The stereoselectivity of the Clostridium perfringens phospholipase C: hydrolysis of thiophosphate analogs of phosphatidylcholine

Thiophosphate containing analogs of phosphatidylcholine have been synthesized with varying degrees of structural complexity. These analogs have been used in a continuous spectrophotometric assay for phospholipase C from Clostridium perfringens in order to examine the requirement for substrate ester functionalities and the stereoselectivity of the enzyme. The substrate analogs with ester groups in the nonpolar portion of the molecule were acceptable substrates for phospholipase C, while those analogs without ester functionalities were not hydrolyzed. Substrate analogs with chiral centers were resolved using the stereospecificity of phospholipase A2 from Crotalus atrox venom. These resolved substrates were used to study the biphasic hydrolytic time courses observed when rac-dioctanoylphosphatidylthiocholine was used as substrate. The "naturally occurring" enantiomer with R absolute configuration was rapidly hydrolyzed in the presence of phospholipase C while the "nonnaturally occurring" enantiomer with S configuration was slowly hydrolyzed only after a long induction or "lag" period. The selectivity for the R enantiomer over the S enantiomer can be lessened by altering the composition of the substrate micelles resulting in accelerated rates of hydrolysis of the S enantiomer.     

Mechanism and stereochemical course at phosphorus of the reaction catalyzed by a bacterial phosphotriesterase

The reaction mechanism for the phosphotriesterase from Pseudomonas diminuta has been examined. When paraoxon (diethyl 4-nitrophenyl phosphate) is hydrolyzed by this enzyme in oxygen-18-labeled water, the oxygen-18 label is found exclusively in the diethyl phosphate product. The absolute configurations for the (+) and (-) enantiomers of O-ethyl phenylphosphonothioic acid have been determined by X-ray diffraction structural determination of the individual crystalline 1-phenylethylamine salts. The (+) enantiomer of the free acid corresponds to the RP configuration. The RP enantiomer of O-ethyl phenylphosphonothioic acid has been converted to the SP enantiomer of EPN [O-ethyl O-(4-nitrophenyl) phenylphosphonothioate]. (SP)-EPN is hydrolyzed by the phosphotriesterase to the SP enantiomer of O-ethyl phenylphosphonothioic acid. The enzymatic reaction therefore proceeds with inversion of configuration. These results have been interpreted as an indication of a single in-line displacement by an activated water molecule directly at the phosphorus center of the phosphotriester substrate. (RP)-EPN is not hydrolyzed by the enzyme at an appreciable rate.     

Synthetic phospholipids as substrates for phospholipase C from Bacillus cereus.

The substrate requirement of phospholipids for hydrolysis with phospholipase C from Bacillus cereus was studied with synthetic lipids well-defined in structure and configuration. For optimal activity, the glycerol molecule must contain three substituents: phosphocholine in sn-3-, an ester bond in sn-2- and an ether- or ester bond in sn-1-position. The length of the ester or ether chains is of minor importance. Any deviation from these structural requirements results in a large decrease in the hydrolysis rate. These essential structural and configurational elements for optimal activity for the B. cereus enzyme are perfectly combined in the platelet activating factor, 1-O-hexadecyl-2-acetyl-sn-glycero-3- phosphocholine. This molecule is one of the best substrates for hydrolysis with the bacterial phospholipase C.     

(2R,3S)-(+)- and (2S,3R)-(-)-Halofuginone lactate: synthesis, absolute configuration, and activity against Cryptosporidium parvum

The trans-enantiomers of the commercially important anti-protozoal compound Halofuginone have been prepared and characterized, and the absolute configuration was assigned by X-ray crystallography. The activity of both enantiomers against Cryptosporidium parvum was determined in vitro and related to acute toxicity in vivo. It was shown that both the activity and the toxicity are properties of the (2R,3S)-enantiomer. We conclude that with respect to broadening the therapeutic window there is no advantage in application of one enantiomer over the application of the racemic mixture in the treatment of C. parvum infections.     

