Effect of change in nucleoside structure on the activation and antiviral activity of phosphoramidate derivatives

Changing the nucleoside group of a series of phosphoramidate derivatives affects the enzyme mediated hydrolysis rate of the compounds. d4T and AZT-substituted analogs were activated by enzymes such as lipases, esterases, and proteases. On the other hand, 3dT-substituted derivatives were comparatively less prone to hydrolysis under similar experimental conditions. From the experimental results, we propose that the most preferable nucleoside group for enzyme activation is d4T rather than AZT or 3dT. Additionally, we also observed that depending on the enzymes used the chiral selectivity of the enzymes for the phosphorus center of these phosphoramidate derivatives differed, demonstrating the importance of the nucleoside structure for this class of compounds.     

Effect of alkyl groups on the cellular hydrolysis of stavudine phosphoramidates

We examined the effect of cellular metabolism of three alkyl-substituted amino acid ester phosphoramidate derivatives of stavudine in different cell lines. Marked cell-to-cell differences were found in both the rate of hydrolysis and chiral selectivity. This selectivity implies that different enzymes may be involved in the metabolism of these compounds depending on the cell type involved. Notably, both the methyl and ethyl substituted derivatives underwent hydrolysis in presence of various cell lines, whereas the tert-butyl substituted compound was resistant to hydrolysis implying that steric hindrance associated with this group along with electron density may play a key role in the hydrolysis profile of these compounds. Additionally we found this mimicked the hydrolysis profiles obtained for bacterial enzymes. Furthermore, our results suggest that the site of attack of the cellular enzymes is confined to the ester side chain of the molecule. This result is also consistent with our earlier observation using bacterial enzymes as well as using 'd' isomers.     

Stereoselectivity of lipases. I. Hydrolysis of enantiomeric glyceride analogues by gastric and pancreatic lipases, a kinetic study using the monomolecular film technique

In the present study, porcine pancreatic lipase, rabbit gastric lipase, and human gastric lipase stereospecificity toward enantiomeric glyceride derivatives was kinetically investigated using the monomolecular film technique. Pseudoglycerides such as enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol, enantiomeric 1(3)-alkyl-2-acyl-sn-glycerol, or enantiomeric 1(3)-acyl-2-acylamino-2-deoxy-sn-glycerol were synthesized in order to assess the lipase stereoselectivity during the hydrolysis of either the primary or the secondary ester position of these glycerides analogues. The cleaved acyl moiety was the same in both enantiomers, thereby excluding the possibility of effects occurring due to fatty acid specificity. We observed a porcine pancreatic lipase sn-3 stereoselectivity when using the enantiomeric 1(3)-alkyl-2-acylamino-2-deoxy-sn-glycerol (diglyceride analogue) which contrasted with the lack of stereoselectivity observed when using the enantiomeric 1(3)-alkyl-2,3(1,2)-diacyl-sn-glycerol (triglyceride analogue). The gastric lipases, in contrast to the pancreatic lipase, preferentially catalyze the hydrolysis of the primary sn-3 ester bond of the enantiomeric monoakyl-diacyl pair tested. From these kinetic data, high hydrolysis rates and no chiral discrimination were observed in the case of rabbit gastric lipase, whereas low rates and a clear chiral discrimination was noticed in the case of human gastric lipase during hydrolysis of the acyl chain from the secondary ester bond of 1(3)-alkyl-2-acyl enantiomers. It is particularly obvious that in the case of human gastric lipase decreasing the lipid packing increases the lipase sn-3 stereopreference during hydrolysis of the primary ester bond of the enantiomeric 2-acylamino derivatives (diglyceride analogue).     

Lipase-mediated stereoselective hydrolysis of stampidine and other phosphoramidate derivatives of stavudine

Enzymatic hydrolysis of stampidine and other aryl phosphate derivatives of stavudine were investigated using the Candida Antarctica Type B lipase. Modeling studies and comparison of the hydrolysis rate constants revealed a chiral preference of the lipase active site for the putative S-stereoisomer. The in vitro anti-HIV activity of these compounds correlated with their susceptibility to lipase- (but not esterase-) mediated hydrolysis. We propose that stampidine undergoes rapid enzymatic hydrolysis in the presence of lipase according to the following biochemical pathway: During the first step, hydrolysis of the ester group results in the formation of carboxylic acid. Subsequent step involves an intramolecular cyclization at the phosphorous center with simultaneous elimination of the phenoxy group to form a cyclic intermediate. In the presence of water, this intermediate is converted into the active metabolite Ala-d4T-MP. We postulate that the lipase hydrolyzes the methyl ester group of the l-alanine side chain to form the cyclic intermediate in a stereoselective fashion. This hypothesis was supported by experimental data showing that chloroethyl substituted derivatives of stampidine, which possess a chloroethyl linker unit instead of a methyl ester side chain, were resistant to lipase-mediated hydrolysis, which excludes the possibility of a direct hydrolysis of stampidine at the phosphorous center. Thus, our model implies that the lipase-mediated formation of the cyclic intermediate is a key step in metabolism of stampidine and relies on the initial configuration of the stereoisomers.     

