ARYL NUCLEOSIDE H-PHOSPHONATES AS A TOOL FOR INVESTIGATION OF STEREOSPECIFICITY DURING COUPLING

It was found that in stereoselective condensations of ribonucleoside 3′-H-phosphonates with alcohols, the major diastereomer of the produced H-phosphonate diesters is formed from the minor diastereomer of the intermediate phosphonic-pivalic anhydride.     

The stereochemical course of the reaction catalyzed by soluble bovine lung guanylate cyclase

The stereochemical course of the reaction catalyzed by the soluble form of bovine lung guanylate cyclase has been investigated using [alpha-18O]guanosine 5'-triphosphate (Rp diastereomer) and guanosine 5'-O-(1-thiotriphosphate) (Sp diastereomer) as substrates. The product from the 3-thiomorpholino-1',1'-dioxide sydnonimine-stimulated enzymatic cyclization of [alpha-18O] guanosine 5'-triphosphate was esterified with diazomethane. 31P NMR analysis of the triesters indicated that all of the 18O label was present in the axial position. Guanosine 5'-O-(1-thiotriphosphate) (Sp diastereomer) was cyclized under stimulated and basal enzyme activities and, in both cases, the Rp diastereomer of guanosine 3',5'-cyclic phosphorothioate was formed. This was determined by direct comparison with material synthesized chemically from guanosine 5'-phosphorothioate. The results from these experiments show that the reaction catalyzed by guanylate cyclase proceeds with inversion of configuration at phosphorus and this indicates that the reaction proceeds by way of a single direct displacement reaction.     

Stereochemical course of the reaction catalyzed by the cyclic GMP phosphodiesterase from retinal rod outer segments

The stereochemical course of hydrolysis catalyzed by the cyclic GMP phosphodiesterase from bovine retinal rod outer segments was determined. The Sp diastereomer of guanosine 3',5'-cyclic monophosphorothioate was hydrolyzed by cyclic GMP phosphodiesterase in H2(18)O to give [16O,18O]guanosine 5'-monophosphorothioate. This isotopomer was reacted with diphenyl phosphorochloridate to form the two diastereomers of P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate. The 31P NMR spectrum of this mixture of diastereomers was identical to that obtained from [16O,18O]guanosine 5'-monophosphorothioate resulting from the hydrolysis of the Rp diastereomer of guanosine 5'-p-nitrophenyl phosphorothioate by snake venom phosphodiesterase. This finding indicates that the 18O is bridging in the Rp diastereomer of the P1-(5'-guanosyl) P2-(diphenyl) 1-thiodiphosphate and nonbridging in the Sp diastereomer. As the snake venom phosphodiesterase reaction is known to proceed with retention of configuration, it follows that hydrolysis by retinal rod cyclic GMP phosphodiesterase proceeds with inversion of configuration at the phosphorus atom.     

Stereochemistry of internucleotide bond formation by polynucleotide phosphorylase from Micrococcus luteus.

Polynucleotide phosphorylase catalyzes the formation of polynucleotides from the Sp diastereomer of adenosine 5'-O-(l-thiodiphosphate) ADPalphaS), whereas the Rp diastereomer is a competitive inhibitor. The absolute configuration of the phosphorothioate diester bond in the polymer was determined by copolymerizing ADPalpha S, Sp isomer with UDP and degrading the resulting copolymer with R Nase A and spleen phosphodiesterase to give, inter alia, uridine 2',-3'-cyclic phosphorothioate. The latter product was shown to be the endo isomer by high-performance liquid chromatography. No evidence for the presence of the exo isomer was obtained. It can thus be concluded that the Sp diastereomer of ADPalphaS polymerizes with inversion of configuration at phosphorus without racemization to give a phosphorothioate diester bond with the Rp configuration.     

