Diastereoselective synthesis of aminobacteriohopanetetrol, a biomarker for methanotrophic bacteria: confirmation of the absolute configuration

A short and convenient strategy was developed for the first stereoselective chemical synthesis of aminobacteriohopanetetrol (= (1R,2R,3S,4S)-5-amino-1-[(22R)-hopan-30-yl]pentane-1,2,3,4-tetrol; 1), a typical biomarker for methanotrophic bacteria. Comparison of the NMR spectra of the synthetic and natural (peracetylated) product enabled us to unambiguously corroborate the absolute configuration of the functionalized pentyl side chain of 1.     

Combinatorial synthesis by nature: volatile organic sulfur-containing constituents of Ruta chalepensis L

Ongoing interest in discovering new natural fragrance and flavor ingredients prompted us to examine a solvent extract of sulfurous-sweaty smelling Ruta chalepensis L. (Rutaceae) plant material more closely. Twenty-one sulfur-containing constituents of similar structures were identified by GC/MS techniques. Amongst them, 14 have never been described to occur in nature. The compounds 1-18 belong to a family of natural flavor and fragrance molecules having a 1,3-positioned O,S moiety in common. The identities of the natural constituents were confirmed by comparison with synthetic reference samples, and the organoleptic properties of the latter were studied. The relative and absolute configurations of the four stereoisomers of 4-methyl-3-sulfanylhexan-1-ol (5) were established by stereoselective synthesis. The natural isomers consisted of a 65 : 35 mixture of (3R,4S)-5 and (3S,4S)-5.     

Inhibition by prostacyclin and carbacyclins of endothelin-induced DNA synthesis in cultured vascular smooth muscle cells

Effects of prostacyclin and carbacyclins on endothelin-induced DNA synthesis were investigated in vascular smooth muscle cells. DNA synthesis was estimated by [3H]thymidine incorporation. Five carbacyclins used in this report were 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-3-hydroxy-1-octenyl]bicyclo [3.3.0]oct-2-en-3-yl) pentanoic acid (TEI-7165), methyl 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-3-hydroxy-1-octenyl]bicyclo[3.3.0]oct-2-en-3- yl]pentanoate (TEI-9090), 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(3S, 5S)-3-hydroxy-5-methyl-1-nonenyl]bicyclo[3.3.0]oct-2-en-3-yl)penta noic acid (TEI-9063), methyl 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(3S, 5S)-3-hydroxy-5-methyl-1- nonenyl]bicyclo[3.3.0]oct-2-en-3-yl)pentanoate (TEI-1324), 5-[(1S, 5S, 6R, 7R)-7-hydroxy-6-[(E)-(S)-4-hydroxy-4-methyl-1- octenyl]bicyclo[3.3.0]oct-2-en-3-yl] pentanoic acid (TEI-3356). Prostacyclin and the carbacyclins inhibited the endothelin-induced DNA synthesis within the nanomolar range. These results suggest that prostacyclin and carbacyclins are possibly effective in inhibiting the proliferation of vascular smooth muscle cells under some situations in vivo.     

Isomerisation and conformation studies of (+)- and (-)6-hydroxy-5, 6-dihydrouridine.

(1) "Uridine hydrates" i.e. (+)- and (-)6-hydroxy-5, 6-dihydrouridine were formed under gamma irradiation in a deaerated aqueous solution of uridine. (2) The structures of two diastereoisomers were determined by spectroscopic measurements (infrared, ultraviolet and NMR) and verified by stereospecific synthesis; uridine hydrates were prepared by mild reduction of trans(+)- and (-)iodohydrins with acetic acid and zinc power. (3) The carbon 6 epimerisation of uridine hydrates 6R or 6S was performed in triated water (pH 5.5, 30 degrees C) and at the same time tritium incorporation on carbon 5 was noted. The mechanism of these reactions could be explained by the opening of the N1-C6 bond of the pyrimidine ring, followed by ketoenolisation reaction of carbons 4 and 5. (4) The 250 MHz NMR analysis has allowed us to determine the nucleoside conformations. Nucleosides had mainly the S(C2' endo) conformation. A slight preference of gauche-gauche (gg) rotamer of the exocyclic hydroxymethyl group was noted and the aglycone was in the anti conformation.     

