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In order to investigate the involvement of amino acids in the catalytic mechanism of the soluble epoxide hydrolase, different mutants of the murine enzyme were produced using the baculovirus expression system. Our results are consistent with the involvement of Asp-333 and His-523 in a catalytic mechanism similar to that of other alpha/beta hydrolase fold enzymes. Mutation of His-263 to asparagine led to the loss of approximately half the specific activity compared to wild-type enzyme. When His-332 was replaced by asparagine, 96.7% of the specific activity was lost and mutation of the conserved His-523 to glutamine led to a more dramatic loss of 99.9% of the specific activity. No activity was detectable after the replacement of Asp-333 by serine. However, more than 20% of the wild-type activity was retained in an Asp-333-->Asn mutant produced in Spodoptera frugiperda cells. We purified, by affinity chromatography, the wild-type and the Asp-333-->Asn mutant enzymes produced in Trichoplusia ni cells. We labeled these enzymes by incubating them with the epoxide containing radiolabeled substrate juvenile hormone III (JH III). The purified Asp-333-->Asn mutant bound 6% of the substrate compared to the wild-type soluble epoxide hydrolase. The mutant also showed 8% of the specific activity of the wild-type. Preincubation of the purified Asp-333-->Asn mutant at 37 degrees C (pH 8), however, led to a complete recovery of activity and to a change of isoelectric point (pI), both of which are consistent with hydrolysis of Asn-333 to aspartic acid. This intramolecular hydrolysis of asparagine to aspartic acid may explain the activity observed in this mutant. Wild-type enzyme that had been radiolabeled with the substrate was digested with trypsin. Using reverse phase-high pressure liquid chromatography, we isolated four radiolabeled peptides of similar polarity. These peptides were not radiolabeled if the enzyme was preincubated with a selective competitive inhibitor of soluble epoxide hydrolase 4-fluorochalcone oxide. This strongly suggested that these peptides contained a catalytic amino acid. Each peptide was characterized with N-terminal amino acid sequencing and electrospray mass spectrometry. All four radiolabeled peptides contained overlapping sequences. The only aspartic acid present in all four peptides and conserved in all epoxide hydrolases was Asp-333. These peptides resulted from cleavage at different trypsin sites and the mass of each was consistent with the covalent linkage of Asp-333 to the substrate.

18O-Labeled epoxides of trans-1,3-diphenylpropene oxide (tDPPO) and cis-9,10-epoxystearic acid were synthesized and used to determine the regioselectivity of sEH. The nucleophilic nature of sEH catalysis was demonstrated by comparing the enzymatic and nonenzymatic hydrolysis products of tDPPO. The results from single turnover experiments with greater or equal molar equivalents of sEH:substrate were consistent with the existence of a stable intermediate formed by a nucleophilic amino acid attacking the epoxide group. Tryptic digestion of sEH previously subjected to multiple turnovers with tDPPO in H 2 18 O resulted in the isolation and purification of a tryptic fragment containing Asp-333. Electrospray mass spectrometry of this fragment conclusively illustrated the incorporation of 18 O. After complete digestion of the latter peptide it was shown that Asp-333 of sEH exhibited an increased mass. The attack by Asp-333 initiates enzymatic activity, leading to the formation of an ␣-hydroxyacyl-enzyme intermediate. Hydrolysis of the acyl-enzyme occurs by the addition of an activated water to the carbonyl carbon of the ester bond, after which the resultant tetrahedral intermediate collapses, yielding the active enzyme and the diol product.Mammalian epoxide hydrolases (E.C. 3.3.2.3) have been implicated in the metabolism of epoxide containing xenobiotics, many of which are believed to be mutagenic and/or carcinogenic (1-3). During the past 20 years the mammalian microsomal epoxide hydrolase (mEH) 1 has received a great deal of attention partly due to its higher selectivity for cyclic and arene oxides (4 -6). A great deal of work has provided a clear picture of the regio-, stereo-, and enantiospecificity of mEH (7-10). Most researchers agree that mEH hydrolyzes the epoxide via an anti (commonly referred to as trans) opening of the oxirane, where attack of the nucleophile usually takes place at the least sterically hindered carbon. These results are substantiated by substituent effects on the rate of hydrolysis as investigated by Dansette et al. (11) and kinetic solvent isotope studies as reported by Armstrong et al. (12). By the use of single turnover experiments in H 2 18 O (13), Lacourciere and Armstrong have also postulated that the hydrolysis of epoxides by mEH proceeds via the intermediary of a nucleophilic amino acid, yielding an acyl-enzyme intermediate, which is hydrolyzed further to release the diol product and the active enzyme. This is in contrast with the previously more accepted hypothesis in which an activated water molecule was thought to be responsible for direct attack on the epoxide.Much less work has been completed on sEH, but the preliminary data closely parallel those for mEH. Because of the critical role of sEHs in the metabolism of xenobiotics and their possible involvement in the biosynthesis of metabolites (14-16, 18), 2 a detailed understanding of the catalytic mechanism of sEH is imperative. The anti opening of epoxides has been demonstrated on a select few substrates along with H 2 18 O studi...