A molecular mechanism for lipopolysaccharide protection of Gram-negative bacteria from antimicrobial peptides

Cationic antimicrobial peptides serve as the first chemical barrier between all organisms and microbes. One of their main targets is the cytoplasmic membrane of the microorganisms. However, it is not yet clear why some peptides are active against one particular bacterial strain but not against others. Recent studies have suggested that the lipopolysaccharide (LPS) outer membrane is the first protective layer that actually controls peptide binding and insertion into Gram-negative bacteria. In order to shed light on these interactions, we synthesized and investigated a 12-mer amphipathic alpha-helical antimicrobial peptide (K(5)L(7)) and its diastereomer (4D-K(5)L(7)) (containing four d-amino acids). Interestingly, although both peptides strongly bind LPS bilayers and depolarize bacterial cytoplasmic membranes, only the diastereomer kills Gram-negative bacteria. Attenuated total reflectance Fourier transform infrared, CD, and surface plasmon resonance spectroscopies revealed that only the diastereomer penetrates the LPS layer. In contrast, K(5)L(7) binds cooperatively to the polysaccharide chain and the outer phosphate groups. As a result, the self-associated K(5)L(7) is unable to traverse through the tightly packed LPS molecules, revealed by epifluorescence studies with LPS giant unilamellar vesicles. The difference in the peptides' modes of binding is further demonstrated by the ability of the diastereomer to induce LPS miscellization, as shown by transmission electron microscopy. In addition to increasing our understanding of the molecular basis of the protection of bacteria by LPS, this study presents a potential strategy to overcome resistance by LPS, and it should help in the design of antimicrobial peptides for future therapeutic purposes.     

Microbiological analysis of 5-formyltetrahydrofolic acid and other folates using an automatic 96-well plate reader

The growth of auxotrophic bacteria remains the method of choice for the determination of biologically active folate metabolites in plasma. This report describes a microbiological assay for folates adapted to use disposable 96-well plates and an automatic plate reader. The modifications in the assay decreased reagent costs and made the analysis of hundreds of samples per day possible with a sensitivity limit of 10 fmol of (6S)-5-formyltetrahydrofolic acid. This limit compares favorably with that of previously reported, more laborious methods. The unnatural 6R diastereomer of 5-formyltetrahydrofolic acid did not interfere with the microbiological assay of the natural 6S diastereomer.     

Synthesis and evaluation of eight-membered cyclic pseudo-dipeptides

In the course of the development of calpain inhibitors, we report the synthesis of eight-membered cyclic pseudo dipeptides closely related to the known inhibitor SJA6017. The ring closure was effected by metathesis of the diallyl-substituted dipeptides 6 and 7. The formation of the dipeptides under kinetic control leads to the preferential formation of the unlike diastereomer 7 over the like diastereomer 6. The relative configuration of the diastereomers was determined by NMR and modeling studies of the related cyclic compounds 8 and 9 and their derivatives. The compounds proved not to inhibit calpain.     

Hydration of C-H groups in natural dithymidine nucleotide and its methylphosphonate analogues.

In this paper we report our preliminary studies on the hydration pattern of selected C-H groups in natural thymidyl(3'-5)thymidine and its Rp and Sp-methylphosphonate analogues using Molecular Dynamic simulations in aqueous solutions. The methyl groups attached to the phosphorus center (P-Me) in methylphosphonate analogues are hydrated by water molecules as efficiently as the hydrophilic P=O group in the natural dithymidine nucleotide and better than the neutral P=O functions in these compounds, although the nature of the hydration is different. The C5-Me centers of the 3'-yl units seem to be better hydrated in the methylphosphonate analogues than in the natural dithymidine phosphate and than other centers of the thymine bases in methylphosphonate analogues. Due to chirality of the phosphorus center, the C5-Me group of the 5'-yl unit in the Sp diastereomer coordinates more water than that in the Rp diastereomer. The C6-H group in the 5'-yl unit of the Sp diastereomer exhibits a specific interaction with water.     