(6R)-5,6,7,8-tetrahydro-L-monapterin from Escherichia coli, a novel natural unconjugated tetrahydropterin

The structure of the major tetrahydropterin in Escherichia coli was determined as (6R)-5,6,7,8-tetrahydro-L-monapterin, i. e. (6R)-2-amino-5,6,7,8-tetrahydro-6-[(1S,2S)-1,2,3-trihydroxypropyl]pteridin-4(3H)-one. Although the stereochemical structure of the trihydroxypropyl side chain has been determined previously by fluorescence detected circular dichroism analysis on its aromatic derivative, the most important configuration at C(6) has not been clarified. The major difficulties for the determination of the chirality were instability toward air oxidation and very low concentration of the tetrahydropterin derivative. In the present study, the C(6)-configuration was determined as R by comparing its stable hexaacetyl derivative with authentic (6R)- and (6S)-hexaacetyl-5,6,7,8-tetrahydro-L-monapterins by high performance liquid chromatography (HPLC) and HPLC-mass spectrometry (LC-MS). (6R)-5,6,7,8-Tetrahydro-L-monapterin is a new unconjugated tetrahydropterin from natural sources.     

Racemic LTA4 methyl ester bioconversion into LTC4 methyl ester by various glutathione S-transferases

It has been shown that various glutathione transferases can synthesize leukotriene C4, or its methyl ester, from glutathione and leukotriene A4. We questioned whether the same enzymes could be used to resolve racemic leukotriene A4 methyl ester (more easily prepared than the optically active enantiomer) and to produce leukotriene C4 methyl ester selectively. We present in this paper a study of the enantioselectivity of some rat liver glutathione transferase isozymes and of the glutathione transferase of human placenta for the leukotriene A4 methyl ester isomers. The rat liver 3-4 glutathione transferase exhibited the highest conversion rate but preferentially converted the (5R, 6R) leukotriene A4 methyl ester. The placental enzyme was fairly selective for the natural (5S, 6S) enantiomer but the rate of conversion was low.     

C(15) Acetogenins from the red alga Laurencia obtusa.

Four C(15) acetogenins, 13-epilaurencienyne (3Z) (1), 13-epipinnatifidenyne (3E) (2), (3E, 6S(*), 7R(*), 9S(*), 10S(*), 12R(*))-9-chloro-13-bromo-6:12-epoxy-7, 10-diacetoxypentadec-3-en-1-yne (3), (3Z, 6S(*), 7R(*), 9S(*), 10S(*), 12R(*))-9-chloro-13-bromo-6:12-epoxy-7, 10-diacetoxypentadec-3-en-1-yne (4), along with the known 13-epilaurencienyne (3E) (5), have been isolated from the organic extract of the red alga Laurencia obtusa, collected in the Aegean Sea, Greece. The structures of the new natural products, as well as their relative stereochemistry, were established by means of spectral data analysis, including 2D NMR spectroscopic experiments. Some of the new metabolites exhibited significant insecticidal activity.     

Characterization of a novel, potent, and specific inhibitor of serine palmitoyltransferase.

