Synthesis of a water-soluble prodrug of entacapone

Entacapone was reacted with phosphorous oxychloride in dry pyridine to yield a phosphate ester. The phosphate promoiety increased aqueous solubility of the parent drug by more than 1700- and 20-fold at pH 1.2 and 7.4, respectively. The phosphate ester provides adequate stability (t(1/2) = 2227 h; pH 7.4) towards chemical hydrolysis, and allowed for release of the parent drug via enzymatic hydrolysis in liver homogenate.     

Dextran–estrone conjugate: synthesis and in vitro release study

A novel hormone conjugate has been prepared by a coupling reaction between modified estrone and dextran. In order to provide a suitable reactive estrone derivative for coupling with dextran and a spacer between the drug carrier and the hormone, the steroidal sex hormone was succinylated by reaction with succinic anhydride. Subsequently, the carboxylic acid terminal of succinylated estrone was further reacted with thionyl chloride to replace the hydroxy group with chlorine to make a better leaving group. The ester bond was employed as labile linkage between the hormone and the biopolymer carrier backbone so that the coupled estrogen could be released from the conjugate via ester hydrolysis. Structures of the modified estrone and dextran–estrone conjugate were determined by elemental analysis and by FTIR, ¹H and ¹³C NMR spectroscopies. The degree of substitution (D.S.) per anhydroglucose (AHG) unit was 0.33 (11.0 mol-% of estrone moieties), as calculated from the ¹H NMR spectrum. In vitro hydrolysis of the conjugate in aqueous phosphate buffer/ethanol solutions at pH 8.0 and 7.4 and 37°C released estrone was completed within a few days and followed zero-order kinetics.     

Synthesis and stability of two indomethacin prodrugs

The purpose of this study was to synthesize and study the in vitro enzymatic and non-enzymatic hydrolysis of indomethacin-TEG ester and amide prodrugs. It was found that the ester conjugate 10 was comparatively stable between pH 3 and 6 (half-life>90h), with a half-life equal to 5.2h in 80% buffered plasma. In contrast, the amide conjugate 12 appeared to be stable over the entire pH range studied with the only observed degradation being cleavage of the indolic N-4-chlorobenzoyl moiety.     

Synthesis of pH-sensitive amphotericin B-poly(ethylene glycol) conjugates and study of their controlled release in vitro

New intravenous conjugates of amphotericin B (AMB) with poly(ethylene glycols) (PEG) (M=5000, 10,000, 20,000) have been synthesized and characterised. The intermediate PEGs possess a 1,4-disubstituted benzene ring with aldehyde group at the end of the chain. The benzene ring is connected with PEG at its 4-position (with respect to the aldehyde group) by various functional groups (ether, amide, ester). Reaction of terminal aldehyde group of the substituted PEGs with AMB gave conjugates containing a pH-sensitive imine linkage, which can be presumed to exhibit antimycotic effect at sites with lowered pH value. All types of the conjugates are relatively stable in phosphate buffer at physiological conditions of pH 7.4 (37 degrees C), less than 5 mol% AMB being split off from them within 24 h. For a model medium of afflicted tissue was used a phosphate buffer (pH 5.5, 37 degrees C), in which controlled release of AMB from the conjugates takes place. The imine linkage is split to give free AMB with half-lives of 2-45 min. The rate of acid catalysed hydrolysis depends upon substitution of the benzene ring; however, it does not depend on molecular weights of the PEGs used. The conjugates with ester linkage undergo enzymatic splitting in human blood plasma and/or blood serum at pH 7.4 (37 degrees C) with half-lives of 2-5 h depending on molecular weights of the PEGs used (M = 5000, 10,000, 20,000). At first, the splitting of ester linkage produces the relatively stable pro-drug, that is, 4-carboxybenzylideniminoamphotericin B, which is decomposed to AMB and 4-formylbenzoic acid in a goal-directed manner only at pH 7 (t1/2 = 2 min, pH 5.5, 37 degrees C). A goal-directed release of AMB is only achieved by acid catalysed hydrolysis of imine linkage, either from the polymeric conjugate or from the pro-drug released thereof. The LD50 values determined in vivo (mouse) are 20.7 mg/kg and 40.5 mg/kg for the conjugates with ester linkage (M = 10,000 and 5000, respectively), which means that they are ca. 6-11 times less toxic than free AMB.     