Synergistic inhibition of hepatic glycogenolysis in the presence of insulin and a cAMP antagonist

The effects of insulin on the ability of the specific intracellular cAMP-dependent protein kinase antagonist, the Rp diastereomer of adenosine cyclic 3',5'-phosphorothioate, to inhibit glycogenolysis induced by the Sp diastereomer was studied in hepatocytes isolated from fed rats. Addition of the cAMP agonist, (Sp)-cAMPS, to hepatocytes resulted in a concentration-dependent increase in glycogenolytic glucose production concomitant with the cAMP-dependent activation of phosphorylase and inhibition of glycogen synthase. Activity curves were shifted to the right in the presence of the cAMP antagonist, (Rp)-cAMPS. Preincubation of the hepatocytes with a maximally effective concentration of insulin did not affect the concentration of (Sp)-cAMPS required for half-maximal activation of phosphorylase but did result in a 10-fold shift in the concentration of (Sp)-cAMPS required for half-maximal inactivation of glycogen synthase. Preincubation of hepatocytes with a combination of the cAMP antagonist, (Rp)-cAMPS, and insulin resulted in synergistic inhibition of (Sp)-cAMPS-induced phosphorylase activation, glycogen synthase inactivation, and glycogenolytic glucose production. Since neither phosphorothioate diastereomer was hydrolyzed significantly during the course of the experiments, the synergistic effects of insulin are postulated to be working through a mechanism subsequent to the phosphodiesterase activation step.     

Preparation of the pure diastereomeric forms of S- (5′-deoxy-5′-adenosyl)-1-ammonio-4-methylsulfonio-2- cyclopentene and their evaluation as irreversible inhibitors of S-adenosylmethionine decarboxylase from Escherichia coli

The conformationally restricted S-adenosylmethionine analogue AdoMac (S-(5′-deoxy-5′-adenosyl)-1-ammonio-4-methylsulfonio-2-cyclopentene has been shown to act as an enzyme activated, irreversible inhibitor of theEscherichia coli form of the enzyme S-adenosylmethionine decarboxylase. Inactivation of the enzyme is presumably initiated by formation of an imine linkage between the inhibitor and the terminal pyruvate of the enzyme, followed by base-catalyzed elimination of methylthioadenosine and generation of a latent electrophile. Removal of the driving force for the elimination of methylthioadenosine resulted in a reversibly binding inhibitor. Thus, the thioether analogue corresponding to AdoMac, and the corresponding dihydro derivative (H₂-AdoMac), reversibly inhibit the enzyme. AdoMac was resolved into its four pure diastereomeric forms, and each diastereomer was evaluated as an irreversible inhibitor of the enzyme. The K_I values for the individual diastereomers range between 3.83 and 39.6 μM, with the cis-1S,4R diastereomer being the most potent inhibitor. However, the k_inact values for the four diastereomers are not significantly different, suggesting that the binding of each diastereomer to the enzyme is configuration-dependent, while the subsequent inactivation likely proceeds through a single intermediate which is formed from each of the four diastereomers. Since each pure diastereomer represents a distinct conformational mimic exhibiting restricted sidechain rotation, the data suggests that these and related analogues may be useful as conformational probes for the catalytic site of AdoMet-DC.     

Activation of an adrenergic pro-drug through sequential stereoselective action of tandem target enzymes.

The synthetic amino acid, 3,4-dihydroxyphenylserine (DOPS) has been of great interest for many years as an adrenergic pro-drug, since the L-threo diastereomer of DOPS can be a precursor of R-(-)-norepinephrine, the natural form of this neurotransmitter. We now report bioactivation of DOPS to the potent pharmacological agent, noradrenalone (arterenone), via sequential stereoselective action by two target enzymes--dopamine beta-monooxygenase (DBM) and L-aromatic amino acid decarboxylase (AADC)--acting in tandem. Enzymatic activation is stereospecific, with only the L-erythro DOPS diastereomer producing noradrenalone; this is consistent with the known stereospecificities of AADC and DBM. These results provide a heretofore unrecognized rationale for the bioactivity of L-erythro DOPS and provide a basis for the design of new adrenergic pro-drugs.     