Cyclopentanoid analogs of phosphatidylcholine: susceptibility to phospholipase A2

Six isomers of dipalmitoylcyclopentanetriol phosphocholine (cyclopentano-lecithin) were tested as potential substrates for phospholipase A2. Since each of these analogs possesses a configuration that mimics a narrow range of conformations of a glycerophospholipid molecule, the analogs were used to assess the enzyme's conformational requirements. Studies showed that all of the analogs containing the phosphocholine at the C-1 (or C-3) position could be hydrolyzed, while only one of the three analogs that contains the polar head group at the C-2 position was susceptible. Kinetic studies, however, revealed that only the all-trans-(1,3/2-1P)-cyclopentano-lecithin gave initial rates of hydrolysis that were measurable by pH-stat. Acyl group specificity of the enzyme towards the all-trans isomer was determined with an analog was acyl groups were distinguishable. The synthesis of this mixed-acid-cyclopentano-PC is described herein. When this analog was enzymatically assayed, results unequivocally showed the enzyme to be specific for C-2 acyl hydrolysis. This specificity, and data showing that the all-trans analog is stereospecifically hydrolyzed, indicate that it is acted on in an analogous manner to dipalmitoylphosphatidylcholine. These studies indicate that although the configuration of the analog is not necessarily a prerequisite for hydrolysis, there does appear to be an optimal spatial orientation for enzymatic activity. The analogy between the susceptibilities of all-trans-(1,3/2-1P)-cyclopentano-lecithin and glycero-lecithin suggests that the conformation of the glycero-lecithin during phospholipase A2-mediated hydrolysis may be best simulated by the all-trans orientation of C-O bonds in the artificial substrate.     

Absolute configuration of hydroxycitric acid produced by microorganisms

Optical resolution for (2S,3R) and (2R,3S)-hydroxycitric acid (HCA) enantiomers was developed using chiral column chromatography. HCA from Bacillus megaterium G45C and Streptomyces sp. U121, newly isolated in our previous study, was analyzed to determine the absolute configuration. These results indicate that both strains generate optically pure (2S,3R)-hibiscus type HCA enantiomer.     

Therapeutic agent for treating dysuria (NC-1800): absolute configuration of the enantiomers

The absolute configuration of an enantiomer of the title compound, (+/-)-1. fumaric acid salt, was determined by X-ray crystallographic analysis of (+)-1 [salt of (+)-tartaric acid [(+)-2]]. This salt for X-ray crystallographic analysis was prepared by a simple method. The analysis of (+)-1 [salt of (+)-2] showed that this enantiomer has the 5-S and 1-R absolute configuration. The final R and Rw values were 0.0614 and 0.0713, respectively.     

Stereoselective metabolism of anthracene and phenanthrene by the fungus Cunninghamella elegans.

The fungus Cunninghamella elegans oxidized anthracene and phenanthrene to form predominately trans-dihydrodiols. The metabolites were isolated by reversed-phase high-pressure liquid chromatography for structural and conformational analyses. Comparison of the circular dichroism spectrum of the fungal trans-1,2-dihydroxy-1,2-dihydroanthracene to that formed by rat liver microsomes indicated that the major enantiomer of the trans-1,2-dihydroxy-1,2-dihydroanthracene formed by C. elegans had an S,S absolute stereochemistry, which is opposite to the predominately 1R,2R dihydrodiol formed by rat liver microsomes. C. elegans oxidized phenanthrene primarily in the 1,2-positions to form trans-1,2-dihydroxy-1,2-dihydrophenanthrene. In addition, a minor amount of trans-3,4-dihydroxy-3,4-dihydrophenanthrene was detected. Metabolism at the K-region (9,10-positions) of phenanthrene was not detected. Comparison of the circular dichroism spectra of the phenanthrene trans-1,2- and trans-3,4-dihydrodiols formed by C. elegans to those formed by mammalian enzymes indicated that each of the dihydrodiols formed by C. elegans had an S,S absolute configuration. The results indicate that there are differences in both the regio- and stereoselective metabolism of anthracene and phenanthrene between the fungus C. elegans and rat liver microsomes.     

