How Cell Physiology Affects Enantioselectivity of the Biotransformation of Ethyl 4-chloro-acetoacetate with Saccharomyces cerevisiae

Saccharomyces cerevisiae (baker's yeast) reduces ethyl 4-chloro-acetoacetate enantioselectively to ( R )- or ( S )-ethyl 4-chloro-3-hydroxybutyrate depending on the reaction conditions and the physiological state of the yeast cells. The ( S )-enantiomer is obtained under batch conditions with resting cells (55%, enantiomeric excess [ee]), and 4-chloro-acetate fed-batch actively metabolising yeast affords the ( R )-isomer (54%, ee). The enantioselective reduction of the substrate is accompanied by competing enzyme actions. Of the metabolites formed from the substrate, chloroacetone and the target compound ( R )-ethyl 4-chloro-3-hydroxybutyrate emerged as most important effectors of enantioselectivity of the microbial reduction. As a minor side-reaction, an aerobic reductive dehalogenation of the substrate was observed. The unusual high enantiopurity of the dehalo-product ( S )-ethyl 3-hydroxybutyrate confirms the stereodirecting effect of chloroacetone impressively. Hence, with S. cerevisiae either enantiomer can be obtained by variation of reaction conditions. The yeast further turned out to be a promising biocatalyst for dehalogenations.     

How Cell Physiology Affects Enantioselectivity of the Biotransformation of Ethyl 4-chloro-acetoacetate with Saccharomyces cerevisiae

Saccharomyces cerevisiae (baker's yeast) reduces ethyl 4-chloro-acetoacetate enantioselectively to ( R )- or ( S )-ethyl 4-chloro-3-hydroxybutyrate depending on the reaction conditions and the physiological state of the yeast cells. The ( S )-enantiomer is obtained under batch conditions with resting cells (55%, enantiomeric excess [ee]), and 4-chloro-acetate fed-batch actively metabolising yeast affords the ( R )-isomer (54%, ee). The enantioselective reduction of the substrate is accompanied by competing enzyme actions. Of the metabolites formed from the substrate, chloroacetone and the target compound ( R )-ethyl 4-chloro-3-hydroxybutyrate emerged as most important effectors of enantioselectivity of the microbial reduction. As a minor side-reaction, an aerobic reductive dehalogenation of the substrate was observed. The unusual high enantiopurity of the dehalo-product ( S )-ethyl 3-hydroxybutyrate confirms the stereodirecting effect of chloroacetone impressively. Hence, with S. cerevisiae either enantiomer can be obtained by variation of reaction conditions. The yeast further turned out to be a promising biocatalyst for dehalogenations.     

Enzymes of l-(+)-3-hydroxybutyrate metabolism in the rat

The three enzymes required for the production and utilization of l-(+)-3-hydroxybutyrate were sought in various tissues of the rat. All tissues examined contained substantial amounts of (No. 1) l-(+)-3-hydroxybutyryl CoA dehydrogenase (EC 1.1.1.35). The specific activity of (No. 2) l-(+)-3-hydroxybutyryl CoA deacylase (EC 3.1.2) was highest in liver (3.8 mU/mg in mitochondrial matrix (1 U = 1 μmol/min). Brain, heart, and skeletal muscle contained < 20% of this activity. The chromatography of liver mitochondrial “matrix” preparations on DEAE-cellulose resolved the deacylase into two peaks. Peak I hydrolyzed 2- or 3- carbon acylCoA esters more efficiently than l-(+)-3-hydroxybutyrate CoA, while Peak II activity was highest using l-(+)-3-hydroxybutyryl CoA. The K_m(app) for Peak II deacylase with l-(+)-3-hydroxybutyryl CoA was 19 μm. Acyl CoA synthetase (EC 6.2.1.2) (No. 3) was assayed with sorbate (sorboyl CoA ligase) or l-(+)-3-hydroxybutyrate (l-(+)-3-hydroxybutyryl CoA ligase). The highest specific activity for l-(+)-3-hydroxybutyryl CoA ligase was associated with brain mitochondria (8.3 mU/mg). In the “matrix” fraction of rat liver mitochondria the activities of these two acyl CoA synthetases were distinguished chromatographically and by their stability at various pH values. Heart and skeletal muscle mitochondria contained <10% of the liver activities of both ligases. These data implicate the liver as a site of l-(+)-3-hydroxybutyrate production.     

