Vinyl sulfide derivatives of truncated oxidosqualene as selective inhibitors of oxidosqualene and squalene-hopene cyclases

Various vinyl sulfide and ketene dithioacetal derivatives of truncated 2,3-oxidosqualene were developed. These compounds, having the reactive functions at positions C-2, C-15 and C-19 of the squalene skeleton, were studied as inhibitors of pig liver and Saccharomyces cerevisiae oxidosqualene cyclases (OSC) (EC 5.4.99.7) and of Alicyclobacillus acidocaldarius squalene hopene cyclase (SHC) (EC 5.4.99.-). They contain one or two sulfur atoms in α-skeletal position to carbons considered to be cationic during enzymatic cyclization of the substrate and should strongly interact with enzyme nucleophiles of the active site. Most of the new compounds are inhibitors of the OSC and of SHC, with various degrees of selectivity. The methylthiovinyl derivative, having the reactive group at position 19, was the most potent and selective inhibitor of the series toward S. cerevisiae OSC, with a concentration inhibiting 50% of the activity of 50 nM, while toward the animal enzyme it was 20 times less potent. These results could offer new insight for the design of antifungal drugs.     

2,3-oxidosqualene cyclase: From azasqualenes to new site-directed inhibitors

2,3-Oxidosqualene cyclases (OSC) are enzymes which convert 2,3-oxidosqualene (OS) into polycyclic triterpenoids such as lanosterol, cycloartenol, and α-and β-amyrin. Our interest in the study of OSC is the development of new OSC inhibitors for potential use as hypocholesterolemic, antifungal, or phytotoxic drugs. In particular, we describe the biological activity and the mechanism of a series of acyclic azasqualene derivatives mimicking the C-2, C-8, and C-20 carbonium ions formed during OS cyclization. Some of these carbonium ion analogues are very promising as specific hypocholesterolemic agents. The toxicity, the biodistribution, and the pharmacokinetics of different azasqualene derivatives in mice are also presented. In order to obtain new, site-directed irreversible inhibitors of OSC, a series of squalene derivatives containing functional groups that can link covalently to an active-site thiol group was designed. Among these compounds, squalene maleimide was the most active toward mammalian OSC, whereas squalene Ellman behaved as an irreversible inhibitor of OSC from yeast Based on a paper presented at the symposium on the “Regulation of Biosynthesis and Function of Isopentenoids” Atlanta, Georgia, May 1994.     

Conjugated methyl sulfide and phenyl sulfide derivatives of oxidosqualene as inhibitors of oxidosqualene and squalene-hopene cyclases

Various (1E,3E)- and (1Z,3E)-conjugated methylthio derivatives of oxidosqualene (OS) and conjugated and non-conjugated phenylthio derivatives of OS were obtained. These compounds, designed as inhibitors of pig liver and Saccharomyces cerevisiae 2,3-oxidosqualene-lanosterol cyclases (OSC) (EC 5.4.99.7) and of Alicyclobacillus acidocaldarius squalenehopene cyclase (SHC) (EC 5.4.99.-), contain the reactive function adjacent to carbons involved in the formation of the third and the fourth cycle during OS cyclization. All the new compounds are inhibitors of OSC and SHC, with various degrees of selectivity. The conjugated methylthio derivatives behaved as potent inhibitors of S. cerevisiae OSC, whereas most of the phenylthio derivatives were especially active toward SHC.     

Identification of the active site of vertebrate oxidosqualene cyclase

Active site mapping of rat liver oxidosqualene cyclase (OSC), a 78 kDa membrane-bound enzyme, was carried out using the mechanism-based irreversible inhibitor, [³H]29-methylidene-2,3-oxidosqualene. The amino acid sequence of the radiolabeled CNBr peptide fragment showed unexpectedly high similarity to the yeast OSC, plant OSC, and bacterial squalene cyclases. Further, radio analysis established that the two adjacent Asp residues in the highly conserved region (Asp-Asp-Thr-Ala-Glu-Ala, or DDTAEA) were equally labeled by the irreversible inhibitor. This result provided the first information on the structural details of the active site of OSC, and showed for the first time the ancient lineage of this vertebrate enzyme to ancestral eukaryotic and prokaryotic cyclases. Interestingly, the covalently-modified DDXX(D/E) sequence of rat liver OSC showed surprising similarity to the putative allylic diphosphate binding site sequence of other terpene cyclases and prenyl transferases. The Asp-rich motif may act as a point charge to stabilize incipient cationic charge. Based on a paper presented at the Symposium on the “Regulation of Biosynthesis and Function of Isopentenoids,” Atlanta, Georgia, May 1994.     