Sulfurization of dinucleoside phosphite triesters with chiral disulfides

Sixteen chiral analogues of phenylacetyl disulfide (PADS) and 5-methyl-3H-1,2,4-dithiazol-3-one (MEDITH) were used to sulfurize five dithymidine phosphite triesters, each incorporating a β-cyanoethoxy or siloxy group. Each mixture of S(P):R(P) phosphite triester diastereomers was combined with approximately one fourth of an equivalent of each of the sulfurizing reagents, and the R(PS):S(PS) diastereomer ratios of the resulting phosphite sulfides or phosphorothioates were determined by reverse-phase HPLC. Diastereoselectivities and corresponding diastereomeric excess (de) values were calculated by correcting for the starting triester diastereomer ratios. The highest de values for R(PS) and S(PS) phosphorothioates were 14.7% and 7.9%, respectively, both using MEDITH analogues.     

Synthesis and kinase inhibitory activity of 3'-(S)-epi-K-252a

The 3'-epi diastereomer of K-252a was synthesized with the goal of evaluating the stereochemical requirements of the 3'-sugar alcohol on kinase inhibitory activity. Inverting the 3'-alcohol resulted in a 20 nM inhibitor of VEGFR2 and a 1 nM inhibitor of TrkA tyrosine kinase.     

Epilentinsäure,ein neuer aroma- und geruchs-precursor in tricholoma arten

The investigation of three further Tricholoma species afforded in addition to lentinic acid a diastereomer epilentinic acid. The structure was derived from NMR spectral data and degradation. The mechanism of flavour formation is discussed.     

Antimalarial activity of thiophenyl- and benzenesulfonyl-dihydroartemisinin

Each diastereomer of 10-thiophenyl- and 10-benzenesulfonyl-dihydroartemisinin was synthesized from artemisinin in three steps, and screened against chloroquine-resistance and chloroquine-sensitive Plasmodium falciparum. Three of the four tested compounds were found to be effective. Especially, 10 beta-benzenesulfonyl-dihydroartemisinin showed stronger antimalarial activity than artemisinin.     

Noncovalent intercalative complex formation and kinetic flow linear dichroism of racemic syn- and anti-benzo[a]pyrenediol epoxide-DNA solutions

In order to gain a better understanding of the molecular basis underlying the differences in biological activities of the diastereomeric syn and anti diol epoxides of benzo[a]pyrene (trans-7,8-dihydroxy-syn-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene and trans-7,8-dihydroxy-anti-9,10-epoxy-7,8,9,10-tetrahydrobenzo[a]pyrene, respectively), their interactions with DNA in aqueous solutions were studied and compared. Kinetic flow linear dichroism experiments indicate that both diastereomers (racemic mixtures) form intercalation complexes immediately after mixing; the association constant (23 degrees C, ionic strength approximately 0.005) is significantly smaller (5200 M-1) in the case of the syn than in the case of the anti diastereomer (12 200 M-1). This difference is attributed to the greater bulkiness of the 7,8,9,10 ring of the syn stereoisomer, which is in the quasi-diaxial conformation as compared to the less bulky quasi-diequatorial conformation of the anti diastereomer. In contrast, the intercalative association constants of the tetraols derived from the hydrolysis of the two diol epoxides are similar in value. Upon formation of noncovalent syn-BPDE-DNA intercalation complexes, the reaction rate constant for the formation of tetraols (approximately 98%) and covalent adducts (approximately 2%) increases from 0.009 to 0.05 s-1 at pH 9.5 in 5 mM tris(hydroxymethyl)aminomethane buffer. The conformations of the aromatic chromophores of BPDE were followed by the kinetic flow dichroism technique as a function of reaction time; while the anti diastereomer changes conformation from an intercalative to an apparently external binding site, the syn diol epoxide molecules do not appear to undergo any measurable reorientation during or after the covalent binding reaction.(ABSTRACT TRUNCATED AT 250 WORDS)     