We have examined the mechanism of action of two natural products identified as broad spectrum antifungal agents (VanMiddlesworth, F., Dufresne, C., Wincott, F. E., Mosley, R. T., and Wilson, K. E. (1992) Tetrahedron Lett., in press; VanMiddlesworth, F., Giacobbe, R. A., Lopez, M. Garrity, G., Bland, J. A., Bartizal, K., Fromtling, R. A., Polishook, J., Zweerink, M. M., Edison, A. M., Rozdilsky, W., Wilson, K. E., and Monaghan, R. L. (1992) J. Antibiot. (Tokyo) 45, 861-867), designated sphingofungin B (2S-amino-3R,4R,5S,14-tetrahydroxyeicos-6-enoic acid) and sphingofungin C (2S-amino-5S-acetoxy-3R,4R,14-trihydroxyeicos-6-enoic acid), and find they are potent specific inhibitors of serine palmitoyltransferase, which catalyze the committed step of sphingolipid biosynthesis. We used Saccharomyces cerevisiae as a model to investigate the mechanism of the antifungal activity of these compounds. Macromolecular synthesis was not immediately affected by either sphingofungin B or C, synthesis continued for 60-90 min following the addition of drug to growing cultures. Significant loss of viability with sphingofungins required growing cultures and began only after several hours, with greater than 99.9% of drug-treated cells non-viable after 24 h. No lysis or other gross changes in cell morphology were observed in drug-treated cells. The structural similarity of sphingofungin B and C to sphingosine and phytosphingosine prompted us to investigate their effects on sphingolipid synthesis. Nanomolar levels of the drugs inhibited the incorporation of [3H]inositol into sphingolipid before incorporation into the sphingolipid precursor, phosphatidylinositol was affected, suggesting specific inhibition of sphingolipid synthesis. This hypothesis was confirmed by experiments in which the growth inhibitory activity of both drugs was completely ablated by the addition of phytosphingosine, dihydrosphingosine, or ketodihydrosphingosine to the culture medium. Reversal of antifungal activity by ketodihydrosphingosine suggested that serine palmitoyltransferase could be the actual target of these compounds. Direct evidence for this hypothesis was the observation of inhibition of serine palmitoyltransferase activity in crude membrane preparations at nanomolar concentrations of each drug. The potent inhibition of serine palmitoyltransferase coupled with the apparent lack of effect of these compounds on other cellular functions suggests that sphingofungin B and C will prove to be important new tools for studying the role of sphingolipids in yeast and perhaps in other organisms.     

The stereochemistry of trans-4-hydroxynonenal-derived exocyclic 1,N2-2'-deoxyguanosine adducts modulates formation of interstrand cross-links in the 5'-CpG-3' sequence

The trans-4-hydroxynonenal (HNE)-derived exocyclic 1, N(2)-dG adduct with (6S,8R,11S) stereochemistry forms interstrand N(2)-dG-N(2)-dG cross-links in the 5'-CpG-3' DNA sequence context, but the corresponding adduct possessing (6R,8S,11R) stereochemistry does not. Both exist primarily as diastereomeric cyclic hemiacetals when placed into duplex DNA [Huang, H., Wang, H., Qi, N., Kozekova, A., Rizzo, C. J., and Stone, M. P. (2008) J. Am. Chem. Soc. 130, 10898-10906]. To explore the structural basis for this difference, the HNE-derived diastereomeric (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals were examined with respect to conformation when incorporated into 5'-d(GCTAGC XAGTCC)-3' x 5'-d(GGACTCGCTAGC)-3', containing the 5'-CpX-3' sequence [X = (6S,8R,11S)- or (6R,8S,11R)-HNE-dG]. At neutral pH, both adducts exhibited minimal structural perturbations to the DNA duplex that were localized to the site of the adduction at X(7) x C(18) and its neighboring base pair, A(8) x T(17). Both the (6S,8R,11S) and (6R,8S,11R) cyclic hemiacetals were located within the minor groove of the duplex. However, the respective orientations of the two cyclic hemiacetals within the minor groove were dependent upon (6S) versus (6R) stereochemistry. The (6S,8R,11S) cyclic hemiacetal was oriented in the 5'-direction, while the (6R,8S,11R) cyclic hemiacetal was oriented in the 3'-direction. These cyclic hemiacetals effectively mask the reactive aldehydes necessary for initiation of interstrand cross-link formation. From the refined structures of the two cyclic hemiacetals, the conformations of the corresponding diastereomeric aldehydes were predicted, using molecular mechanics calculations. Potential energy minimizations of the duplexes containing the two diastereomeric aldehydes predicted that the (6S,8R,11S) aldehyde was oriented in the 5'-direction while the (6R,8S,11R) aldehyde was oriented in the 3'-direction. These stereochemical differences in orientation suggest a kinetic basis that explains, in part, why the (6S,8R,11S) stereoisomer forms interchain cross-links in the 5'-CpG-3' sequence whereas the (6R,8S,11R) stereoisomer does not.     