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The haterumalides are a series of chlorinated macrolides isolated from an Okinawan sea sponge of the species Ircinia (Figure 1). 1,2 Kigoshi's synthesis of haterumalide NA led to its structural revision. 3 The stereochemistry of the remaining haterumalides is assumed to mimic that of haterumalide NA, and the stereochemistries in Figure 1 have been changed to reflect the corrections made to the initial structure of haterumalide NA.Figure 1 summarizes previous synthetic approaches to haterumalide NA. Kigoshi and coworkers utilized an intramolecular Reformatsky reaction as a key step to form the macrolide. 3 Snider and Gu used an intermolecular Stille coupling reaction followed by a Yamaguchi macrolactonization. 4 Hoye and Wang reported the first total synthesis of the correct enantiomer of haterumalide NA using a Pd-mediated alkyne haloallylation reaction. 5 Most recently, Roulland and Kigoshi, in two separate reports, described the use of variants of Suzuki-Miyaura cross-coupling along with macrolactonizations to achieve the synthesis of haterumalide NA. 6,7 Our approach entailed the construction of the C8-C9 bond, such that any member of the haterumalide family could be accessed. Critical to this assembly would be the ability to construct a nucleophilic Z-vinyl chloride moiety that would participate in a macrocyclization event with a pendant aldehyde. Thus, we explored the utility of the chlorovinylidene chromium carbenoids developed by Falck and Mioskowski (see Figure 1, box). 8,9 To the best of our knowledge, this chemistry has not been utilized in a natural product synthesis and its participation in a macrocyclization has not been reported.Synthesis of 1c commenced with preparation of the tetrahydrofuran-containing aldehyde for intramolecular CrCl 2 -mediated coupling (Scheme 1). 2-Deoxy-D-ribose 2 was converted to the disilylated methyl acetal 3 in high yield. Addition of allyltrimethylsilane to 3 in the presence of tin tetrabromide by the method of Woerpel and co-workers gave the allylated product in excellent diastereoselectivity. 10,11 Desilylation to yield 4 was followed by protection of the primary alcohol as a trityl group, yielding 5 poised for Mitsunobu esterification with carboxylic acid 9c.Preparation of 9c began with the ring-opening of 6 with the anion of chloroform to yield the corresponding alcohol. 12 DMP oxidation furnished the unstable, reactive, and volatile aldehyde 7, which was immediately subjected to a Still-Gennari olefination to yield the desired Z-acrylate. 13 Reduction of the ester to the allylic alcohol 8 was followed by a rapid oxidation to the unsaturated aldehyde and an immediate asymmetric Mukaiyama aldol reaction to furnish 9a. 14 The secondary alcohol was protected as a TBS ether, and the phenyl ester was hydrolyzed to give the desired 1,1,1-trichloroalkane substrate 9c. Mitsunobu esterification of alcohol 5 and carboxylic acid 9c yielded 10a without incident. Both TBS-protected and acetylated C3-OH compounds (10a and 10c) were carried forward for comparative studies in later...