Inhibition of cholesterol synthesis by cyclopropylamine derivatives of squalene in human hepatoblastoma cells in culture

Two squalene derivatives, trisnorsqualene cyclopropylamine and trisnorsqualeneN-methylcyclopropylamine, were synthesized and tested for inhibition of lanosterol and squalene epoxide formation from squalene in rat hepatic microsomes, and for the inhibition of cholesterol syntheses in human cultured hepatoblastoma (HepG2) cells. Trisnorsqualene cyclopropylamine inhibited [³H]-squalene conversion to [³H]squalene epoxide in microsomes (IC₅₀=5.0 μM), indicating that this derivative inhibited squalene mono-oxygenase. Trisnorsqualenen-methylcyclopropylamine inhibited [³H]squalene conversion to [³H]lanosterol (IC₅₀=12.0 μM) and caused [³H]-squalene epoxide to accumulate in microsomes, indicating that this derivative inhibited 2,3-oxidosqualene cyclase. Cholesterol biosynthesis from [¹⁴C]acetate in HepG2 cells was inhibited by both derivatives (IC₅₀=1.0 μM for trisnorsqualene cyclopropylamine; IC₅₀=0.5 μM for trisnorsqualeneN-methylcyclopropylamine). Cells incubated with trisnorsqualene cyclopropylamine accumulated [¹⁴C]squalene, while cells incubated with trisnorsqualeneN-methylcyclopropylamine accumulated [¹⁴C]squalene epoxide and [¹⁴C]squalene diepoxide. The concentration range of inhibitor which caused these intermediates to accumulate coincided with that which inhibited cholesterol synthesis. The results indicate that cyclopropylamine derivatives of squalene are effective inhibitors of cholesterol synthesis, and that substitutions at the nitrogen affect enzyme selectivity and thus the mechanism of action of the compounds.     

Analogs of squalene and oxidosqualene inhibit oxidosqualene cyclase of <Emphasis Type="Italic">Trypanosoma cruzi</Emphasis> expressed in <Emphasis Type="Italic">Saccharomyces cerevisiae</Emphasis>

Recently, a number of inhibitors of the enzyme oxidosqualene cyclase (OSC; EC 5.4.99.7), a key enzyme in sterol biosynthesis, were shown to inhibit in mammalian cells the multiplication of Trypanosoma cruzi, the parasite agent of Chagas’ disease. The gene coding for the OSC of T. cruzi has been cloned and expressed in Saccharomyces cerevisiae. The expression in yeast cells could be a safe and easy model for studying the activity and the selectivity of the potential inhibitors of T. cruzi OSC. Using a homogenate of S. cerevisiae cells expressing T. cruzi OSC, we have tested 19 inhibitors: aza, methylidene, vinyl sulfide, and conjugated vinyl sulfide derivatives of oxidosqualene and squalene, selected as representative of different classes of substrate analog inhibitors of OSC. The IC₅₀ values of inhibition (the compound concentration at which the enzyme is inhibited by 50%) are compared with the values obtained using OSC of pig liver and S. cerevisiae. Many inhibitors of pig liver and S. cerevisiae OSC show comparable IC₅₀ for T. cruzi OSC, but some phenylthiovinyl derivatives are 10–100 times more effective on the T. cruzi enzyme than on the pig or S. cerevisiae enzymes. The expression of proteins of pathogenic organisms in yeast seems very promising for preliminary screening of compounds that have potential therapeutic activity.     