Synthesis and functional activity of initiation regions of mRNA translation. II. RNA-ligase synthesis of hepta- and decaribonucleotides--components of 20-base polyribonucleotide templates

Preparative RNA-ligase synthesis of decaribonucleotides, the 5'-and 3'-constituent parts to be used for the synthesis of 20-base polyribonucleotides] simulating minimal translation initiation regions for phage RNA was carried out. The decamers were obtained via appropriate heptamers also by RNA-ligase catalyzed synthesis. Apart from decamers used to prepare the functionally active 20-base polyribonucleotide, the minimal translation initiation region of the replicase gene (R) in MS2 and fr phage--sequence R(-17----3) and two its variants, decanucleotides for other template modification were also synthesized. Three 5'-terminal decamers were isolated and identified including the natural decamer ApApApCpApUpGpApGpG (-17----(-)8) and those with G(-9)----A(-9) and U(-12)----C(-12) nucleotide substitutions, as well as three 3'-terminal products differing from the natural region ApUpUpCpCpCpApUpG (-7----3) in MS2 RNA by U(-6)----A(-6), U(-6)U(-5)----A(-6)A(-5) and CCC----UUU (-3----(-)1) substitutions.     

Enzymatic characterization of human mitochondrial C1-tetrahydrofolate synthase

A human mitochondrial isozyme of C1-tetrahydrofolate (THF) synthase was previously identified by its similarity to the human cytoplasmic C1-THF synthase. All C1-THF synthases characterized to date, from yeast to human, are trifunctional, containing the activities of 5,10-methylene-THF dehydrogenase, 5,10-methenyl-THF cyclohydrolase, and 10-formyl-THF synthetase. Here we report on the enzymatic characterization of the recombinant human mitochondrial isozyme. Enzyme assays of purified human mitochondrial C1-THF synthase protein revealed only the presence of 10-formyl-THF synthetase activity. Gel filtration and crosslinking studies indicated that human mitochondrial C1-THF synthase exists as a homodimer in solution. Steady-state kinetic characterization of the 10-formyl-THF synthetase activity was performed using (6R,S)-H4-PteGlu1, (6R,S)-H4-PteGlu3, and (6R,S)-H4-PteGlu5 substrates. The (6R,S)-H4-PteGlun Km dropped from greater than 500 microM for the monoglutamate to 15 microM and 3.6 microM for the tri- and pentaglutamates, respectively. The Km values for formate and ATP also are lowered when THF polyglutamates are used. The formate Km dropped 79-fold and the ATP Km dropped more than 5-fold when (6R,S)-H4-PteGlu5 was used as the substrate in place of (6R,S)-H4-PteGlu1.     

Synthesis of 2',3'-dideoxy-2'-fluoro-3'-thioarabinothymidine and its 3'-phosphoramidite derivative

