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.     

Facile entry to (-)-(R)- and (+)-(S)-mexiletine

The title compounds, 1a and 1b, have been synthesized in a three-step sequence starting from (-)-(S) and (+)-(R)-propylene oxide, respectively, in acceptable overall yields. The enantiomeric excess values for 1a and 1b were 96% and 93% respectively, as assessed by HPLC analysis on a chiral stationary phase of the corresponding N-acetyl derivatives. The synthetic route herein presented may represent a facile entry to highly enriched mexiletine enantiomers, alternative to those previously reported in the literature.     

Biomimetic synthesis of acid-sensitive (-)- and (+)-caparrapi oxides, (-)- and (+)-8-epicaparrapi oxides, and (+)-dysifragin induced by artificial cyclases

Asymmetric total syntheses of acid-sensitive (-)- and (+)-caparrapi oxides (1) and (+)-8-epicaparrapi oxide (2) from farnesol (10) are achieved using Sharpless-Katsuki epoxidation and Lewis acid-assisted chiral Br?nsted acid (chiral LBA)-induced polyene cyclization as key steps. The relative configuration of (+)-dysifragin (4) is determined by a single-crystal X-ray diffraction and its total synthesis is accomplished by the diastereoselective epoxidation of (+)-1. Furthermore, (-)-1 can be directly synthesized from (S)-nerolidol (3) and (R)-LBA with 88% ds by reagent control, which overcame substrate control, while (-)-2 is obtained from (R)-3 and (R)-LBA with >99% ds by the double asymmetric induction.     

Syntheses of (+)- and (-)-dihydropinidine and (+)- and (-)-epidihydropinidine by using yeast reduction of methyl (2-oxocyclohexyl)acetate

(+) and (-)-Dihydropinidine and (+)- and (-)-epidihydropinidine were synthesized from hydroxy esters 1 and 2 which had been prepared by yeast reduction of methyl (2-oxocyclohexyl)acetate. The enantiomeric excess at the C-1 positions of 1 and 2 were both determined as more than 99% ee.     

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.     

Phosphorylation of the salivary Na(+)-K(+)-2Cl(-) cotransporter

Westudied the phosphorylation of the secretoryNa⁺-K⁺-2Cl cotransporter (NKCC1)in rat parotid acinar cells. We have previously shown that NKCC1activity in these cells is dramatically upregulated in response to-adrenergic stimulation and that this upregulation correlates withNKCC1 phosphorylation, possibly due to protein kinase A (PKA). We showhere that when ATP is added to purified acinar basolateral membranes(BLM), NKCC1 is phosphorylated as a result of membrane-associatedprotein kinase activity. Additional NKCC1 phosphorylation is seen whenPKA is added to BLMs, but our data indicate that this is due to aneffect of PKA on endogenous membrane kinase or phosphatase activities,rather than its direct phosphorylation of NKCC1. Also, phosphopeptidemapping demonstrates that these phosphorylations do not take place atthe site associated with the upregulation of NKCC1 by -adrenergicstimulation. However, this upregulatory phosphorylation can be mimickedby the addition of cAMP to permeabilized acini, and this effect can beblocked by a specific PKA inhibitor. These latter results provide good evidence that PKA is indeed involved in the upregulatoryphosphorylation of NKCC1 and suggest that an additional factor presentin the acinar cell but absent from isolated membranes is required to bring about the phosphorylation.

     

Formation of glycine conjugate and (-)-(R)-enantiomer from (+)-(S)-2-phenylpropionic acid suggesting the formation of the CoA thioester intermediate of (+)-(S)-enantiomer in dogs.