Hydrolysis of phosphate monoesters: a biological problem with multiple chemical solutions.

Formation of phosphate esters by kinases has long been recognized as an important process in biochemistry, but the reverse reaction, hydrolysis of phosphate esters by phosphatases, has attracted less attention. Recent work suggests that phosphatases are as important as kinases in regulatory processes, and that they constitute a diverse group of enzymes that utilize a variety of chemical means to accelerate phosphate ester hydrolysis.     

Inhibitors of neutral cholesteryl ester hydrolase

p-Nitrophenyl N-butyl, N-octyl, and N-dodecyl carbamates and a newly synthesized diethyl phosphate compound were studied as potential inhibitors of the cholesteryl ester hydrolases of Fu5AH rat hepatoma cells. Whole homogenates of Fu5AH cells were used as an enzyme source for the assay of cholesteryl ester hydrolase activity. All four compounds led to marked inhibition (70-80%) of neutral cholesteryl ester hydrolase activity (assayed at pH 7) at concentrations where the activity of acid cholesteryl ester hydrolase (assayed at pH 4) was unaffected. Cholesteryl ester hydrolysis was also evaluated in intact cultured cells induced to accumulate cholesteryl esters in cytoplasmic lipid droplets by exposure to cholesterol-rich phospholipid dispersions. Hydrolysis was then assessed during subsequent incubations in the presence of an inhibitor of cholesterol esterification. All compounds caused significant inhibition of cholesterol ester hydrolysis with the diethyl phosphate being the most effective. At a concentration that caused greater than 90% inhibition of the hydrolysis of cytoplasmic cholesteryl esters, the compound had only a minimal effect on lysosomal hydrolysis of cholesteryl esters. These results suggest that diethyl phosphates and N-alkylcarbamates may be of value in future studies on the substrate specificities, regulation, and physiological role(s) of cholesteryl ester hydrolases.     

Phosphate ester hydrolysis is catalyzed by a bacterial transferrin: potential implications for in vivo iron transport mechanisms

Two synergistic anions, p-nitrophenyl phosphate ester (NPP) and SO(4)(2-), were found to form new stable assemblies with Fe(3+) and a bacterial transferrin, FbpA (FbpA=ferric binding protein). Fe(3+)FbpA-SO(4) undergoes rapid anion exchange in the presence of NPP to form Fe(3+)FbpA-NPP. Formation of Fe(3+)FbpA-NPP was found to accelerate the rate of hydrolysis of the bound phosphate ester (k(hyd)=1.6 x 10(-6) s(-1) at 25 degrees C and pH 6.5) by >10(3) fold over the uncatalyzed reaction. These findings suggest a dual function for FbpA in vivo: transport of Fe(3+) across the periplasmic space to the inner membrane in certain gram-negative bacteria and hydrolysis of periplasmic polyphosphates.     

Pharmacologic properties of a phosphate ester of dopamine

The 3 or 4 phosphate ester of dopamine (PD) was hydrolyzed by homogenates of rat tissues to give inorganic phosphate (Pi) and dopamine. The rate of hydrolysis of PD by kidney homogenates was increased by exogenous MgCl2 but not CaCl2 or KCl. The activity of brain, heart or liver homogenates was insensitive to the added salts. Several lines of evidence indicate that alkaline phosphatase activity contributes to the high rate of PD hydrolysis by the kidney but not brain homogenate. The intravenous infusion of PD at 12 mumole/kg in one hr to anesthetized rats increased the dopamine content of the plasma, kidney and heart without altering brain or liver dopamine. The results suggest that PD may be more effective than dopamine for increasing dopamine levels of the kidney. In addition, the hydrolysis of PD by brain homogenates, which is independent of alkaline phosphatase activity, suggests that specific enzymes exist for the metabolism of PD.     