N-glycohydrolysis of adenosine diphosphoribosyl arginine linkages by dinitrogenase reductase activating glycohydrolase (activating enzyme) from Rhodospirillum rubrum

The reaction catalyzed by the activating enzyme for dinitrogenase reductase from Rhodospirillum rubrum has been studied using an ADP-ribosyl hexapeptide, obtained from proteolysis of inactive dinitrogenase reductase, and synthetic analogs such as N alpha-dansyl-N omega-ADP-ribosylarginine methyl ester. The activating enzyme catalyzed N-glycohydrolysis of the ribosyl-guanidinium linkage releasing ADP-ribose and regenerating an unmodified arginyl guanidinium group. Optimal glycohydrolysis of the low molecular weight substrates occurred at pH 6.6 and required 1 mM MnCl2, but did not require ATP. The ADP-ribosyl hexapeptide (Km 11 microM), N alpha-dansyl-N omega-ADP-ribosylarginine methyl ester (Km 12 microM), N alpha-dansyl-N omega-ADP-ribosylarginine (Km 12 microM), N alpha-dansyl-N omega-1,N6-etheno-ADP-ribosylarginine methyl ester (Km 11 microM), and N alpha-dansyl-N omega-GDP-ribosylarginine methyl ester (Km 11 microM) were comparable substrates. N omega-ADP-ribosylarginine (Km 2 mM) was a poor substrate, and the activating enzyme did not catalyze N-glycohydrolysis of N alpha-dansyl-N omega-5'-phosphoribosylarginine methyl ester or N alpha-dansyl-N omega-ribosylarginine methyl ester. 13C NMR of N alpha-tosyl-N omega-ADP-ribosylarginine methyl ester established that the activating enzyme specifically hydrolyzed the alpha-ribosyl-guanidinium linkage. The beta-linked anomer was hydrolyzed only after anomerization to the alpha configuration. We recommend [arginine(N omega-ADP-alpha-ribose)]dinitrogenase reductase N-glycohydrolase (dinitrogenase reductase activating) and dinitrogenase reductase activating glycohydrolase as the systematic and working names for the activating enzyme.     

Synthesis and dopaminergic properties of the two enantiomers of 3-(3,4-dimethylphenyl)-1-propylpiperidine, a potent and selective dopamine D4 receptor ligand

The synthesis of the two enantiomers of 3-(3,4-dimethylphenyl)-1-propylpiperidine 1, a potent and selective D4 dopaminergic ligand, was performed. The 3-(3,4-dimethylphenyl)- 1-propylpiperidine with the R configuration showed an affinity for the D4 receptors 6-fold higher than the corresponding enantiomer with the S configuration. Furthermore, the (R)-1 enantiomer proved to be highly selective for D4 receptors with respect to D2-D3 receptors, with a Ki ratio higher than 25,000, while the (S)-1 enantiomer was about 100-fold less selective than the (R)-1 one.     

Absolute configuration of tropane alkaloids bearing two alpha,beta-unsaturated ester functions using electronic CD spectroscopy: application to (R,R)-trans-3-hydroxysenecioyloxy-6-senecioyloxytropane

The absolute configuration of heterocyclic natural products substituted with two mobile alpha,beta-unsaturated esters was studied using electronic circular dichroism (CD) spectroscopy. The conformational flexibility of the side chains imposed the use of density functional theory calculation to determine the set of the most probable conformations in solution. The electronic CD and UV spectra were calculated by Boltzmann-weighted average of the simulated spectra using the results of the excited states calculation of a set of simplified structures. Comparison with the experimental CD spectrum allowed to determine whether the calculations were made with the right enantiomer. The method was applied to the determination of the absolute configuration of (R,R)-trans-3-hydroxysenecioyloxy-6-senecioyloxytropane.     

Analogs of Substance P modified at the C-terminus which are both agonist and antagonist of the NK-1 receptor depending on the second messenger pathway.