An efficient method for the synthesis of 5'-O-monomethoxytrityl-2',3'-dideoxy-2'-fluoro-3'-thioarabinothymidine [(5'MMT)araF-T(3'SH), (5)] and its 3'-phosphoramidite derivative (6) suitable for automated incorporation into oligonucleotides, is demonstrated. A key step in the synthesis involves reaction of 5'-O-MMT-2,3'-O-anhydrothymidine (4) (Eleuteri, A.; Reese, C.B.; Song, Q. J. Chem. Soc. Perkin Trans. 1 1996, 2237 pp.) with sodium thioacetate to give (5'-MMT)araF-T(3'SAc) (5) (Elzagheid, M.I.; Mattila, K.; Oivanen, M.; Jones, B.C.N.M.; Cosstick, L?nnberg, H. Eur. J. Org. Chem. 2000, 1987-1991). This nucleoside was then converted to its corresponding phosphoramidite derivative, 6, as described previously ((a) Sun, S.; Yoshida, A.; Piccirilli, J.A. RNA, 1997, 3, 1352-1363; (b) Matulic-Adamic, J.; Beigelman, L. Helvetica Chemica Acta 1999, 82, 2141-2150: (c) Fettes, K.J.; O'Neil, I.; Roberts, S.M.; Cosstick, R. Nucleosides, Nucleotides and Nucl. Acids 2001, 20, 1351-1354).     

Efficient synthesis and biological evaluation of all A-ring diastereomers of 1alpha,25-dihydroxyvitamin D3 and its 20-epimer

An improved synthesis of the diastereomers of 1alpha,25-dihydroxyvitamin D3 (1) was accomplished utilizing our practical route to the A-ring synthon. We applied this procedure to synthesize for the first time all possible A-ring diastereomers of 20-epi-1alpha,25-dihydroxyvitamin D3 (2). Ten-step conversion of 1-(4-methoxyphenoxy)but-3-ene (6), including enantiomeric introduction of the C-3 hydroxyl group to the olefin by the Sharpless asymmetric dihydroxylation, provided all four possible stereoisomers of A-ring enynes (3). i.e., (3R,5R)-, (3R,5S)-, (3S,5R)- and (3S,5S)-bis[(tert-butyldimethylsilyl)oxy]oct-1-en-7-yne, in good overall yield. Palladium-catalyzed cross-coupling of the A-ring synthon with the 20-epi CD-ring portion (5), (E)-(20S)-de-A,B-8-(bromomethylene)cholestan-25-ol, followed by deprotection, afforded the requisite diastereomers of 20-epi-1alpha,25-dihydroxyvitamin D3 (2). The biological profiles of the synthesized stereoisomers were assessed in terms of affinities for vitamin D receptor (VDR) and vitamin D binding protein (DBP). HL-60 cell differentiation-inducing activity and in vivo calcium-regulating potency in comparison with the natural hormone.     

Regio- and stereoselective cyclizations of dianhydro sugar alcohols catalyzed by a chiral (salen)Co(III) complex

The (salen)Co(III)OAc ((R,R)-1 and (S,S)-1) catalyzed cyclizations of the chiral dianhydro sugars, 1,2:5,6-dianhydro-3,4-di-O-methyl-D-glucitol (2), 1,2:5,6-dianhydro-3,4-di-O-methyl-D-mannitol (3), 1,2:5,6-dianhydro-3,4-di-O-methyl-L-iditol (4), and 1,2:4,5-dianhydro-3-O-methyl-L-arabinitol (5), is a facile method for the synthesis of anhydroalditol alcohols. Cyclization of 2 using (R,R)-1 and (S,S)-1 proceeded diastereoselectively to form 2,5-anhydro-3,4-di-O-methyl-D-mannitol (6) and 2,5-anhydro-3,4-di-O-methyl-L-iditol (7), respectively. The cyclization of 3 and 5 is a novel method for obtaining 1,6-anhydro-3,4-di-O-methyl-D-mannitol (11) and a stereoselective route to 1,5-anhydro-3-O-methyl-L-arabinitol (13). It is proposed that the reaction occurs via endo-selective cyclization of an epoxy alcohol produced by the endo-selective ring-opening of one of the two epoxide moieties in the starting material.     