Design of high energy intermediate analogues to study sterol biosynthesis in higher plants

Several enzymes of plant sterol biosynthesis involve during their catalysis postulated or demonstrated carbocationic high energy intermediates (HEI). The aim of this study was to interfere with plant sterol biosynthesis by means of rationally designed species able to mimic these carbocationic HEI. It has been demonstrated previously that the design of transition state (TS) or HEI analogues could lead to powerful and specific inhibitors of enzymes. We applied this approach to the following target enzymes: 2,3-epoxy-2,3-dihydroqualene cyclase, AdoMet-cycloartenol-C-24-methyltransferase (AdoMet CMT), cycloeucalenol-obtusifoliol isomerase (COI) and Δ⁸-Δ⁷-sterol isomerase. Very potent inhibitors have been obtained in the four cases. As an example, analogues of cycloartenol substituted at C-25 by a charged heteroatom (N, As, S) have been synthesized and shown to be able to mimic the C-25 carbocationic HEI involved in the reaction catalyzed by the AdoMet CMT. These compounds were shown to be very potent and specific inhibitors of this enzyme both in vitro (Ki=2.10⁻⁸ M, Ki/Km=10⁻³) and in vivo. The potent inhibitors described are powerful tools to control in vivo the sterol profile of plant cells and therefore to study the structural and functional roles of sterols in cell membranes. Moreover, these compounds constitute leader molecules of a new class of rationally designed inhibitors which could be of value in plant protection.     

The squalene-2,3-epoxide cyclase as a model for the development of new drugs

The 2,3-oxido squalene (SO) cyclases represent a group of enzymes which convert SO into polycyclic triterpenoids such as lanosterol, cycloartenol, cucurbitadienol and β-amyrin. Taking into account the postulated model of the enzymatic cyclization of SO, we have investigated the possibility of designing compounds that would be selective and potent inhibitors of SO cyclases. Due to the fundamental role of sterols in animal, higher plant and fungal tissues, these inhibitors might behave as very selective (ipocholesterolemic, antifungal or phytotoxic) drugs. Our first approach was the synthesis and biological evaluation of 2-aza-2,3-dihydrosqualene and its derivatives which, being protonated at physiological pH, would present some similarities to the C-2 carbon ion generated by the opening of the oxirane ring of SO. Microsomes from different sources (germinated pea cotyledons, maize seedlings, rat liver and yeasts) were utilized to determine the inhibition values (I₅₀: concentration of inhibitor producing 50% inhibition at a given substrate concentration). From the results obtained so far we conclude that 2-aza-2-dihydrosqualene and its derivatives strongly inhibited the cyclases, the site of the enzyme responsible for binding to the inhibitor is quite sensitive to the steric hindrance, and the degree of the inhibitory activity is greater in higher plants than in rat liver or fungi.     

On the specificity of allene oxide cyclase

Jasmonic acid is a carbocyclic fatty acid that is biosynthesized from α-linolenic acid in several steps. The formation of the ring structure of jasmonic acid is catalyzed by the enzyme allene oxide cyclase (EC 5.3.99.6) and involves the cyclization of an unstable allene oxide into the cyclopentenone 12-oxo-10,15(Z)-phytodienoic acid. In this study, a number of allene oxides were generated, and their enzymatic and nonenzymatic cyclization into cyclopentenones was investigated. Nonenzymatic cyclization was observed with allene oxides having one pair of conjugated double bonds and an additional isolated double bond in the β,γ position relative to the epoxide group, i.e., the partial structure 4,5-epoxy-1,3,7-octatriene. Enzymatic cyclization took place provided that this structural element was inserted in the fatty acid chain with its epoxide group in the n−6,7 position and the isolated double bond in the n−3 position. A number of oxygenated fatty acids having structural features in common with the natural allene oxides were tested as inhibitors of allene oxide cyclase. Fatty acids having an allene oxide structure in the n−6,7 position but lacking the double bond in the n−3 position, as well as fatty acids having a saturated epoxide group in the n−6,7 position, served as competitive inhibitors of the enzyme. Data on the substrate specificity of allene oxide synthase (EC 4.2.1.92) from corn seeds indicated that fatty acid hydroperoxides with a double bond at n−3 and with the hydroperoxide function at n−6 exhibit the highest affinity but the slowest reaction velocity.     

Umbelliferone aminoalkyl derivatives as inhibitors of oxidosqualene cyclases from <Emphasis Type="Italic">Saccharomyces cerevisiae,Tripanosoma cruzi</Emphasis>, and <Emphasis Type="Italic">Pneumocystis carinii</Emphasis>

A series of umbelliferone aminoalkyl derivatives, previously studied as inhibitors of squalene-hopene cyclase, were tested as inhibitors of yeast (Saccharomyces cerevisiae) oxidosqualene cyclase (OSC) and OSC from Trypanosoma cruzi and Pneumocystis carinii expressed in yeast. Enzymes from these pathogens were included in this study to provide a preliminary screening for antiparasitic activity. Tests were carried out both on cell homogenates incubated with radiolabeled oxidosqualene and on spheroplasts incubated with radiolabeled acetate. Derivatives bearing a methylallylamino group were the most effective on all of the three enzymes. The P. carinii enzyme was the most susceptible to the action of the inhibitors, with IC₅₀ values for almost all of them ranging from 0.1 to 1μM. The T. cruzi enzyme was appreciably inhibited (IC₅₀ 4–5 μM) only by derivatives bearing a methylallylaminoalkyl flexible chain. Results identify a particularly promising new family of OSC inhibitors, for the development of novel antiparasitic agents.     