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.     

Artificial peptides with unnatural components designed for materializing protein function

In order to design functional peptides, we employed two strategies. The first one is to incorporate rigid unnatural amino acids into peptides to make the peptide backbone rigid. Functions were expected to appear through the conformational control by the strategy. A series of cyclic peptides constituted of alternating natural amino acids and 3-aminobenzoic acid, used as an unnatural amino acid, were synthesized. These cyclic peptides were found to function as strong binders for phosphomonoester, catalysts for ester hydrolysis, and/or ion channels. The second strategy is to conjugate peptides with unnatural and inherently functional molecules. Following this strategy, oligo(L-leucine)- or oligo(L-phenylalanine)-modified ruthenium tris(bipyridine) complexes were synthesized. Distance dependence of the photoinduced electron transfer from the ruthenium complexes and the function as sensors for phosphate anion (H(2)PO(-)(4)) are discussed.     

Studies on the chemical stability of propylene glycol alginate esters

The chemical stability of propylene glycol alginates (PGAs) has been examined. Under acidic conditions the ester groups in PGA are stable to hydrolysis but hydrolytic degradation of the glycosidic linkages in the polysaccharide backbone occurs. Under alkaline conditions the ester groups are hydrolysed with the primary 2-hydroxyprop-1-yl ester groups being more susceptible than secondary 1-hydroxyprop-2-yl ester groups, with little degradation of the polysaccharide backbone. Sodium carbonate-bicarbonate buffer was a much more effective hydrolysing reagent than sodium hydroxide at the same concentration and pH, and the rate of hydrolysis was greatly accelerated by increasing the hydrolysis temperature. Acetate, citrate and phosphate ions accelerated the rate of hydrolysis of the ester groups in PGA when added to the sodium hydroxide hydrolysing reagent. Hydrolysis of the ester groups in PGA with sodium hydroxide was unaffected by the addition of imidazole. However hydrolysis of the ester groups in PGA with sodium hydroxide in the presence of 1-aminobutane led to the formation of an alginate amide in which only the primary 2-hydroxylprop-1-yl ester groups were present, suggesting that a nucleophilic substitution of primary ester groups by amine groups is involved in the reaction.     

Calcineurin-catalyzed reaction with phosphite and phosphate esters of tyrosine.

A convenient synthesis is reported for the preparation of the phosphite ester of tyrosine methyl ester. By use of calcineurin, at 30 degrees C, a phosphite ester was hydrolyzed with a VM value [119 nmol/(min.micrograms of E)] approximately 500 times greater than that obtained with tyrosine phosphate [0.23 nmol/(min.microgram of E)] as substrate, but with similar KM values (12 mM for Tyr-PH ME, 11 mM for Tyr-P). Acid phosphatase, on the other hand, hydrolyzed the phosphite ester with a VM and KM value lower than those obtained with tyrosyl phosphate. The temperature dependence of the kinetic parameters (KM and VM) was evaluated, and the activation parameters were obtained with both substrates. The entropy of activation associated with the enzymatic hydrolysis of tyrosine phosphate agrees with the entrophy change for the hydrolysis of the monoanion of phosphate monoesters. The energy of activation for both substrates was in agreement with the energy change for hydrolysis of the oxygen-phosphorous linkage of phosphate monoester monoanions and phosphite esters. These results are consistent with a scheme of general acid catalysis in the action of calcineurin.     