Biosynthesis of monoterpenes. Enantioselectivity in the enzymatic cyclization of (+)- and (-)-linalyl pyrophosphate to (+)- and (-)-bornyl pyrophosphate

Enzymes from Salvia officinalis and Tanacetum vulgare leaf epidermis catalyze the conversion of the acyclic precursor geranyl pyrophosphate to the cyclic monoterpenes (+)- and (-)-bornyl pyrophosphate, respectively. The antipodal cyclizations are considered to proceed by the initial isomerization of the substrate to the respective bound tertiary allylic intermediates (-)-(3R)- and (+)-(3S)-linalyl pyrophosphate. [(3R)-8,9-14C,(3RS)-1E-3H] Linalyl pyrophosphate (3H:14C = 5.22) was tested as a substrate with the cyclases from both sources to determine the configuration of the cyclizing intermediate. This substrate yielded (-)-bornyl pyrophosphate with 3H:14C ratio greater than 31, indicating specific utilization of (+)-(3S)-linalyl pyrophosphate as predicted. With the (+)-bornyl pyrophosphate cyclase, the 3H:14C ratio of the product was about 4.16, indicating a preference for the (-)-(3R)-enantiomer, but the ability also to utilize (+)-(3S)-linalyl pyrophosphate. (3R)- and (3S)-[1Z-3H]Linalyl pyrophosphate were separately compared to the achiral precursors [1-3H] geranyl pyrophosphate and [1-3H]neryl pyrophosphate (cis-isomer) as substrates for the cyclizations. All functional precursors afforded optically pure (-)-(1S,4S)-bornyl pyrophosphate with the T. vulgare-derived cyclase (as determined by chromatographic separation of diastereomeric ketals of the derived ketone camphor), and (+)-(3S)-linalyl pyrophosphate was the preferred substrate. With the (+)-bornyl pyrophosphate cyclase from S. officinalis, geranyl, neryl, and (-)-(3R)-linalyl pyrophosphates gave the expected (+)-(1R,4R)-stereoisomer as the sole product, and (-)-(3R)-linalyl pyrophosphate was the preferred substrate. However, (3S)-linalyl pyrophosphate yielded (-)-(1S,4S)-bornyl pyrophosphate, albeit at lower rates, indicating the ability of this enzyme to catalyze the anomalous enantiomeric cyclization.     

Bacillus megaterium WZ009催化合成(R)-4-氯-3-羟基丁酸乙酯和(S)-3-羟基-γ-丁内酯

以外消旋4-氯-3-羟基丁酸乙酯为唯一C源的富集培养筛选得到一株菌株WZ009,经16S rDNA测序鉴定为巨大芽胞杆菌(Bacillus megaterium)。B.megaterium WZ009静息细胞可以立体选择性催化(S)-4-氯-3-羟基丁酸乙酯水解和脱氯反应得到光学纯的(R)-4-氯-3-羟基丁酸乙酯(e.e.≥99%)和(S)-3-羟基-γ-丁内酯(e.e.≥95%)。笔者对B.megaterium WZ009不对称催化反应影响因素(温度、pH、中和剂、底物浓度、时间进程以及细胞重复利用)进行优化研究,确定了该反应体系最优条件:底物浓度200 mmol/L,中和剂氨水,pH 7.2,40℃反应12 h,转化率达到50.6%,底物对映体过量值为99.6%。该生物催化合成(R)-4-氯-3-羟基丁酸乙酯和(S)-3-羟基-γ-丁内酯过程具有良好的工业化应用前景。     

Partial and incomplete oxidation of palmitate by cultured beating cardiac cells from neonatal rats.