3-carboxy-4-nitrophenyl-dithio-1,1′,2-trisnorsqualene: A site-directed inactivator of yeast oxidosqualene cyclase

The role and location of essential thiol groups in 2,3-oxidosqualene cyclase fromSaccharomyces cerevisiae was examined (i) by comparing inactivation properties of two known thiol reagents, 5,5′-dithiobis(2-nitrobenzoic acid) (DTNB) and 2-nitro-5-thiocyanobenzoic acid (NTCB), with 3-carboxy-4-nitrophenyl-dithio-1,1′,2-trisnorsqualene (CNDT-squalene), a new thiol reagent designed as a sitedirected inactivator of oxidosqualene cyclase and (ii) by testing the ability of the substrate to protect the enzyme against inactivation by the reagents. All reagents gave a time-dependent inactivation following pseudo-first order kinetics. DTNB and CNDT-squalene showed comparable inactivation ability (K_i=0.67 and 1.21 mM), whereas NTCB was less effective (K_i=15.6 mM). Strong differences between the two most active inhibitors, DTNB and CNDT-squalene, were observed when the enzyme was saturated with substrate prior to incubation with the thiol reagent. While substrate did not protect the enzyme against the inactivation caused by DTNB, a reduction in the inactivation ability of CNDT-squalene was observed under protection conditions. The data suggest that the squalene-like inactivator modifies a thiol group located at the active site of the enzyme.     

Alicyclobacillus acidocaldarius Squalene‐Hopene Cyclase: The Critical Role of Steric Bulk at Ala306 and the First Enzymatic Synthesis of Epoxydammarane from 2,3‐Oxidosqualene

The acyclic molecule squalene ( 1 ) is cyclized into 6,6,6,6,5‐fused pentacyclic hopene ( 2 ) and hopanol ( 3 ; ca. 5:1) through the action of Alicyclobacillus acidocaldarius squalene‐hopene cyclase (AaSHC). The polycyclization reaction proceeds with regio‐ and stereochemical specificity under precise enzymatic control. This pentacyclic hopane skeleton is generated by folding 1 into an all‐chair conformation. The Ala306 residue in AaSHC is conserved in known squalene‐hopene cyclases (SHCs); however, increasing the steric bulk (A306T and A306V) led to the accumulation of 6,6,6,5‐fused tetracyclic scaffolds possessing 20R stereochemistry in high yield (94 % for A306V). The production of the 20R configuration indicated that 1 had been folded in a chair‐chair‐chair‐boat conformation; in contrast, the normal chair‐chair‐chair‐chair conformation affords the tetracycle with 20S stereochemistry, but the yield produced by the A306V mutant was very low (6 %). Consequently, bulk at position 306 significantly affects the stereochemical fate during the polycyclization reaction. The SHC also accepts (3R) and (3S)‐2,3‐oxidosqualenes (OXSQs) to generate 3α,β‐hydroxyhopenes and 3α,β‐hydroxyhopanols through polycyclization initiated at the epoxide ring. However, the Val and Thr mutants generated epoxydammarane scaffolds from (3R)‐OXSQ; this indicated that the polycyclization cascade started in these instances at the terminal double bond position. This work is the first to report the polycyclization of oxidosqualene starting at the terminal double bond.     