Assay and inhibition of diacylglycerol lipase activity

A series of N-formyl-α-amino acid esters of β-lactone derivatives structurally related to tetrahydrolipstatin (THL) and O-3841 were synthesized that inhibit human and murine diacylglycerol lipase (DAGL) activities. New ether lipid reporter compounds were developed for an in vitro assay to efficiently screen inhibitors of 1,2-diacyl-sn-glycerol hydrolysis and related lipase activities using fluorescence resonance energy transfer (FRET). A standardized thin layer chromatography (TLC) radioassay of diacylglycerol lipase activity utilizing the labeled endogenous substrate [1″-(14)C]1-stearoyl-2-arachidonoyl-sn-glycerol with phosphorimaging detection was used to quantify inhibition by following formation of the initial product [1″-(14)C]2-arachidonoylglycerol and further hydrolysis under the assay conditions to [1-(14)C]arachidonic acid.     

Synthesis and biological evaluation of aryl phosphoramidate prodrugs of fosfoxacin and its derivatives

Aryl phosphoramidate prodrugs of fosfoxacin derivatives 15a-b and 8a-b were synthesized and investigated for their ability to target bacteria. No growth inhibition was observed neither for Mycobacterium smegmatis nor for Escherichia coli on solid medium, demonstrating the absence of release of the active compounds in the bacterial cells. Investigation of the stability of the prodrugs and their multienzymatic cleavage in abiotic and biotic conditions showed that the use of aryl phosphoramidate prodrug approach to deliver non-nucleotides compounds is not obvious and might not be appropriate for an antimicrobial drug.     

Design and synthesis of vidarabine prodrugs as antiviral agents

5′-O-d- and l-amino acid derivatives and 5′-O-(d- and l-amino acid methyl ester phosphoramidate) derivatives of vidarabine (ara-A) were synthesized as vidarabine prodrugs. Some compounds were equi- or more potent in vitro than vidarabine against two pox viruses and their uptake by cultured cells was improved compared to the parent drug.     

Rapid synthesis and lipase inhibition activity of some new benzimidazole and perimidine derivatives

This study presents a synthesis of new series of some benzimidazole, bisbenzimidazole and perimidine derivatives via microwave technique, which, leads to the good product yields and short reaction times. The structure of newly synthesized compounds was confirmed by ¹H NMR and ¹³C NMR spectra. These compounds were screened for their lipase inhibition activity. Then, all compounds were evaluated with regard to pancreatic lipase activity, and some of the 2-substituted perimidines, bisperimidine and bisbenzimidazole derivatives showed lipase inhibition at various concentrations.     

Preparation of 3-deacetyl cephalosporins by Aspergillus niger lipase

Lipase from Aspergillus niger was used for the selective hydrolysis of the 3-O-acetate of cephalosporin C to give an intermediate useful for further chemical elaborations. This lipase was purified to homogeneity and its properties compared with previously published data that present some discrepancies. The lipase proved to be very effective in catalyzing 3-O-acetate hydrolysis and versatile toward substitution on the beta-lactamic ring. In fact, as an example, two other cephalosporinic derivatives, cephalotin and cefotaxime, were efficiently deacetylated. The lipase was immobilized on Eupergit C and employed continuously in either a column or a batch reactor for 2 months without appreciable loss of activity. (c) 1996 John Wiley & Sons, Inc.     