A model ternary heparin conjugate by direct covalent bond strategy applied to drug delivery system

A model ternary heparin conjugate by direct covalent bond strategy has been developed, in which modified heparin using active mix anhydride as intermediate conjugates with model drug molecule and model specific ligand, respectively. Designed ester bonds between model drug and heparin facilitate hydrolysis kinetics research. The strategy can be extended to design and synthesize a targeted drug delivery system. The key point is to use mixed anhydride groups as activating intermediates to mediate the synthesis of the ternary heparin conjugate. Formation of mixed anhydride is detected by the conductimetry experiment. The ternary heparin conjugate is characterized by ¹³C NMR, FT-IR and GPC, respectively. The decreased trend on degree of substitution (DS) is consistent with that of introduced anticancer drug and specific ligand in drug delivery system. Moreover, their anticoagulant activity is evaluated by measuring activated partial thromboplastin time (APTT) and anti-factor Xa activity. The results show that model ternary heparin conjugate with reduced anticoagulant activity may avoid the risk of severe hemorrhagic complication during the administration and is potential to develop a safe and effective drug delivery system on anticancer research.     

Fluoride inhibition of bovine spleen purple acid phosphatase: characterization of a ternary enzyme-phosphate-fluoride complex as a model for the active enzyme-substrate-hydroxide complex.

Purple acid phosphatases (PAPs) employ a dinuclear Fe(3+)Fe(2+) or Fe(3+)Zn(2+) center to catalyze the hydrolysis of phosphate monoesters. The interaction of fluoride with bovine spleen purple acid phosphatase (BSPAP) has been studied using a combination of steady-state kinetics and spectroscopic methods. For FeZn-BSPAP, the nature of the inhibition changes from noncompetitive at pH 6.5 (K(i(comp)) approximately K(i(uncomp)) approximately 2 mM) to uncompetitive at pH 5.0 (K(i(uncomp)) = 0.2 mM). The inhibition constant for AlZn-BSPAP at pH 5.0 (K(i) = 3 microM) is approximately 50-70-fold lower than that observed for both FeZn-BSAP and GaZn-BSPAP, suggesting that fluoride binds to the trivalent metal. Fluoride binding to the enzyme-substrate complex was found to be remarkably slow; hence, the kinetics of fluoride binding were studied in some detail for FeZn-, AlZn-, and FeFe-BSPAP at pH 5.0 and for FeZn-BSPAP at pH 6.5. Since the enzyme kinetics studies indicated the formation of a ternary enzyme-substrate-fluoride complex, the binding of fluoride to FeZn-BSPAP was studied using optical and EPR spectroscopies, both in the presence and absence of phosphate. The characteristic optical and EPR spectra of FeZn-BSPAP. F and FeZn-BSPAP.PO(4).F are similar at pH 5.0 and pH 6.5, indicating the formation of similar fluoride complexes at both pHs. A structural model for the ternary enzyme-(substrate/phosphate)-fluoride complexes is proposed that can explain the results from both the spectroscopic and the enzyme kinetics experiments. In this model, fluoride binds to the trivalent metal replacing the water/hydroxide ligand that is essential for the hydrolysis reaction to take place, while phosphate or the phosphate ester coordinates to the divalent metal ion.     

Bis-ketol nucleoside triesters as prodrugs of the antiviral nucleoside triphosphate analogues of 3'-deoxythymidine and 3'-deoxy-2',3'-didehydrothymidine

Derivatives of 3'-deoxythymidine (ddT) and 3'-deoxy-2',3'-didehydrothymidine (d4T) were prepared in which the 5'-hydroxyl group of the nucleoside was esterified to a bis-ketol phosphate. The resulting phosphate triesters are postulated to be prodrugs of the corresponding 5'-mononucleotides, which are formed intracellularly by the hydrolysis of the two ketol ester groups. The triesters were tested for anti-HIV activity with the result that those derived from ddT showed enhanced antiviral activity when compared to the parent nucleoside.     