It has been proposed that the chiral inversion of the 2-arylpropionic acids is due to the stereospecific formation of the (-)-R-profenyl-CoA thioesters which are putative intermediates in the inversion. Accordingly, amino acid conjugation, for which the CoA thioesters are obligate intermediates, should be restricted to those optical forms which give rise to the (-)-R-profenyl-CoA, i.e., the racemates and the (-)-(R)-isomers. We have examined this problem in dogs with respect to 2-phenylpropionic acid(2-PPA). Regardless of the optical configuration of 2-phenylpropionic acid administered, the glycine conjugate was the major urinary metabolite and this was shown to be exclusively the (+)-(S)-enantiomer by chiral HPLC. Both (-)-(R)- and (+)-(S)-2-phenylpropionic acid were present in plasma after the administration of either antipode, and further evidence of the chiral inversion of both enantiomers was provided by the presence of some 25% of the opposite enantiomer in the free 2-phenylpropionic acid and its glucuronide excreted in urine after administration of (-)-(R)- and (+)-(S)-2-phenylpropionic acid. The (+)-(S)-enantiomer underwent chiral inversion to the (-)-(R)-antipode when incubated with dog hepatocytes. These data suggests that both enantiomers of 2-phenylpropionic acid are substrates for canine hepatic acyl CoA ligase(s) and thus undergo chiral inversion, but that the CoA thioester of only (+)-(S)-2-phenylpropionic acid is a substrate for the glycine N-acyl transferase. These studies are presently being extended to the structure and species specificity of the reverse inversion and amino acid conjugation of profen NSAIDs.     

Synthesis and Biological Properties of (+) and (-)-(E)-5-(2-bronovinyl)-21 -deoxy-11 a-Carbauridine

Abstract

Carbocyclic (+)- and (-)-(E)-5- (2-bromovinyl)-2′-deoxyuridlne have been prepared from (+)- and (-)-endo-norborn-5-en-2-y1 butyrate. In cell cultures both (+)- and (-)-C-BVDU showed activity against herpes simplex virus types 1 and 2, (+)-C-BVDU being only slightly less active than BVDU itself. (-)-C-BVDU gave a smaller but still significant antiviral effect. A nomenclature for carbocyclic nucleosides is proposed.     

(+)-3-(3-hydroxyphenyl)-N-(1-propyl)piperidine [(+)-3-PPP] but not (-)-3-PPP produces (+)-N-allylnormetazocine-like (SKF 10,047) discriminative stimuli

In rats trained to discriminate the prototypic sigma receptor agonist, (+)-N-Allylnormetazocine [(+)-N-Allylnormetazocine [(+)-NANM/SKF 10,047], from saline, the (+)- but not the (-)-isomer of 3-(3-hydroxyphenyl)-N-(1-propyl)piperidine (3-PPP) produced (+)-NANM-like discriminative stimuli. (+)-3-PPP binds stereo selectively to the (+)-NANM binding site, but not to the phencyclidine binding site. Additionally, phencyclidine was found to produce (+)-NANM-like discriminative stimuli. Although the 3-PPP isomers were shown to produce changes in central dopaminergic activity (Hjorth et al. Life Sci 37, 673, 1985), the discriminative stimulus properties of (+)-3-PPP are apparently not mediated via the dopaminergic system. This hypothesis is supported by the fact that apomorphine did not produce (+)-NANM-like discriminative stimuli. These stimuli are thus non-dopaminergic and may be due to the (+)-3-PPP actions at the sigma binding site. However, it is possible that (+)-NANM, PCP, and (+)-3-PPP may have common non-sigma pharmacologic properties that account for the similar discriminative stimulus properties of these compounds.     

Formation of psi (+) and psi (-) DNA

DNA molecules can be organized into ordered aggregates of opposite handedness by complexation with polylysine and other polypeptides; we have investigated the conditions under which ψ(+) and ψ(?) structures are produced with double-helical synthetic polynucleotides. Both poly(dGdC)·poly(dGdC) and poly(dAdT)·poly(dAdT) readily form ψ(?) structures with polylysine, although the method of preparation can alter the CD spectra. The GC copolymer, which is more susceptible to conversion into A or Z conformers, forms ψ(+) structures with lysine–alanine copolypeptides more readily than the AT copolymer. Nucleotide base modifications that favor the Z structure, such as bromination and methylation, also favor ψ(+) formation, and the Co(NH₃)₆Cl₃ reagent that readily induces the Z structure also leads to ψ(+). Thus, the production of the ψ(+) structure seems to be frequently correlated with susceptibility to A or Z formation, although there are some cases in which the B conformer also leads to ψ(+). Polyethylene glycol generally produces a ψ(?) structure; the differentiation between ψ(+) and ψ(?) structures seems to require electrically charged polymers.     