Synthesis of "glycospirostanes"--steroid sapogenins with a sugar-like ring F

The synthesis of two "glycospirostanes" from 23-oxotigogenin acetate is described. (23S,24S,25R)-5alpha-Spirostane-3beta,23,24,25-tetraol was obtained by dehydrogenation followed by stereoselective reduction of the 23-oxo group and OsO(4) dihydroxylation of the C24-C25 double bond. Allylic hydroxylation with SeO(2) of 3beta-acetoxy-5alpha-spirost-23-ene obtained from 23-oxotigogenin acetate followed by OsO(4) dihydroxylation of the C23-C24 double bond afforded (23R,24S,25R)-5alpha-spirostane-3beta,23,24,25-tetraol.     

Stereoselective formation and hydration of benzo[c]phenanthrene 3,4- and 5,6-epoxide enantiomers by rat liver microsomal enzymes

The K-region 5,6-epoxides, formed in the metabolism of benzo[c]phenanthrene (BcPh) in the presence of an epoxide hydrolase inhibitor 3,3,3-trichloropropylene 1,2-oxide (TCPO) by liver microsomes from untreated, phenobarbital-treated, 3-methylcholanthrene-treated, and polychlorinated biphenyls (Aroclor 1254)-treated rats of the Sprague-Dawley and the Long-Evans strains, were found by chiral stationary phase high-performance liquid chromatography analyses to be enriched (58-72%) in the 5S, 6R enantiomer. In the absence of TCPO, the metabolically formed BcPh trans-5,6-dihydrodiol was enriched (78-86%) in the 5S,6S enantiomer. The major enantiomer of the BcPh 3,4-epoxide metabolite was found to be enriched in the 3S,4R enantiomer which undergoes racemization under the experimental conditions. The major enantiomer of the 5,6-dihydrodiol metabolite was elucidated by the exciton chirality circular dichroism (CD) method to have a 5S,6S absolute stereochemistry. Absolute configurations of enantiomeric methoxylation products derived from each of the two BcPh 5,6-epoxide enantiomers. Optically pure BcPh 5S,6R-epoxide was enzymatically hydrated exclusively at the C6 position to form an optically pure BcPh 5S,6S-dihydrodiol. However, optically pure BcPh 5R,6S-epoxide was hydrated at both C5 and C6 positions to form a BcPh trans-5,6-dihydrodiol with a (5S,6S):(5R,6R) enantiomer ratio of 32:68.     

Total synthesis of junionone, a natural monoterpenoid from Juniperus communis L., and determination of the absolute configuration of the naturally occurring enantiomer by ROA spectroscopy

Recently, we reported a novel access to 2,2-diethyl-3-[(E/Z)-prop-1-en-1-yl]cyclobutanone by an intramolecular nucleophilic substitution with allylic rearrangement (S(N)i') of (E)-6-chloro-3,3-diethylhept-4-en-2-one. The ring closure reaction was found to proceed with selective syn-displacement of the leaving group. This method was now applied to the total synthesis of junionone, an olfactorily interesting cyclobutane monoterpenoid isolated from Juniperus communis, L. S(N)i' Ring closure of the ketone enolate of (E)-3,3-dimethyl-5-[(2R,3R)-3-methyloxiran-2-yl]pent-4-en-2-one (R,R)-(E)-4' proceeded only after the epoxide moiety had been activated by Lewis acid and led to the junionone precursors (3R)- and (3S)-3-[(1E,3R)-3-hydroxybut-1-en-1-yl]-2,2-dimethylcyclobutanone (S/R,R)-(E)-3. The ratio of syn- and anti-conformers in the transitory molecular arrangement was found to depend on the nature of the Lewis acid. The absolute configuration of both the synthetic as well as the natural junionone, isolated from juniper berry oil, was determined by Raman Optical Activity (ROA) spectroscopy. Our experiments led to a novel synthetic route to both (+)- and (-)-junionone, the first determination of the absolute configuration of natural junionone, and to the development of a practical ROA procedure for measuring milligram quantities of volatile liquids.     