Chemical and functional characterization of metal-binding pseudotripeptides with different functionalized N-alkyl residues Dedicated to Professor Dr. Ernst-Gottfried Jaeger on occasion of his 65th birthday

Pseudotripeptide ligands with 4 different N-functionalized glycine residues were qualitatively, semiquantitatively and quantitatively tested for their complexation of the bivalent transition metal ions Zn²⁺, Cu²⁺, Co²⁺, Ni²⁺ and Mn²⁺. The functional side chains have different length and different groups available for complexation. MALDI-MS and ESI-MS were used for more qualitative or semiquantitative estimation of the complex formation tendencies. The found ranking differs by these two methods only for Zn²⁺ and Ni²⁺. For one of the pseudotripeptide ligands, the ligand L1, complex formation with certain transition metal was estimated quantitatively by potentiometric titration. The Zn-complex of that ligand polarizes bound water strongly, resulting in a low pK_a-value. Complexes of pseudotripeptide ligand L1 with certain metal ions were tested for their hydrolytic activity. The pseudo first order rate constants of the hydrolysis of the substrates 4-nitrophenyl acetate and bis(4-nitrophenyl)phosphate were compared to complexes with the same metal ions formed with a very well studied ligand from the literature, the 1,4,7,10-tetraaza cyclododecane (cyclen). The hydrolysis of the phosphate ester occurs very slowly compared to the acetate ester. No correlation exists between the estimated pK_a values of complexes formed from ligand L1 with different metal ions and the phosphate ester hydrolysis. The Ni ions give totally different hydrolytic activities for pseudotripeptide ligand L1 and cyclen. With one exception, the Ni-cyclen complex, all other complexes have only a low or moderate catalytic activity.     

Purification and partial characterization of a collagenolytic enzyme from Pseudomonas aeruginosa.

A proteinase from Pseudomonas aeruginosa exhibiting collagenolytic activity was purified 1575-fold with a recovery of 24% by use of chemical and chromatographic technics. The enzyme preparation appeared to be homogeneous when subjected to chromatographic, electrophoretic and ultracentrifugational analyses. A standard state sedimentation coefficient of 2.10 S was calculated and further analyses indicated that the enzyme had a molecular weight of 17 500 and dimerizes under certain conditions to yield an apparent molecular weight of 34 000. In addition to insoluble collagen, the enzyme catalyzed the hydrolysis of congocoll, azocoll, soluble collagen and casein, but did not attack orcein-elastin, azoalbumin, p-toluene eulfonyl-L-arginine methyl ester, benzoyl-L-tyrosine ethyl ester, and the hexapeptide N-benzyloxycarbonyl-glycyl-L-prolyglycylglycyl-L-prolyl-L-alanine. Enzymatic activity against congocoll was 6-fold greater at pH 7.5 in Tris with HCl than in phosphate buffer at the same ionic strength. Cobalt, and to a lesser extent, Zn2+ appeared to activate the enzyme, especially in phosphate buffer. NcCN and p-chloromercuribenzoate did not appreciably inhibit enzyme activity, while (NH4)2 SO4, EDTA and cysteine displayed a significant inhibitory effect under certain conditions.     

Protein kinase C-mediated gonadotropin-releasing hormone receptor sequestration is associated with uncoupling of phosphoinositide hydrolysis

Gonadotropin-releasing hormone (GnRH) regulates pituitary gonadotropin release by a Ca2+-dependent mechanism involving receptor-mediated phosphoinositide hydrolysis. Previous studies indicate that activation of pituitary protein kinase C (PKC), while not required for acute gonadotropin release in response to GnRH, is likely involved in the chronic regulation of gonadotrope responsiveness. Studies from our laboratory have shown that activation of PKC by phorbol esters produces both the uncoupling of GnRH-stimulated phosphoinositide hydrolysis and the selective enhancement of GnRH agonist binding in pituitary cell cultures. In the present work, we have examined the possibility that these processes are related in mechanism. Dissociation of bound agonist radioligand at 23 degrees C was found to be reduced in the presence of phorbol esters, and ligand bound in the presence of phorbol ester was resistant to displacement by competing ligands at 4 degrees C. However, agonist bound in the presence of phorbol ester was dissociable by subsequently washing cells at pH 3. Receptor photoaffinity labeling studies confirmed that agonist association with membrane component(s) identified as the GnRH receptor was increased in the presence of phorbol ester. These results suggest that, in the presence of a phorbol ester PKC activator, agonist-occupied GnRH receptors remain at the cell surface, but are sequestered in some manner. In other experiments, cell preloaded with [3H]inositol were treated with GnRH agonist ligand and phorbol ester at 4 degrees C to form a pool of sequestered, agonist-occupied receptors, and then displaceable (nonsequestered) agonist was removed by incubation with antagonist ligand. After addition of LiCl and warming to 37 degrees C, [3H]inositol phosphate production (an index of phosphoinositide hydrolysis) in phorbol ester-treated cells was reduced to 67% of vehicle control, although residual specific agonist binding had been increased to greater than 300% of control. The appearance of sequestered receptors and inhibition of [3H]inositol phosphate production had similar phorbol ester concentration dependencies. These results suggest that the same agonist-occupied GnRH receptors sequestered as a result of PKC activation also are preferentially uncoupled from phosphoinositide hydrolysis.     