The discrepancy in the rate of [14C]O2 formation from either [1-14C]- or [16-14C]palmitate is demonstrated and could be explained by the preferential formation of L-(+)-3-hydroxybutyrate from the four carbon atoms at the omega terminus. The identity of this product as L(+)-3-hydroxybutyrate was established and shown to be the major component of the radioactive products in the extracellular medium from palmitate based on (a) ion-exchange chromatographical properties, (b) gas-liquid chromatography, (c) mass spectrometric analysis, (d) stereoisomeric separation, and (e) its very low rate of utilization by the cells. We therefore propose a shunt to the oxidation of palmitate in these cells occurring at the stage of L(+)-hydroxybutyryl-CoA which undergoes deacylation causing the product to be transported outside the cell.     

3-Hydroxybutyrate dehydrogenase in tissues from normal and ketonaemic sheep

1. In liver, rumen epithelium and kidney cortex of the sheep, a dehydrogenase active against dl-3-hydroxybutyrate occurred in both the cytosol and particulate fractions of the tissues. In brain, heart, skeletal and smooth muscles, the enzyme occurred only in the particulate fraction. 2. Enzyme activity in the cytoplasmic fraction of liver and rumen epithelium was similar with either d(-)-3-hydroxybutyrate or dl-3-hydroxbutyrate, but was less with acetoacetate as the substrate. The cytosol fraction of kidney cortex showed very little activity with d(-)-3-hydroxybutyrate, confirming that most of the activity with dl-3-hydroxybutyrate was with the l(+) isomer in this tissue. 3. 3-Hydroxybutyrate dehydrogenase activities in the cytosol and particulate fractions of liver, rumen epithelium and kidney cortex and in the particulate fraction of brain tissue were not stimulated by phosphatidylcholine, unlike the enzyme in sheep muscle and in tissues of other species. 4. The activity of 3-hydroxybutyrate dehydrogenase was not increased significantly in any of the tissues of ketonaemic sheep. 5. Comparison of rates of 3-hydroxybutyrate production in vivo with the enzyme activity in ketogenic tissue suggested that in sheep the maximum rate of production might be limited by this activity.     

Cosubstrate-induced dynamics of D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida

D-3-Hydroxybutyrate dehydrogenase from Pseudomonas putida belongs to the family of short-chain dehydrogenases/reductases. We have determined X-ray structures of the D-3-hydroxybutyrate dehydrogenase from Pseudomonas putida, which was recombinantly expressed in Escherichia coli, in three different crystal forms to resolutions between 1.9 and 2.1 A. The so-called substrate-binding loop (residues 187-210) was partially disordered in several subunits, in both the presence and absence of NAD(+). However, in two subunits, this loop was completely defined in an open conformation in the apoenzyme and in a closed conformation in the complex structure with NAD(+). Structural comparisons indicated that the loop moves as a rigid body by about 46 degrees . However, the two small alpha-helices (alphaFG1 and alphaFG2) of the loop also re-orientated slightly during the conformational change. Probably, the interactions of Val185, Thr187 and Leu189 with the cosubstrate induced the conformational change. A model of the binding mode of the substrate D-3-hydroxybutyrate indicated that the loop in the closed conformation, as a result of NAD(+) binding, is positioned competent for catalysis. Gln193 is the only residue of the substrate-binding loop that interacts directly with the substrate. A translation, libration and screw (TLS) analysis of the rigid body movement of the loop in the crystal showed significant librational displacements, describing the coordinated movement of the substrate-binding loop in the crystal. NAD(+) binding increased the flexibility of the substrate-binding loop and shifted the equilibrium between the open and closed forms towards the closed form. The finding that all NAD(+) -bound subunits are present in the closed form and all NAD(+) -free subunits in the open form indicates that the loop closure is induced by cosubstrate binding alone. This mechanism may contribute to the sequential binding of cosubstrate followed by substrate.     

