester inhibitor, compound 1, exhibited an I 50 of 1.2 × 10 28

M, and the most effective epoxy-ester inhibitor, compound 11, exhibited an

I 50 of 9.4 × 10 28 M. The potency of the inhibitors was found to be dependent on the absolute configuration of the epoxide. In both series of inhibitors, the C-10 R-configuration was found to be significantly more potent that the corresponding C-10 S-configuration. A mechanism for epoxide hydration catalyzed by insect EH is also presented.  2000 Elsevier Science Ltd. All rights reserved. Keywords: Juvenile hormone; Epoxide hydrolase; JH metabolism; Trichoplusia ni; Inhibitors; Enantioselective

1. Introduction

The insect juvenile hormones JHs are methyl esters of farnesoic acid 10,11-epoxide JH III and analogous compounds, which function as important regulatory fac- tors in embryogenesis, larval and adult development, metamorphosis, reproduction, diapause, migration, poly- morphism, and metabolism Nijhout, 1994; Roe and Venkatesh, 1990; de Kort and Granger, 1996; Hammock, 1985. The two primary metabolic degradation pathways of JH in insects are ester hydrolysis by JH esterase and epoxide hydration by an epoxide hydrolase EH. The development of specific inhibitors of JH esterase has been instrumental in demonstrating a biological role for this enzyme in insects Hammock et al., 1982, 1984; Prestwich et al., 1984; Abdel-Aal and Hammock, 1985; Corresponding author. Tel.: + 1-919-515-3616; fax: + 1-919-515- 8920. E-mail address: russell Flindermanncsu.edu R.J. Linderman. 0965-174800 - see front matter  2000 Elsevier Science Ltd. All rights reserved. PII: S 0 9 6 5 - 1 7 4 8 0 0 0 0 0 4 8 - 5 Linderman et al., 1987, 1989. In contrast, significantly less information is available on JH epoxide hydrolase JH-EH Roe and Venkatesh, 1990; de Kort and Granger, 1996. The complete coding region for an EH has been cloned and sequenced by our laboratories from a fat body L5D3 cDNA library derived from last stadium Trichoplusia ni, GenBank accession no. U73680 Roe et al., 1996; Harris et al., 1999. The full length fat body EH cDNA was 1887 bp in length and consisted of an 81 bp 5 9 untranslated region UTR, a 1389 bp ORF encoding 463 amino acids, and a 416 bp 3 9 UTR. The predicted protein amino terminus is hydrophobic sug- gesting that the protein encoded by U73680 is a microso- mal enzyme. A cDNA sequence encoding a JH-EH, showing significant similarity to vertebrate mEH, has also been reported by Prestwich, Hammock and co- workers Wojtasek and Prestwich, 1996; Debernard et al., 1998. The two insect EH cDNAs from T. ni and Manduca sexta appear to code for microsomal enzymes. The T. ni fat body full length EH was 41 and 38 ident- 768 R.J. Linderman et al. Insect Biochemistry and Molecular Biology 30 2000 767–774 ical at the amino acid level with mEH from rat and human, respectively; and 46 identical with M. sexta JH-EH Wojtasek and Prestwich, 1996. The T. ni fat body EH also contains the identical amino acid catalytic triad Glu-403, Asp-226 and His-430 found in M. sexta EH, microsomal EHs from rat Falany et al., 1987, human Skoda et al., 1988 and rabbit Hassett et al., 1989, and a haloalkane dehalogenase HLD1, Xanthob- acter autotrophicus Janssen et al., 1989. The development of inhibitors for mammalian EHs has been somewhat successful. Hammock and co-work- ers developed chalcone oxides as effective inhibitors for mammalian soluble EH sEH Morisseau et al., 1998; Borhan et al., 1995; Pinot et al., 1995. Trichloropropene oxide, cyclohexene oxide and glycidyl derivatives have been shown to be microsomal EH mEH inhibitors Tzeng et al., 1996. Nearly all of the examples of EH inhibition involve epoxides which typically serve as competitive alternative substrate inhibitors, with the exception of recent work by Hammock and co-workers Morisseau et al., 1999 that revealed ureas and carbam- ates could function as inhibitors of murine sEH. The inhibitors of insect EHs are generally not effective and exhibit considerable variation in activity depending on the insect and substrate used for the assay. Hammock and co-workers examined several glycidyl ethers and epoxy-alcohol JH analogs as inhibitors, but only the JH analogs provided significant levels µ M of inhibition Casas et al., 1991; Harshman et al., 1991. Typically, mammalian mEH or sEH inhibitors are not as effective against insect EH. We previously investigated several potential inhibitors for T. ni JH-EH using crude microsomal enzyme prep- arations Roe et al., 1996; Harris et al., 1999. Inhibitors designed to mimic a polarized or ionic transition state were only modest competitive inhibitors of T. ni JH-EH while non-juvenoid long chain aliphatic epoxides were the most potent competitive substrates to JH III. The most effective inhibitor identified in our earlier studies, methyl 10,11-epoxy-11-methyldodecanoate MEMD, exhibited an I 50 of 80 µ M. Mimicry of the length of the JH backbone and of the methyl ester functional group of JH was found to be important in substrate binding. We now report a more extensive structure activity relationship SAR study of glycidol-ester and epoxy- ester inhibitors of JH-EH which further examines epox- ide substitution patterns, and addresses the question of enantioselectivity in epoxide hydration by recombinant T. ni JH-EH.

2. Materials and methods