New oligodeoxynucleotide derivatives containing <Emphasis Type="Italic">N</Emphasis>-(methanesulfonyl)-phosphoramidate (mesyl phosphoramidate) internucleotide group

Novel oligonucleotide derivatives containing N-(methanesulfonyl)-phosphoramidate (mesyl phosphoramidate) group have been described. Solid-phase synthesis of these compounds using an automated DNA synthesizer has been performed for the first time, including the Staudinger reaction between methanesulfonyl azide (mesyl azide) and 3′,5′-dinucleoside 2-cyanoethyl phosphite within an oligonucleotide immobilized on the polymer support, which is a product of phosphoramidite coupling. The mesyl phosphoramidate group is stable to the conditions of oligonucleotide synthesis, in particular, during acidic detritylation and subsequent removal of protecting groups and cleavage of an oligonucleotide from the polymer support by concentrated aqueous ammonia or methylamine at 55°C. It has been shown that the stability of complementary duplexes of oligodeoxynucleotides containing the mesyl phosphoramidate group with a single-stranded DNA is not inferior to the stability of native DNA:DNA duplex. Furthermore, mesyl phosphoramidate oligonucleotides are able to form a complementary duplex with RNA, which is only slightly less stable than the equivalent DNA:RNA duplex. This raises the possibility of their application as potential antisense therapeutic agents.     

Synthesis and anti-HIV activity of some S-acyl-2-thioethyl (SATE) phosphoramidate derivatives of 3'-azido-2',3'-dideoxythymidine.

The synthesis and in vitro anti-HIV activity of tBuSATE phosphoramidate derivatives of AZT incorporating several methyl-esterified alpha-amino acids are reported. The biological evaluation strongly supports the hypothesis that such compounds exert their anti-HIV effects via intracellular delivery of the corresponding 5'-mononucleotide.     

Digestion in vitro of erythritol esters by rat pancreatic juice enzymes

The mechanism of the digestion of erythritol esters was determined using rat pancreatic juice and purified pancreatic lipase (EC 3.1.1.3). Conditions of hydrolysis were used that would selectively activate or inactivate nonspecific lipase or lipase. It was shown that erythritol tetraoleate was hydrolyzed by nonspecific lipase but not by lipase. The initial digestion product was a triester, predominantly erythritol-1,2,3-trioleate. Thus, nonspecific lipase preferentially hydrolyzed the ester of a primary alcohol. In contrast to the results obtained with the tetraester, lipase could remove a fatty acid from the triester but the resulting erythritol-2,3-dioleate was not hydrolyzed by lipase. The selectivity of this hydrolysis and the inability to hydrolyze the diester are attributed to the known specificity of this enzyme to act only on esters of primary alcohols. Nonspecific lipase completely hydrolyzed erythritol tetraoleate to free erythritol in a stepwise manner. The relative rates of these reactions were tetraester --> triester --> diester --> monoester --> erythritol Because of the specificity of pancreatic lipase and the lack of specificity of nonspecific lipase it is likely that this latter enzyme is the primary agent for the hydrolysis of erythritol esters in the intact animal.     

Pseudomonas fluorescens lipase immobilization on polysiloxane–polyvinyl alcohol composite chemically modified with epichlorohydrin

The technique based on sol–gel approach was used to generate silica matrices derivatives by hydrolysis of silane compounds. The present work evaluates a hybrid matrix obtained with tetraethoxysilane (TEOS) and polyvinyl alcohol (PVA) on the immobilization yield of lipase from Pseudomonas fluorescens. The resulting polysiloxane–polyvinyl alcohol (POS–PVA) matrix combines the property of PVA as a suitable polymer to retain proteins with an excellent optical, thermal and chemical stability of the host silicon oxide matrix. Aiming to render adequate functional groups to the covalent binding with the enzyme the POS–PVA matrix was chemically modified using epichlorohydrin. The results were compared with immobilized derivative on POS–PVA activated with glutaraldehyde. Immobilization yield based on the recovered lipase activity depended on the activating agent and the highest efficiency (32%) was attained when lipase was immobilized on POS–PVA activated with epichlorohydrin, which, probably, provided more linkage points for the covalent bind of the enzyme on the support. This was confirmed by determining the morphological properties using different techniques as X-ray diffraction and scanning electron microscopy (SEM). Comparative studies were carried out to attain optimal activities for free lipase and immobilized systems. For this purpose, a central composite experimental design with different combinations of pH and temperature was performed. Enzymatic hydrolysis with the immobilized enzyme in the framework of the Michaelis–Menten mechanism was also reported. Under optimum conditions, the immobilized derivative on POS–PVA activated with epichlorohydrin showed to have more affinity for the substrate in the hydrolysis of olive oil, with a Michaelis–Menten constant value (K_m) of 293 mM, compared to the value of 401 mM obtained for the immobilized lipase on support activated with glutaraldehyde. Data generated by DSC showed that both immobilized derivatives have similar thermal stabilities.     