Deoxythymidine 3', 5'-di-p-nitrophenyl phosphate as a synthetic substrate for bovine pancreatic deoxyribonuclease.

Bovine pancreatic deoxyribonuclease liberates p-nitrophenol from the 3'-group of deoxythymidine 3', 5'-di-p-nitrophenyl phosphate. A similar hydrolysis occurs with deoxythymidine 3'-p-nitrophenyl phosphate 5'-phsophate, but the rate is less than 2% of that with the di-p-nitrophenyl ester. The rate of formation of the p-nitrophenol, measured spectrophotometrically at 400 nm, varies linearly with DNase concentration. The binding of the substrate is not strong (K-m(app) in the 10 mM range), but the hydrolysis is rapid; 1 mug of DNase (free from other phosphodiesterases) can be assayed in 3 min after addition to a 10 mM substrate solution at pH 7.2, 10mM in MnCl2, and 1mM in CaCl2. All four bovine pancreatic DNases (A,B,C, and D) show the same relative activities toward DNA and toward the di-p-nitrophenyl ester; both activities are lost when DNase is inactivated by iodoacetate or by trypsin. The specificity of DNase toward the di-p-nitrophenyl substrate is different from that which has been established for the enzyme's predominant action on DNA or synthetic oligonucleotides, where a monoesterified phosphate group is formed at the 5'-position.     

Conjugation of penicillin acylase with the reactive copolymer of N-isopropylacrylamide: a step toward a thermosensitive industrial biocatalyst

Conjugation of penicillin acylase (PA) to poly-N-isopropylacrylamide (polyNIPAM) was studied as a way to prepare a thermosensitive biocatalyst for industrial applications to antibiotic synthesis. Condensation of PA with the copolymer of NIPAM containing active ester groups resulted in higher coupling yields of the enzyme (37%) compared to its chemical modification and copolymerization with the monomer (9% coupling yield) at the same NIPAM:enzyme weight ratio of ca. 35. A 10-fold increase of the enzyme loading on the copolymer resulted in 24% coupling yield and increased by 4-fold the specific PA activity of the conjugate. Two molecular forms of the conjugate were found by gel filtration on Sepharose CL 4B: the lower molecular weight fraction of ca. 10(6) and, presumably, cross-linked protein-polymer aggregates of MW > 10(7). Michaelis constant for 5-nitro-3-phenylacetamidobenzoic acid hydrolysis by the PA conjugate (20 microM) was found to be slightly higher than that of the free enzyme (12 microM), and evaluation of V(max) testifies to the high catalytic efficiency of the conjugated enzyme. PolyNIPAM-cross-linked PA retained its capacity to synthesize cephalexin from d-phenylglycin amide and 7-aminodeacetoxycephalosporanic acid. The synthesis-hydrolysis ratios of free and polyNIPAM-cross-linked enzyme in cephalexin synthesis were 7.46 and 7.49, respectively. Thus, diffusional limitation, which is a problem in the industrial production of beta-lactam antibiotics, can be successfully eliminated by cross-linking penicillin acylase to a smart polymer (i.e., polyNIPAM).