Degradation of poly(3-hydroxybutyrate) by poly(3-hydroxybutyrate) depolymerase from Alcaligenes faecalis T1

The extracellular poly(3-hydroxybutyrate) depolymerase purified from Alcaligenes faecalis T₁ has two disulfide bonds, one of which appears to be necessary for the full enzyme activity. This depolymerase hydrolyzed not only hydrophobic poly(3-hydroxybutyrate) but also water-soluble trimer and larger oligomers of D-(−)-3-hydroxybutyrate, regardless of their solubilities in water. Kinetic analyses with oligomers of various sizes indicated that the substrate cleaving site of the enzyme consisted of four subsites with individual affinities for monomer units of the substrate. Analyses of the hydrolytic products of oligomers, which had labeled D-(−)-3-hydroxybutyrate at the hydroxy terminus, showed that the enzyme cleaved only the second ester linkage from the hydroxy terminus of the trimer and tetramer, and acted as an endo-type hydrolase toward the pentamer and higher oligomers. The enzyme appeared to have a hydrophobic site which interacted with poly(3-hydroxybutyrate) and determined the affinity of the enzyme toward the hydrophobic substrate.     

Improvements of enzyme activity and enantioselectivity via combined substrate engineering and covalent immobilization

Esterases, lipases, and serine proteases have been applied as versatile biocatalysts for preparing a variety of chiral compounds in industry via the kinetic resolution of their racemates. In order to meet this requirement, three approaches of enzyme engineering, medium engineering, and substrate engineering are exploited to improve the enzyme activity and enantioselectivity. With the hydrolysis of (R,S)-mandelates in biphasic media consisting of isooctane and pH 6 buffer at 55 degrees C as the model system, the strategy of combined substrate engineering and covalent immobilization leads to an increase of enzyme activity and enantioselectivity from V(S)/(E(t)) = 1.62 mmol/h g and V(S)/V(R) = 43.6 of (R,S)-ethyl mandelate (1) for a Klebsiella oxytoca esterase (named as SNSM-87 from the producer) to 16.7 mmol/h g and 867 of (R,S)-2-methoxyethyl mandelate (4) for the enzyme immobilized on Eupergit C 250L. The analysis is then extended to other (R,S)-2-hydroxycarboxylic acid esters, giving improvements of the enzyme performance from V(S)/(E(t)) = 1.56 mmol/h g and V(S)/V(R) = 41.9 of (R,S)-ethyl 3-chloromandelate (9) for the free esterase to 39.4 mmol/h g and 401 of (R,S)-2-methoxyethyl 3-chloromandelate (16) for the immobilized enzyme, V(S)/(E(t)) = 5.46 mmol/h g and V(S)/V(R) = 8.27 of (R,S)-ethyl 4-chloromandelate (10) for free SNSM-87 to 33.5 mmol/h g and 123 of (R,S)-methyl 4-chloromandelate (14) for the immobilized enzyme, as well as V(S)/(E(t)) = 3.0 mmol/h g and V(S)/V(R) = 7.94 of (R,S)-ethyl 3-phenyllactate (11) for the free esterase to 40.7 mmol/h g and 158 of (R,S)-2-methoxyethyl 3-phenyllactate (18) for the immobilized enzyme. The great enantioselectivty enhancement is rationalized from the alteration of ionization constants of imidazolium moiety of catalytic histidine for both enantiomers and conformation distortion of active site after the covalent immobilization, as well as the selection of leaving alcohol moiety via substrate engineering approach.     

Identification of an acetoacetyl coenzyme A synthetase-dependent pathway for utilization of L-(+)-3-hydroxybutyrate in Sinorhizobium meliloti

D-(-)-3-Hydroxybutyrate (DHB), the immediate depolymerization product of the intracellular carbon store poly-3-hydroxybutyrate (PHB), is oxidized by the enzyme 3-hydroxybutyrate dehydrogenase to acetoacetate (AA) in the PHB degradation pathway. Externally supplied DHB can serve as a sole source of carbon and energy to support the growth of Sinorhizobium meliloti. In contrast, wild-type S. meliloti is not able to utilize the L-(+) isomer of 3-hydroxybutyrate (LHB) as a sole source of carbon and energy. In this study, we show that overexpression of the S. meliloti acsA2 gene, encoding acetoacetyl coenzyme A (acetoacetyl-CoA) synthetase, confers LHB utilization ability, and this is accompanied by novel LHB-CoA synthetase activity. Kinetics studies with the purified AcsA2 protein confirmed its ability to utilize both AA and LHB as substrates and showed that the affinity of the enzyme for LHB was clearly lower than that for AA. These results thus provide direct evidence for the LHB-CoA synthetase activity of the AcsA2 protein and demonstrate that the LHB utilization pathway in S. meliloti is AcsA2 dependent.     