Production of omega-3 polyunsaturated fatty acids through hydrolysis of fish oil by Candida rugosa lipase immobilized and stabilized on different supports

This paper describes the fish oil hydrolysis performed to obtain Omega-3 fatty acids using Candida rugosa lipase (CRL) immobilized and stabilized on different supports. The enzyme was successfully immobilized, presenting higher thermal stability than the free enzyme. Besides, the cationic derivatives were more stable than the others derivatives and free enzyme in methanol, propanol and cyclohexane. Reactions of fish oil hydrolysis were carried out in organic aqueous medium using 10?U of biocatalyst per gram of oil, at 37?°C. After 96?h, the CRL immobilized on cyanogen bromide agarose rendered the lower fish oil hydrolysis, producing 218?μM of Omega-3, which was 1.1-fold more than the hydrolysis catalyzed by free enzyme, while the ionic derivatives rendered the highest fish oil hydrolysis producing 582 and 577?μM of Omega-3 using the carboxymethyl and sulfopropyl derivatives, respectively. The carboxymethyl and the sulfopropyl derivatives resulted in a 2.9-fold increase in the hydrolysis of fish oil, making these derivatives attractive for industrial applications.     

Hydrolysis of lipid monolayers and the substrate specificity of hepatic lipase

The substrate specificities of the phospholipase and triglyceridase activities of purified rat liver hepatic lipase were compared using lipid monolayers so that the substrates were presented to the enzyme in a controlled physical state. The rate of hydrolysis of 14C-labeled lipid at constant surface pressure in the presence of hepatic lipase and fatty acid-free bovine serum albumin at 33 degrees C was determined by monitoring the decrease of surface radioactivity. In monolayers of sphingomyelin/cholesterol (2:1, mol/mol) containing either 1 mol% triacylglycerol, 1 mol% phosphatidylethanolamine, or 10 and 20 mol% phosphatidylcholine, hepatic lipase clearly showed a preference for unsaturated over saturated lipids. In addition, with a sphingomyelin/cholesterol (2:1) monolayer containing 1 mol% of lipid substrate, hepatic lipase showed the following preference: triolein = dioleoylphosphatidylethanolamine much greater than dioleoylphosphatidylcholine; the respective rates of hydrolysis were 15.3 +/- 1.2, 14.9 +/- 0.8, and 0.5 +/- 0.1 mumol fatty acid produced/h per mg hepatic lipase. Overall, it appears that when comparing rates of hydrolysis of molecules within a given lipid class, hydrocarbon chain interactions are important. However, when comparing different lipid classes such as phosphatidylcholines and phosphatidylethanolamines, it is apparent that the polar group has a significant influence on the rate of hydrolysis. The rate of [14C]triolein hydrolysis, when mixed at surface concentrations of up to 2 mol% in a sphingomyelin/cholesterol (2:1) monolayer, was significantly faster than when triolein was present in a 1-oleyl-2-palmitylphosphatidylcholine monolayer; the rates of hydrolysis were 47.7 +/- 5.4 and 8.9 +/- 0.8 mumol fatty acid produced/h per mg hepatic lipase, respectively. The monolayer physical state and the miscibility of the substrate in the inert matrix influence the presentation of the substrate to the enzyme, thereby affecting the hydrolysis rate.     

Use of phosphorus oxychloride in synthesizing nucleotides and oligonucleotides

Procedures are described for phosphorylating protected nucleotides, oligonucleotides and phosphoramidate oligonucleotide derivatives at the 3′-hydroxyl group. The conditions (phosphorylation with phosphorus oxychloride and pyridine in dioxane followed by hydrolysis with aqueous pyridine) are sufficiently mild that base labile (trifluoroacetylamino; β-cyanoethyl phosphotriester) and acid labile (O-monomethoxytrityl; phosphoramidate) functions are retained intact. Application of the technique is illustrated by the synthesis of dpT, dTp, d(CF₃CONH)Tp, dTp_NTp, and dTp_NTp_NTp. In addition, the utilization of phosphorus oxychloride in joining thymidine derivatives and dinucleoside phosphotriester blocks via phosphodiester links is described.     