19-Azasqualene-2,3-epoxide and its N-oxide: Metabolic fate and inhibitory effect on sterol biosynthesis in Saccharomyces cerevisiae

19-Azasqualene-2,3-epoxide was more inhibitory than the corresponding N-oxide against 2,3-oxidosqualene cyclase (OSC) solubilized from Saccharomyces cerevisiae (IC₅₀ 7±2 and 25±5 μM, respectively). Both compounds showed a reversible, noncompetitive-type inhibition on solubilized OSC. Different inhibitory properties between the compounds were especially evident when measuring [¹⁴C]acetate incorporation into nonsaponifiable lipids extracted from treated cells. In cells treated with 19-azasqualene-2,3-epoxide at 30 μM, the radioactivity associated with the oxidosqualene fraction, which was negligible in the controls, rose to over 40% of the nonsaponifiable lipids, whereas it remained at a slightly appreciable level in cells treated with the N-oxide derivative under the same conditions. 19-Azasqualene-2,3-epoxide was also more effective than the N-oxide as a cell growth inhibitor (minimal concentration of compound needed to inhibit yeast growth: 45 and >100 μM, respectively). The two inhibitors underwent different metabolic fates in the yeast: while 19-azasqualene-2,3-epoxide did not undergo any transformation, its N-oxide was actively reduced to the corresponding amine in whole and in “ultrasonically stimulated” cells. The N-oxide reductases responsible for this transformation appear to be largely confined within the microsomal fractions and require NADPH for their activity. A possible relationship between the inhibitory properties of the two compounds and their metabolic fates is discussed.     

Analysis of the Catalytic Mechanism of Bifunctional Triterpene/Sesquarterpene Cyclase: Tyr167 Functions To Terminate Cyclization of Squalene at the Bicyclic Step

Onoceroids are a group of triterpenes biosynthesized from squalene or dioxidosqualene by cyclization from both termini. We previously identified a bifunctional triterpene/sesquarterpene cyclase (TC) that constructs a tetracyclic scaffold from tetraprenyl‐β‐curcumene (C₃₅) but a bicyclic scaffold from squalene (C₃₀) in the first reaction. TC also accepts the bicyclic intermediate as a substrate and generates tetracyclic and pentacyclic onoceroids in the second reaction. In this study, we analyzed the catalytic mechanism of an onoceroid synthase by using mutated enzymes. TC^Y167A produced an unnatural tricyclic triterpenol, but TC^Y167L, TC^Y167F, and TC^Y167W formed small quantities of tricyclic compounds, which suggested that the bulk size at Y167 contributed to termination of the cyclization of squalene at the bicyclic step. Our findings provide insight into the unique catalytic mechanism of TC, which triggers different cyclization modes depending on the substrate. These findings may facilitate the large‐scale production of an onoceroid for which natural sources are limited.     

A possible regulatory role of squalene epoxidase in chinese hamster ovary cells

Growth of Chinese Hamster Ovary (CHO) cells in the presence of 20% lipid depleted serum (LDS) for only 2 hr results in an increase in the synthesis of [¹⁴C] sterols from [¹⁴C] mevalonate and from [¹⁴C] squalene compared with cells grown under normal growth conditions in the presence of 10% fetal calf serum (FCS). This enhanced sterol synthesis increases with time of exposure of the cells to LDS. However, exposing these cells for time periods up to 42.5 hr to a growth medium containing 20% LDS did not result in enhanced [¹⁴C] sterol synthesis from [¹⁴C] 2,3-oxidosqualene. Incubation of these cells with [¹⁴C] mevalonate resulted in the accumulation of [¹⁴C] squalene regardless of the presence of either LDS or FCS. These results suggest that squalene epoxidase is a regulatory enzyme in the cholesterol biosynthetic pathway in CHO. This research was supported by Grant 1426 from the Council for Tobacco Research-USA, Inc.     

Access of the substrate to the active site of yeast oxidosqualene cyclase: an inhibition and site-directed mutagenesis approach

A structural model of Saccharomyces cerevisiae oxidosqualene cyclase (SceOSC) suggests that some residues of the conserved sequence Pro-Ala-Glu-Val-Phe-Gly (residues 524-529) belong to a channel constriction that gives access to the active-site cavity. Starting from the SceOSC C457D mutant, which lacks the cysteine residue next to the catalytic Asp456 residue Cys457 has been replaced but Asp456 is still there, we prepared two further mutants where the wild-type residues Ala525 and Glu526 were individually replaced by cysteine. These mutants, especially E526C, were very sensitive to the thiol-reacting agent dodecyl-maleimide. Moreover, both the specific activity and the thermal stability of E526C were severely reduced. A similar decrease of the enzyme functionality was obtained by replacing Glu526 with alanine, while substitution with the conservative residues aspartate or glutamine did not alter catalytic activity. Molecular modeling of the yeast wild-type OSC and mutants on the template structure of human OSC confirms that the channel constriction is an important aspect of the protein structure and suggests a critical structural role for Glu526.     