Carotenoids with a 5,6-dihydro-5,6-dihydroxy-beta-end group, from yellow sweet potato "Benimasari", Ipomoea batatas Lam

A series of carotenoids with a 5,6-dihydro-5,6-dihydroxy-beta-end group, named ipomoeaxanthins A (1), B (2), C1 (3) and C2 (4) were isolated from the flesh of yellow sweet potato "Benimasari", Ipomoea batatas Lam. Their structures were determined to be (5R,6S,3'R)-5,6-dihydro-beta,beta-carotene-5,6,3'-triol (1), (5R,6S,5'R,6'S)-5,6,5',6'-tetrahydro-beta,beta-carotene-5,6,5'6'-tetrol (2), (5R,6S,5'R,8'R)-5',8'-epoxy-5,6,5',8'-tetrahydro-beta,beta-carotene-5,6-diol (3), and (5R,6S,5'R,8'S)-5',8'-epoxy-5,6,5',8'-tetrahydro-beta,beta-carotene-5,6-diol (4) by UV-Vis, NMR, MS and CD data.     

Stereoselective formation and hydration of 12-methylbenz[a]anthracene 5,6-epoxide enantiomers by rat liver microsomal enzymes.

The K-region trans-5,6-dihydrodiols formed in the metabolism of 12-methylbenz[a]anthracene (12-MBA) by liver microsomal preparations from untreated, phenobarbital-treated and 3-methylcholanthrene-treated male Sprague-Dawley rats were found by chiral stationary-phase h.p.l.c. (c.s.p.-h.p.l.c.) analyses to contain (5S,6S)/(5R,6R) enantiomer ratios of 93:7, 88:12 and 97:3 respectively. The absolute stereochemistry of a 12-MBA trans-5,6-dihydrodiol enantiomer was elucidated by the exciton-chirality c.d. method. The 5,6-epoxides formed in the metabolism of 12-MBA by liver microsomal preparations from untreated, phenobarbital-treated and 3-methylcholanthrene-treated male Sprague-Dawley rats in the presence of the epoxide hydrolase inhibitor 3,3,3-trichloropropylene 1,2-oxide were isolated from a mixture of metabolites by normal-phase h.p.l.c., and their (5S,6R)/(5R,6S) enantiomer ratios were found by c.s.p.-h.p.l.c. analyses to be 73:27, 78:22 and 99:1 respectively. The absolute configurations of 12-MBA 5,6-epoxide enantiomers, resolved by c.s.p.-h.p.l.c., were determined via high-resolution (500 MHz) proton-n.m.r. and c.d. spectral analyses of the two isomeric methoxylation products derived from each of the 12-MBA 5,6-epoxide enantiomers. Enantiomeric pairs of the two methoxylation products were resolved by c.s.p.-h.p.l.c. The results indicate that enantiomeric 5S,6R-epoxide and 5S,6S-dihydrodiol were the major enantiomers preferentially formed in the metabolism at the K-region 5,6-double bond of 12-MBA by all three rat liver microsomal preparations. Optically pure 12-MBA 5S,6R-epoxide was hydrated predominantly at the C(6) position (R centre) to form 12-MBA trans-5,6-dihydrodiol with a (5S,6S)/(5R,6R) enantiomer ratio of 97:3. However, optically pure 12-MBA 5R,6S-epoxide was hydrated nearly equally at both C(5) and C(6) positions to form 12-MBA trans-5,6-dihydrodiol with a (5S,6S)/(5R,6R) enantiomer ratio of 57:43.     

Synthesis of the racemate and both enantiomers of massoilactone

A simple and efficient synthesis of (+/-)-massoilactone (1) as a key substance for the butter and milk flavor was accomplished from n-hexanal in only a few steps. Application of its racemic synthesis enabled natural (R)-(-)- and unnatural (S)-(+)-massoilactone (1a, 1b) to be synthesized by starting from commercially available (R)-(+)-1,2-epoxyheptane (5).