Synthesis, structure, and structure-activity relationship analysis of enamines as potential antibacterials

Twenty-four enamines were synthesized and reported for the first time. Their chemical structures were confirmed by means of 1H NMR, ESI mass spectra, and elemental analyses, and four of them were determined by single crystal X-ray diffraction analysis. All of the compounds were assayed for antibacterial (Bacillus subtilis ATCC 6633, Escherichia coli ATCC 35218, Pseudomonas fluorescens ATCC 13525, and Staphylococcus aureus ATCC 6538) and antifungal (Aspergillus niger ATCC 16404, Candida albicans ATCC 10231, and Trichophyton rubrum ATCC 10218) activities by MTT method. Compounds (E)-ethyl 3-(4-hydroxyphenylamino)-2-(4-methoxyphenyl)acrylate (9b), (E)-ethyl 3-(3,5-difluorophenylamino)-2-(4-chlorophenyl)acrylate (11b), (E)-ethyl 3-(3,5-dichlorophenylamino)-2-(4-chlorophenyl)acrylate (12b), and (E)-ethyl 3-(4-methylphenylamino)-2-(4-chlorophenyl)acrylate (15b) showed considerable antibacterial activities against S. aureus ATCC 6538 with MICs of 3.8, 1.9, 1.1, and 0.9 microg/mL, respectively. Structure-activity relationship (SAR) analysis disclosed, generally, an E-isomer exhibited higher antibacterial activity than the corresponding Z-isomer. An electron-withdrawing group on A-ring led to some decrease in activity, while on B-ring, a similar substitution provided higher activity.     

Biosynthesis of monoterpenes. Stereochemistry of the enzymatic cyclizations of geranyl pyrophosphate to (+)-alpha-pinene and (-)-beta-pinene

The conversion of geranyl pyrophosphate to (+)-alpha-pinene and to (-)-beta-pinene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)- and to (+)-(3S)-linalyl pyrophosphate, respectively, and the subsequent cyclization of the anti, endo-conformer of these bound intermediates by mirror-image sequences which should result in the net retention of configuration at C1 of the geranyl precursor. Incubation of (1R)-[2-14C,1-3H]- and (1S)-[2-14C,1-3H]geranyl pyrophosphate with (+)-pinene cyclase and with (-)-pinene cyclase from common sage (Salvia officinalis) gave labeled (+)-alpha- and (-)-beta-pinene of unchanged 3H/14C ratio in all cases, and the (+)- and (-)-olefins were stereoselectively converted to (+)- and (-)-borneol, respectively, which were oxidized to the corresponding (+)- and (-)-isomers of camphor, again without change in isotope ratio. The location of the tritium was determined in each case by stereoselective, base-catalyzed exchange of the exo-alpha-hydrogens of these derived ketones. The results indicated that the configuration at C1 of the substrate was retained in the enzymatic transformations to the (+)- and (-)-pinenes, which is entirely consistent with the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate, transoid to cisoid rotation, and anti, endo-cyclization of the latter. The absolute stereochemical elements of the antipodal reaction sequences were confirmed by the selective enzymatic conversions of (3R)- and (3S)-1Z-[1-3H]linalyl pyrophosphate to (+)-alpha-pinene and (-)-beta-pinene, respectively, and by the location of the tritium in the derived camphors as before. The summation of the results fully defines the overall stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal pinenes.     