Comparative toxicity of (+)-(R)- and (−)-(S)-1,2-dibromo-3-chloropropane

The haloalkane 1,2-dibromo-3-chloropropane (DBCP), an environmental pollutant that was widely used as a soil fumigant, is a carcinogen and a mutagen and displays target-organ toxicity to the testes and the kidneys. Because little is known about effects of stereochemistry on the metabolism and toxicity of halogenated alkyl compounds and because DBCP, which has a chiral center at C-2, may show enantioselectivity in its metabolism and/or toxicities, the optically pure enantiomers of DBCP were tested in vivo in rats for organ toxicity as well as for bacterial mutagenicity. Organ toxicity studies showed that (S)-DBCP was slightly more renal toxic than (R)-DBCP but was not significantly more toxic than the racemate, and that no significant differences were observed in the extents of testicular necrosis and atrophy caused by either enantiomer or the racemate. In contrast, (R)-DBCP was more mutagenic than either (S)-DBCP or the racemate to Salmonella typhimurium (S. typhimurium) strains TA 100 and TA104. However, there was little or no enantioselectivity in glutathione S-transferase (GST)-catalyzed conjugation reactions of glutathione with DBCP based on the lack of selectivity in the rates of disappearance of the enantiomers of DBCP in the presence of glutathione (GSH) and GSTs as monitored by chiral gas chromatography (GC). © 1995 Wiley-Liss, Inc.     

Asymmetric total synthesis of (+)- and (-)-clusianone and (+)- and (-)-clusianone methyl enol ether via ACC alkylation and evaluation of their anti-HIV activity

The total asymmetric synthesis of (+)- and (−)-clusianone and (+)- and (−)-clusianone methyl enol ether is reported. Asymmetric induction is achieved through the use of ACC alkylation, providing the key intermediates with an er of 99:1. The four synthetic compounds were evaluated for their anti-HIV activity. Both (+)- and (−)-clusianone displayed significant anti-HIV activity.     

Biotransformation of (+)- and (-)-camphorquinones by plant cultured cells

Biotransformation of (+)- and (-)-camphorquinones with suspension plant cultured cells of Nicotiana tabacum and Catharanthus roseus was investigated. It was found that the plant cultured cells of N. tabacum and C. roseus reduce stereoselectively the carbonyl group of (+)- and (-)-camphorquinones to the corresponding alpha-keto alcohols.     

New synthesis of (+)-(S)-halothane

A new synthesis of non-racemic halothane (1-bromo-1-chloro-2,2,2-trifluoroethane) is reported. The stereospecificity of the key reaction, decarboxylation of a salt of optically enriched 1-bromo-1-chloro-2,2,2-trifluoropropionic acid to give halothane, is shown to be highly dependent on the nature of the cation. When the cation is trialkylammonium, a high level of stereospecificity is observed. © 1996 Wiley-Liss, Inc.     

Enantiospecific synthesis of carbocyclic (+)-(E)-5-(2-bromovinyl)-2'-deoxyuridine

(+)-1-[(1R, 3S, 4R)-3-hydroxy-4-hydroxymethylcyclopentyl]-5-[(E)-2- bromovinyl]-1H,3H-pyrimidin-2,4-dione 10 was synthesized starting from (+)-endo-5-norbornen-2-yl acetate. This chiral educt was obtained by enzymatic hydrolysis of racemic esters of endo-5-norbornen-2-ol.     