Purification and characterization of an intracellular lipase from pleopods of whiteleg shrimp (Litopenaeus vannamei)

An intracellular lipase present in the whiteleg shrimp Litopenaeus vannamei was detected in pleopods. The lipase from pleopods was purified and characterized by biochemical and kinetic parameters. Purified intracellular lipase has a molecular mass of 196kDa, the polypeptide is assembled by two monomers, 95.26 and 63.36kDa. The enzyme lacks glycosylation, and it has an isoelectric point of 5.0. The enzyme showed the highest activity at a temperature range of 30-40°C at pH 8.0-10.0. Activity was completely inhibited by tetrahydrolipstatin and diethyl p-nitrophenyl phosphate, suggesting that the intracellular lipase is a serine lipase. The lipase hydrolyzes short and long-chain triacylglycerides, as well as naphthol derivatives at comparable rates in contrast to other sources of lipases. Specific activity of 930U mg(-1) and 416.56U mg(-1) was measured using triolein and tristearin at pH 8.0 at 30°C as substrates, respectively. The lipase showed a K(M,app) of 41.03mM and k(cat)/K(M,app) ratio of 4.88 using MUF-butyrate as the substrate. The intracellular lipase described for shrimp has a potential role in hydrolysis of triacylglycerides stored as fat body, as has been shown in humans.     

Synthesis of nucleotide antibiotics having N-acyl phosphoramidate linkages.

This paper reports the synthesis of nucleotide antibiotics having N-acyl phosphoramidate linkages. The key reaction, the construction of the N-acyl phosphoramidate linkage was achieved by the reaction of nucleoside 5'-phosphoramidite derivatives with carboxamide derivatives in the presence of 5-(3,5-dinitrophenyl)-1H-tetrazole as a very effective activator. By use of this activator, Phosmidosine was synthesized by condensation of an appropriately protected 8-oxoadenosine 5'-O-phosphoramidite derivative with an N-protected prolinamide derivative. In the case of Agrocin 84, the two P-N bonds were constructed progressively. The N-acyl phosphoramidate linkage at the 5'-position of the ribose moiety was similarly synthesized. After phosphorylation of the amino group of the adenine moiety, a fully protected Agrocin 84 derivative, which would be converted to Agrocin 84, was successfully synthesized.     

The histidines reacting with ethoxyformic anhydride in porcine pancreatic lipase: their relationships with enzyme activity

The activities of porcine pancreatic lipase (449 amino acid residues) toward two different substrates, p-nitrophenylacetate and tributyrylglycerol, and their dependence on histidine ethoxyformylation were studied. In parallel, the ethoxyformylation of the lipase fragment constituting the C-terminal sequence of lipase (residues 336 to 449) was also investigated. This fragment was found to have retained the ability of lipase to catalyse p-nitrophenylacetate hydrolysis. The first histidine to react either in lipase or in the lipase fragment was His-354. The activities of the two compounds toward p-nitrophenyl-acetate were lost but that of the enzyme toward tributyrylglycerol was almost entirely retained. When a larger excess of ethoxyformic anhydride was used for the lipase reaction, 2.8 histidine residues were ethoxyformylated and characterised as His-354, His-156 and His-75, which resulted in an 85% inhibition of the tributyrylglycerol hydrolysis by the enzyme. Hydroxylamine treatment reactivated most of the lipase and lipase fragment. This is the first demonstration that the two lipase activities are not associated with the same active site. The loss of activity toward triacylglycerol hydrolysis suggests that His-156 and/or His-75 belong(s) to the active site or that a conformational change resulting from the ethoxyformylation renders the lipase inactive.     

Phosphoramidate and phosphate prodrugs of (-)-beta-D-(2R,4R)-dioxolane-thymine: synthesis, anti-HIV activity and stability studies

A series of phosphoramidate and phosphate prodrugs of DOT were synthesized via dichlorophosphate or H-phosphonate chemistry and evaluated for their anti-HIV activity against LAI M184V mutants in PBM cells as well as for their cytotoxicity. The antiviral and cytotoxic profiles of the prodrugs were compared with that of the parent compound (DOT), and it was found that four aryl phosphoramidates 5, 18, 20, and 26 showed a significant enhancement (8- to 12-fold) in anti-HIV activity without cytotoxicity. Chemical stability of these prodrugs was evaluated in phosphate buffer at pH values of biological relevance (i.e., pH 2.0 and 7.4). Enzymatic hydrolysis was also studied in esterase or lipase in buffer solution. Chemical stability studies indicate that the phosphoramidates have good chemical stability at pH 2.0 and at pH 7.4 phosphate buffer. Phosphoramidate prodrugs were hydrolyzed in vitro by esterase or lipase and found to be better substrates for lipases than for esterases. 1,3-Diol cyclic phosphates showed potent anti-HIV activity without increasing the cytotoxicity compared with that of DOT and have good chemical and enzymatic stability. Long-chain lipid phosphates, although showed potent anti-HIV activity, exhibited increased cytotoxicity.