Biosynthesis of monoterpenes. Enantioselectivity in the enzymatic cyclization of (+)- and (-)-linalyl pyrophosphate to (+)- and (-)-pinene and (+)- and (-)-camphene

Cyclase I from Salvia officinalis leaf catalyzes the conversion of geranyl pyrophosphate to the stereo-chemically related bicyclic monoterpenes (+)-alpha-pinene and (+)-camphene and to lesser quantities of monocyclic and acyclic olefins, whereas cyclase II from this plant tissue converts the same acyclic precursor to (-)-alpha-pinene, (-)-beta-pinene and (-)-camphene as well as to lesser amounts of monocyclics and acyclics. These antipodal cyclizations are considered to proceed by the initial isomerization of the substrate to the respective bound tertiary allylic intermediates (-)-(3R)- and (+)-(3S)-linalyl pyrophosphate. [(3R)-8,9-14C,(3RS)-1E-3H]Linalyl pyrophosphate (3H:14C = 5.14) was tested as a substrate with both cyclases to determine the configuration of the cyclizing intermediate. This substrate with cyclase I yielded alpha-pinene and camphene with 3H:14C ratios of 3.1 and 4.2, respectively, indicating preferential, but not exclusive, utilization of the (3R)-enantiomer. With cyclase II, the doubly labeled substrate gave bicyclic olefins with 3H:14C ratios of from 13 to 20, indicating preferential, but not exclusive, utilization of the (3S)-enantiomer in this case. (3R)- and (3S)-[1Z-3H]linalyl pyrophosphate were separately compared to the achiral precursors [1-3H]geranyl pyrophosphate and [1-3H]neryl pyrophosphate (cis-isomer) as substrates for the cyclizations. With cyclase I, geranyl, neryl, and (3R)-linalyl pyrophosphate gave rise exclusively to (+)-alpha-pinene and (+)-camphene, whereas (3S)-linayl pyrophosphate produced, at relatively low rates, the (-)-isomers. With cyclase II, geranyl, neryl, and (3S)-linalyl pyrophosphate yielded exclusively the (-)-isomer series, whereas (3R)-linalyl pyrophosphate afforded the (+)-isomers at low rates. These results are entirely consistent with the predicted stereochemistries and additionally revealed the unusual ability of these enzymes to catalyze antipodal cyclizations when presented with the unnatural linalyl enantiomer.     

Anthocyanidin synthase from Gerbera hybrida catalyzes the conversion of (+)-catechin to cyanidin and a novel procyanidin

Anthocyanidins were proposed to derive from (+)-naringenin via (2R,3R)-dihydroflavonol(s) and (2R,3S,4S)-leucocyanidin(s) which are eventually oxidized by anthocyanidin synthase (ANS). Recently, the role of ANS has been put into question, because the recombinant enzyme from Arabidopsis exhibited primarily flavonol synthase (FLS) activity with negligible ANS activity. This and other studies led to the proposal that ANS as well as FLS may select for dihydroflavonoid substrates carrying a "beta-face" C-3 hydroxyl group and initially form the 3-geminal diol by "alpha-face" hydroxylation. Assays with recombinant ANS from Gerbera hybrida fully supported the proposal and were extended to catechin and epicatechin isomers as potential substrates to delineate the enzyme specificity. Gerbera ANS converted (+)-catechin to two major and one minor product, whereas ent(-)-catechin (2S,3R-trans-catechin), (-)-epicatechin, ent(+)-epicatechin (2S,3S-cis-epicatechin) and (-)-gallocatechin were not accepted. The K(m) value for (+)-catechin was determined at 175 microM, and the products were identified by LC-MS(n) and NMR as the 4,4-dimer of oxidized (+)-catechin (93%), cyanidin (7%) and quercetin (trace). When these incubations were repeated in the presence of UDP-glucose:flavonoid 3-O-glucosyltransferase from Fragariaxananassa (FaGT1), the product ratio shifted to cyanidin 3-O-glucoside (60%), cyanidin (14%) and dimeric oxidized (+)-catechin (26%) at an overall equivalent rate of conversion. The data appear to identify (+)-catechin as another substrate of ANS in vivo and shed new light on the mechanism of its catalysis. Moreover, the enzymatic dimerization of catechin monomers is reported for the first time suggesting a role for ANS beyond the oxidation of leucocyanidins.     