Carbonic Anhydrase Interaction With Lipothioars Enites: A Novel Class of Isozymes I and II Inhibitors

The interaction of carbonic anhydrase (CA) isozymes I and II with a series of As(III) derivatives, dialkyl and diaryl rac-2,3-dimyristoyloxypropyldithioarsonites, was investigated kinetically and spectrophotometrically, utilizing the native and Co(II)-substituted enzymes. Depending on the substitution pattern at the -As(SR)(2) moiety of the investigated derivatives, inactive compounds were found for R = phenyl or naphthyl, and active ones for derivatives containing carboxyl groups (R = CH(2)COOH, cysteinyl and glutathionyl). Together with the arsonolipids previously investigated, the active compounds of this series - the "lipothioarsenites"- constitute a novel class of CA inhibitors that bind to the metal ion within the enzyme active site, as proved by changes in the electronic spectra of adducts of such inhibitors with Co(II)CA.     

Recent Developments in Enzymatic Asymmetric C?C Bond Formation

Some of the recent developments in enzymatic asymmetric C C bond formation are described in this review. The close relationship of biocatalysis and biosynthesis is highlighted with a special emphasis on diversity and biogenesis. One focus of this review is the creation of tetrasubstituted carbon stereocenters. Members of the supposedly well‐known aldolase and hydroxynitrile lyase enzyme families possess the ability to catalyze the formation of tertiary alcohols. In the case of aldolases, this can occur through intramolecular cyclization or intermolecular asymmetric C C bond formation. Thiamine diphosphate‐dependent YerE has been identified as a potent catalyst for the acyloin condensation with ketones as acceptor substrates. C₁ transformations such as methylation or carboxylation are catalyzed in an asymmetric manner by enzymes from different classes, for example S‐adenosylmethionine‐dependent (radical) enzymes or NADPH‐dependent oxidoreductases. Insights from biosynthetic and mechanistic studies of enzymatic reactions proceeding via radical intermediates give valuable hints towards possible applications in biocatalysis. Still, the oxygen sensitivity of many of these biocatalysts poses a considerable challenge for practical applications.     

Photoactivation of an inhibitor of the 12/15-lipoxygenase pathway

Lipoxygenases are lipid-peroxidizing enzymes that have been implicated in the pathogenesis of inflammatory diseases and lipoxygenase inhibitors may be developed as anti-inflammatory drugs. Structure comparison with known lipoxygenase inhibitors has suggested that (2Z)-2-(3-benzylidene)-3-oxo-2,3-dihydrobenzo[b]thiophene-7-carboxylic acid methyl ester might inhibit the lipoxygenase pathway but we found that it exhibited only a low inhibitory potency for the pure 12/15-lipoxygenase (IC(50) = 0.7 mM). However, photoactivation, which induces a Z-to-E isomerization of the double bond, strongly augmented the inhibitory potency and an IC(50) value of 0.021 mM was determined for the pure E isomer. Similar isomer-specific differences were observed with the recombinant enzyme and its 12-lipoxygenating Ile418Ala mutant, as well as in intracellular lipoxygenase activity. Structure modeling of the enzyme/inhibitor complex suggested the molecular reasons for this isomer specificity. Since light-induced isomerization may proceed in the skin, such photoreactive compounds might be developed as potential drugs for inflammatory skin diseases.     

The Application of 3-Amino-2H-Azirines as Amino Acid Equivalents

3-amino-2H-azirines undergo a number of ring opening reactions. The cleavage of the azirine-N(1), C(3) double bond leads thereby to synthons which can be considered as amino acid equivalents. The reaction of 3-amino-2H-azirines with carboxylic acids yields N-acyl amino acid amides, which can be converted by a selective amide cleavage to the corresponding N-acyl amino acids. 2-Oxazolin-5-ones are intermediates of this amide cleavage. This reaction sequence has been used for the extension of peptide chains as well as for a number of heterocycle syntheses. Likewise, the described synthesis of cyclic depsipeptides and lactones by direct amide cyclization proceeds via 2-oxazolin-5-one intermediates. The selective amide cleavage was also applied to a novel method for the resolution of enantiomeric amino acid derivatives.