The initial goal of this study was to analyze, using photolabeling, the interactions between Substance P and its tachykinin NK-1 receptor. Therefore, the photoreactive amino acid para-benzoyl-phenylalanine (pBzl)Phe was incorporated into the Substance P sequence from position 4 to 11 leading to Bapa0[(pBzl)Phex]SP analogs. Biotinyl sulfone-5-aminopentanoic acid (Bapa) was introduced in order to purify the covalent complex. These photoreactive SP analogs were first assayed for their affinity for the two binding sites associated with the NK-1 receptor, as well as for their potency in activating the phospholipase C and adenylate cyclase pathways. All analogs photoreactive from position 4 to 11 have moderate to high affinity for the two NK-1 receptor-binding sites, except for the analog modified at position 7. This affinity could be correlated to their potency to activate the phospholipase C and adenylate cyclase pathways, except for the analog photoreactive at position 11. Bapa0[(pBzl)Phe11]SP was found to be an agonist in the phospholipase C pathway and an antagonist in the adenylate cyclase pathway, other analogs modified at position 11 were therefore analyzed. Among these, Bapa0[Pro9, (pBzl)Hcy(O2)11]SP is a partial agonist, whereas Bapa0[Hcy(ethylaminodansyl)11]SP is a full agonist in the phospholipase C pathway, the two analogs being antagonist in the adenylate cyclase pathway. These results show that analogs of SP can be simultaneously agonist at one binding site and antagonist at the other binding site associated with the NK-1 receptor.     

Stereochemical and positional specificity of the lipase/acyltransferase produced by Aeromonas hydrophila.

Aeromonas species secrete a glycerophospholipid-cholesterol acyltransferase (GCAT) which shares many properties with mammalian plasma lecithin-cholesterol acetyltransferase (LCAT). We have studied the stereochemical and positional specificity of GCAT against a variety of lipid substrates using NMR spectroscopy as well as other assay methods. The results show that both the primary and secondary acyl ester bonds of L-phosphatidylcholine can be hydrolyzed but only the sn-2 fatty acid can be transferred to cholesterol. The enzyme has an absolute requirement for the L configuration at the sn-2 position of phosphatidylcholine. The secondary ester bond of D-phosphatidylcholine cannot be hydrolyzed, and this lipid is not a substrate for acyl transfer. In contrast to the phospholipases, but similar to LCAT, the enzyme does not interact stereochemically with the phosphorus of phosphatidylcholine. In fact, the phosphorus is not required for enzyme activity, as GCAT will also hydrolyze monolayers of diglyceride, although at much lower rates.     

Enantiomers of a nonylphenol isomer: absolute configurations and estrogenic potencies

Enantiomers of 4-(1,1,2-trimethylhexyl)phenol, a chiral isomer of the endocrine disrupting chemical nonylphenol, have been resolved and isolated by preparative chiral HPLC. The absolute configurations of the enantiomers were then determined by an X-ray crystallographic study of the (-)-camphanoyl derivative of the first eluted enantiomer NP(35)E1. The first enantiomer (NP(35)E1) and the second enantiomer (NP(35)E2) eluted were found to have the S and R absolute configurations, respectively. The estrogenic potencies of the S and R enantiomers were tested by the E-screen assay. A slight difference was observed in the relative proliferative effect between the S enantiomer and R enantiomer in the E-screen assay.     

The dirhodium-method in the determination of absolute configurations of phospholene chalcogenides

The 1H, 13C, and 31P NMR signals of six chiral phospholene chalcogenides (X = O, S, Se) are duplicated in the presence of one mole equivalent of the chiral auxiliary Rh2[(R)-MTPA]4 (diastereomeric dispersion Deltanu; in Hz). The samples were investigated as nonracemic mixtures of enantiomers with known absolute configurations so that signs can be attributed to the Deltanu-values and each signal set can be assigned to the respective enantiomer. The signs are uniform--in particular those of 1H nuclei--and nearly independent of the nature of the chalcogen atom. Thus, if the absolute configuration of one compound is known, it is possible to derive absolute configurations in the whole series (correlation method).     

X-ray structure determination of mexiprostil, a new gastroprotective 16-methoxy-16-methyl-PGE1 analogue

Mexiprostil is a new gastroprotective 16-methoxy-16-methyl-PGE1 methyl ester. To assign the absolute configuration at C-15, a crystalline high-melting C-1 ester analog 5 11,15-dihydroxy-16-methoxy-16-methyl-9-oxoprost-13-en-1-oic acid 4-(4-bromobenzamide)phenyl ester (15R, 16R) was prepared and submitted to single crystal X-ray analysis. Since C-8, C-11, C-12 and C-16 are shown to have R configurations, the X-ray diffraction results established that the configuration at C-15 is also R.     