Biosynthesis of monoterpenes: stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to camphane and isocamphane monoterpenes

The conversion of geranyl pyrophosphate to (+)-bornyl pyrophosphate and (+)-camphene is considered to proceed by the initial isomerization of the substrate to (-)-(3R)-linalyl pyrophosphate and the subsequent cyclization of this bound intermediate. In the case of (-)-bornyl pyrophosphate and (-)-camphene, isomerization of the substrate to the (+)-(3S)-linalyl intermediate precedes cyclization. The geranyl and linalyl precursors were shown to be mutually competitive substrates (inhibitors) of the relevant cyclization enzymes isolated from Salvia officinalis (sage) and Tanacetum vulgare (tansy) by the mixed substrate analysis method, demonstrating that isomerization and cyclization take place at the same active site. Incubation of partially purified enzyme preparations with (3R)-[1Z-3H]linalyl pyrophosphate plus [1-14C]geranyl pyrophosphate gave rise to double-labeled (+)-bornyl pyrophosphate and (+)-camphene, whereas incubation of enzyme preparations catalyzing the antipodal cyclizations with (3S)-[1Z-3H]-linalyl pyrophosphate plus [1-14C]geranyl pyrophosphate yielded double-labeled (-)-bornyl pyrophosphate and (-)-camphene. Each product was then transformed to the corresponding (+)- or (-)-camphor without change in the 3H:14C isotope ratio, and the location of the tritium label was deduced in each case by stereoselective, base-catalyzed exchange of the exo-alpha-hydrogen of the derived ketone. The finding that the 1Z-3H of the linalyl precursor was positioned at the endo-alpha-hydrogen of the corresponding camphor in all cases, coupled to the previously demonstrated retention of configuration at C1 of the geranyl substrate in these transformations, confirmed the syn-isomerization of geranyl pyrophosphate to linalyl pyrophosphate and the cyclization of the latter via the anti,endo- conformer. These relative stereochemical elements, in combination with the observed enantiospecificities of the enzymes for the linalyl intermediates, allows definition of the overall absolute stereochemistry of the coupled isomerization and cyclization of geranyl pyrophosphate to the antipodal camphane (bornane) and isocamphane monoterpenoids.     

Synthesis, in vitro pharmacology, and pharmacokinetic profiles of 2-[1-amino-1-carboxy-2-(9H-xanthen-9-yl)-ethyl]-1-fluorocyclopropanecarboxylic acid and its 6-heptyl ester, a potent mGluR2 antagonist

In this paper, we describe the synthesis of (+)-(1R( *),2R( *))-2-[(1S( *))-1-amino-1-carboxy-2-(9H-xanthen-9-yl)-ethyl]-1-fluorocyclopropanecarboxylic acid (+)-16a, a compound, that is, fluorinated at the alpha position of the carboxylic acid in the cyclopropane ring of a group II mGluRs antagonist, 1 (LY341495), using a previously reported stereoselective cyclopropanation reaction. The fluorinated compound (+)-16a exhibited almost the same affinity (IC(50)=3.49 nM) for mGluR2 as 1 but had a superior pharmacokinetic profile. Furthermore, a marked elevation of the plasma levels of (+)-16a was observed following the administration of a prodrug, (+)-17.     

Gluconeogenesis by isolated guinea-pig liver parenchymal cells

1. Guinea-pig hepatocytes were prepared by collagenase digestion of the perfused liver. 2. The highest rates of gluconeogenesis were obtained from fructose, followed by pyruvate, xylitol and lactate, glycerol and propionate in that order. Maximum rates of gluconeogenesis were attained at 6-10mm substrate. 3. An initial 15-min lag period occurred during gluconeogenesis from lactate. This lag was abolished by preincubating the cells or by preincubation plus the addition of NH(4)Cl or lysine. 4. The lactate/pyruvate and 3-hydroxybutyrate/acetoacetate ratios were increased during the lag and adjusted to values favouring rapid gluconeogenesis from lactate after 15min. 5. The data suggest that the low glucose synthesis during the lag resulted from a limitation of the glutamate-aspartate shuttle and from the unusual redox state of the NAD(+) couple prevailing during this period. 6. At 0.1mm, amino-oxyacetate, a transaminase inhibitor, decreased gluconeogenesis from lactate by 80%, but had a negligible effect on glucose production from pyruvate. Gluconeogenesis from lactate was also inhibited (20%) by 10mm-dl-3-hydroxybutyrate.