Isolation of basidiomycetous yeast Pseudozyma tsukubaensis and production of glycolipid biosurfactant, a diastereomer type of mannosylerythritol lipid-B

The producers of glycolipid biosurfactant, mannosylerythritol lipid-B (MEL-B), were isolated from leaves of Perilla frutescens on Ibaraki in Japan. Four isolates, 1D9, 1D10, 1D11, and 1E5, were identified as basidiomycetous yeast Pseudozyma tsukubaensis by rDNA sequence and biochemical properties. The structure of MEL-B produced by these strains was analyzed by ¹H nuclear magnetic resonance and gas chromatography–mass spectrometry methods, and was determined to be the same as the diastereomer MEL-B produced by P. tsukubaensis NBRC 1940. Of these isolates, P. tsukubaensis 1E5 (JCM 16987) is capable of producing the largest amount of the diastereomer MEL-B from vegetable oils. In order to progress the diastereomer MEL-B production by strain 1E5, factors affecting the production, such as carbon and organic nutrient sources, were further examined. Olive oil and yeast extract were the best carbon and nutrient sources, respectively. Under the optimal conditions, a maximum yield, productivity, and yield coefficient of 73.1 g/L, 10.4 g L⁻¹ day⁻¹, and 43.5 g/g were achieved by feeding of olive oil in a 5-L jar-fermenter culture using strain 1E5.     

Diastereomer characterizations of nitroxide-labeled nucleic acids

Site-directed spin labeling (SDSL) obtains structural and dynamic information of a macromolecule using a site-specifically attached stable nitroxide radical. SDSL studies of arbitrary DNA and RNA sequences can be achieved using an efficient phosphorothioate labeling scheme, where a nitroxide is attached to a phosphorothioate substituted at a target site during chemical synthesis. The chemically introduced phosphorothioate contains two diastereomers (Rp and Sp), and nitroxides attached to each diastereomer may experience different local environments. Here, we report work on using anion-exchange HPLC to separate and characterize diastereomers in three DNA oligonucleotides and one RNA oligonucleotide. In all oligonucleotides studied, the Rp diastereomer was found to elute earlier than the Sp in the unlabeled oligonucleotides, while a reversal in the elution order (Sp earlier than Rp) was observed for nitroxide-labeled oligonucleotides. The results enable a one-step purification procedure for preparing diastereomerically pure nitroxide-labeled oligonucleotides. This expands the score of nucleic acids SDSL.     

Mechanistic studies on deoxyribonucleic acid dependent ribonucleic acid polymerase from Escherichia coli using phosphorothioate analogues. 1. Initiation and pyrophosphate exchange reactions.

The diastereomers of adenosine 5'-O-(1-thiotriphosphate) (ATP alpha S) and adenosine 5'-O-(2-thiotriphosphate) (ATP beta S) can replace adenosine triphosphate (ATP) in the initiation reaction catalyzed by deoxyribonucleic acid (DNA) dependent ribonucleic acid (RNA) polymerase from Escherichia coli. In both cases, the Sp diastereomer is a better initiator than the Rp isomer. The diasteromers of 3'-uridyl 5'-adenosyl ,O-phosphorothioate [Up(S)A] can replace UpA in the primed initiation reaction catalyzed by RNA polymerase; however, the Rp diastereomer is a better initiator than the Sp isomer. By using ATP or CpA as initiator and UTP alpha S, isomer A, as substrate, we determined the stereochemical courses of both the initiation and primed initiation reactions, respectively, with T7 DNA template and found them to proceed with inversion of configuration. Determination of the stereochemical course of the pyrophosphate exchange reaction catalyzed by RNA polymerase provides evidence that this reaction is the reverse of the phosphodiester bond-forming reaction.