Toxic doses of rac-, (−)-(S)- and (+)-(R)-propranolol in rats and rabbits

The contribution of the individual enantiomers ([+]-[R]- and [−]-[S]-propranolol) to rac-propranolol intoxication was studied in anaesthetized, spontaneously breathing (SB) rats and artificially ventilated (AV) rats and rabbits. In the SB rat, propranolol (30 mg.kg⁻¹.h⁻¹ i.v.) decreased heart rate and mean arterial blood pressure and caused hypoventilation, serious hypoxaemia, respiratory acidosis, and death by respiratory arrest. Survival time (ST) in the (+)-(R)-propranolol group (ST 91 ± 5 min) was significantly longer than in the rac-propranolol group (ST 68 ± 6 min). In AV rats and rabbits toxic doses of rac-, (−)-(S)- and (+)-(R)-propranolol, 30 mg.kg⁻¹.h⁻¹ and 15 mg.kg⁻¹.h⁻¹ i.v., respectively, induced comparable effects on haemodynamic variables as in the SB rat. Artificial ventilation lengthened ST by a factor of three to four in rats. In the AV rat, ST's were not significantly different between the rac-, (−)-(S)- and (+)-(R)-propranolol groups. In the rabbit, as in the SB rat, ST in the (+)-(R)-propranolol group was significantly longer than ST's in the rac- and (−)-(S)-propranolol groups. The acute respiratory acidosis in SB rats and the prolonged ST in AV rats suggest that respiratory failure is the primary and cardiovascular failure the secondary cause of death in propranolol intoxication. The potentiation of the toxic effect of the enantiomers observed after dosing the racemate instead of the pure enantiomers could not be explained by a stereoselective difference in plasma propanolol concentration. © 1996 Wiley-Liss, Inc.     

Dynamic regulation of Na(+)-K(+)-2Cl(-) cotransporter surface expression by PKC-{epsilon} in Cl(-)--secretory epithelia

In secretory epithelia, activation of PKC by phorbol ester and carbachol negatively regulates Cl^– secretion, the transport event of secretory diarrhea. Previous studies have implicated the basolateral Na⁺-K⁺-2Cl^– cotransporter (NKCC1) as a target of PKC-dependent inhibition of Cl^– secretion. In the present study, we examined the regulation of surface expression of NKCC1 in response to the activation of PKC. Treatment of confluent T84 intestinal epithelial cells with the phorbol ester 12-O-tetradecanoylphorbol-13-acetate (PMA) reduced the amount of NKCC1 accessible to basolateral surface biotinylation. Loss of cell surface NKCC1 was due to internalization as shown by 1) the resistance of biotinylated NKCC1 to surface biotin stripping after incubation with PMA and 2) indirect immunofluorescent labeling. PMA-induced internalization of NKCC1 is dependent on the -isoform of PKC as determined on the basis of sensitivity to a panel of PKC inhibitors. The effect of PMA on surface expression of NKCC1 was specific because PMA did not significantly alter the amount of Na⁺-K⁺-ATPase or E-cadherin available for surface biotinylation. After extended PMA exposure (>2 h), NKCC1 became degraded in a proteasome-dependent fashion. Like PMA, carbachol reduced the amount of NKCC1 accessible to basolateral surface biotinylation in a PKC--dependent manner. However, long-term exposure to carbachol did not result in degradation of NKCC1; rather, NKCC1 that was internalized after exposure to carbachol was recycled back to the cell membrane. PKC--dependent alteration of NKCC1 surface expression represents a novel mechanism for regulating Cl^– secretion. endocytosis; recycling; ion transporters     

Ca(2+)-dependent K(+) channels and Na(+)-K(+)-ATPase mediate H(2)O(2)- and superoxide-induced relaxations in canine trachealis.

We examined the ionic mechanisms underlying the responses of canine trachealis to superoxide (generated in vitro by using xanthine oxidase or added exogenously) and peroxide (generated spontaneously in vitro by the dismutation of superoxide or added exogenously). Although neither had any effect on resting tone, both triggered relaxations in carbachol-precontracted tissues. These relaxations were eliminated by catalase but were much less sensitive to the hydroxyl radical scavenger dimethylthiourea, indicating they were mediated primarily by peroxide. These relaxations were decreased in magnitude and/or slowed by nifedipine (10(-6) M), ouabain (10(-6) M), or tetraethylammonium (25 mM), but not by 4-aminopyridine (5 mM), and were small or absent in tissues precontracted with 30 mM KCl. Finally, peroxide triggered membrane hyperpolarization and elevated cytosolic concentration of Ca(2+) (primarily via release from the internal store). Thus peroxide-mediated relaxations seem to involve Ca(2+) release, opening of Ca(2+)-dependent K(+) channels, hyperpolarization, closure of Ca(2+) channels, and relaxation. In addition, some other free radical (hydroxyl radical?) may activate the Na(+)-K(+) pump, also hyperpolarizing the membrane and causing relaxation.