Activities of native and tyrosine-69 mutant phospholipases A2 on phospholipid analogues. A reevaluation of the minimal substrate requirements

The role of Tyr-69 of porcine pancreatic phospholipase A2 in substrate binding was studied with the help of proteins modified by site-directed mutagenesis and phospholipid analogues with a changed head-group geometry. Two mutants were used containing Phe and Lys, respectively, at position 69. Modifications in the phospholipids included introduction of a sulfur at the phosphorus (thionophospholipids), removal of the negative charge at phosphorus (phosphatidic acid dimethyl ester), and reduction (phosphonolipids) or extension (diacylbutanetriol choline phosphate) of the distance between the phosphorus and the acyl ester bond. Replacement of Tyr-69 by Lys reduces enzymatic activity, but the mutant enzyme retains both the stereospecificity and positional specificity of native phospholipase A2. The Phe-69 mutant not only hydrolyzes the Rp isomer of thionophospholipids more efficiently than the wild-type enzyme, but the Sp thiono isomer is hydrolyzed too, although at a low (approximately 4%) rate. Phosphonolipids are hydrolyzed by native phospholipase A2 about 7 times more slowly than natural phospholipids, with retention of positional specificity and a (partial) loss of stereospecificity. The dimethyl ester of phosphatidic acid is degraded efficiently in a calcium-dependent and positional-specific way by native phospholipase A2 and by the mutants, indicating that a negative charge at phosphorus is not an absolute substrate requirement. The activities on the phosphatidic acid dimethyl ester of native enzyme and the Lys-69 mutant are lower than those on the corresponding lecithin, in contrast to the Phe-69 mutant, which has equal activities on both substrates.(ABSTRACT TRUNCATED AT 250 WORDS)     

A versatile method for functionalization and grafting of 2-hydroxyethyl cellulose (HEC) via Click chemistry

This article describes a versatile method for the modification of 2-hydroxyethyl cellulose (HEC) involving azide-alkyne cycloaddition reaction to impart neutral (ester) and ionic (carboxylic acid and 1(ry) amine) functionalities. The synthetic approach involved, first the introduction of the azide functionality to HEC and then followed by its cycloaddition reaction with several alkyne terminated compounds: namely ethyl propiolate, 5-hexynoic acid and propargyl amine. Sequential Click reactions were also demonstrated to be feasible by the successful synthesis of polydimethylsiloxane (PDMS) grafted HEC containing neutral (ester) and ionic (carboxylic acid and 1(ry) amine) functionalities. The Click chemistry was then further utilized similarly to graft poly(lactic acid) (PLA) and poly(ethylene glycol) (PEG) segments to HEC to access its hydrophobic and hydrophilic analogs, respectively. AFM analysis revealed that while HEC itself formed uniform oval features, the PLA grafted HEC exhibited a brushlike architecture. The formation of these brushlike structures suggested that the HEC backbone exhibits an extended conformation with the side chains stretched out. The resulting polymeric materials were characterized by solution and solid state (13)C NMR and FTIR spectroscopy.     

Influence of the phospholipid structure on the stability of liposomes in serum

The effect of serum on the structural integrity of liposomes consisting of ether and/or carbamyl analogs of 1,2-diester phosphatidylcholine (PC) has been evaluated by measuring both the efflux of the entrapped 6-carboxyfluorescein and the lipid transfer to serum proteins, and the results have been compared with the egg PC liposomes. Replacement of the C-1 ester bond in PC by an ether linkage did not significantly enhance the liposome stability, but it was markedly increased upon introducing further structural changes in the C-2 ester region of the resulting 1-ether-2-ester PC. However, the stability was not influenced by altering the steric configuration of the latter phospholipid. These results strongly suggest that lysis of liposomes in serum can be prevented by structurally modifying the ester bond(s) in the phospholipid component of liposomes.     

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.