Human plasma kallikrein. Purification and preliminary characterization

A method is described for the convenient purification of the protease plasma kallikrein from human Cohn fraction IV-1. The enzyme was produced by endogenous activation after acid treatment to remove an inhibitor and was concentrated by the successive use of affinity adsorbents prepared by the immobilization of soybean trypsin inhibitor and aminobenzamidine. The esterase- and kinin-producing activities were enriched about 1100-fold from fraction IV-1.Several properties of plasma kallikrein strengthen the impression that it is related to trypsin, namely, competitive inhibition by benzamidine and the formation of a stable p-guanidinobenzoyl acyl enzyme intermediate. Inactivation by affinity labeling with Z-LysCH₂Cl was successful in contrast to the inertness of Tos-LysCH₂Cl.     

Synthesis, characterization and in vitro anti-cancer activity of N-(ferrocenyl)benzoyl tri- and tetrapeptide esters

N-ortho, N-meta and N-para-(ferrocenyl)benzoyl tri- and tetrapeptide esters (2-7) were prepared by coupling ortho, meta and para-ferrocenyl benzoic acids to the tri- and tetrapeptide ethyl esters of GlyGlyGly(OEt) and GlyGlyGlyGly(OEt) in the presence of N-(3-dimethylaminopropyl)-N′-ethylcarbodiimide hydrochloride and 1-hydroxybenzotriazole. The compounds were characterized by a range of NMR spectroscopic techniques, mass spectrometry and cyclic voltammetry. The anti-proliferative effects of the ortho derivatives 2 and 5 were measured in vitro against H1299 lung cancer cells and both gave IC₅₀ values greater than 50 μM. Therefore, extending the length of the peptide chain had a negative effect on activity, relative to N-(ferrocenyl)benzoyl amino acid and dipeptide derivatives.     

p-Toluenesulfonyldiazoacetates: Reagents for photoaffinity labeling

p-Toluenesulfonyldiazoacetyl chloride and p-nitrophenyl p-toluenesulfonyldiazoacetate have been prepared and offer potential advantages as reagents for photoaffinity labeling. (i) The extinction coefficient for the sulfonyldiazo compounds at 370 nm is about 10 times that for the long wavelength absorption of other diazoesters; this absorption permits reasonably rapid photolysis in the presence of compounds that are destroyed by short wavelength uv radiation. (ii) The two derivatives named above are stable thermally; furthermore, since sulfonyldiazoesters are stable to acid and to weak base, photoaffinity labeling can be conducted over a wide range of pH. (iii) Photolysis of ordinary (i.e., oxygen) esters of sulfonyldiazo compounds in methanol or cyclohexane leads to insertion into the solvent to the exclusion of Wolff rearrangement; photolysis of thioesters at 350 nm in methanol gives about 25% insertion into solvent, accompanied by about 75% Wolff rearrangement; in contrast, photolysis of most thioesters of diazo derivatives leads exclusively to Wolff rearrangement     

Affinity chromatography: purification of bovine trypsin and thrombin

Bovine trypsin has been purified by affinity chromatography on agarose beads containing covalently bound p-aminophenylguanidine, p-aminobenzamidine, or m-aminobenzamidine. Bovine thrombin was purified on a m-aminobenzamidine-agarose column containing a high concentration of the inhibitor. The values of the inhibition constant, K_i, for these inhibitors were determined for both enzymes and found to be 5–10 times poorer for thrombin than for trypsin. Only those benzamidines with low K_i values and coupled in high concentration to the agarose matrix were satisfactory for thrombin purification. Affinity-purified trypsin and thrombin were both greater than 90% active as measured by active site titration.     

Biotransformation of organic sulfides: Part. 10. Formation of chiral ortho- and meta-substituted benzyl methyl sulfoxides by biotransformation using Helminthosporium species NRRL 4671

Benzyl methyl sulfides substituted with methyl, chloro, cyano, bromo, methoxy, nitro and amino groups in the ortho or meta positions of the aromatic ring have been converted to (S) sulfoxides by biotransformation using the fungal biocatalyst Helminthosporium species NRRL 4671. The enantiomeric excesses for meta-substituted examples were high in those cases where the substituent was of a polar nature, and comparable to those observed for the corresponding para-substituted substrates. With one exception (o-amino), the ortho-substituted examples gave sulfoxides of lower enantiomeric purity. The role of a suitably located polar substituent on an aryl ring of the substrate in ensuring a high enantiomeric excess in sulfoxidation by Helminthosporium species has been confirmed by the biotransformations of 4-(methylthiomethyl)benzyl alcohol and 2-(4-nitrophenyl) ethyl methyl sulfide, which give sulfoxides of much higher optical purity than those obtained from the corresponding unsubstituted substrates.     

Inhibition of LPS-stimulated ROS production by fluorinated and hydroxylated chalcones in RAW 264.7 macrophages with structure-activity relationship study

Based on the importance of the previous fluorinated and/or hydroxylated chalcones studies, thirty-six compounds were designed as phenyl or hydroxyphenyl bearing fluoro, trifluoromethyl or trifluoromethoxy phenyl propenones and synthesized by applying modified Claisen-Schmidt condensation reaction as a single step. Inhibitory effects of the synthesized compounds on ROS production stimulated by LPS in RAW 264.7 macrophage were evaluated. Structure-activity relationship (SAR) study revealed that the compounds possessing para-hydroxyphenyl group combined with meta-fluoro or meta-trifluoromethyl phenyl group, and meta/para-hydroxyphenyl group combined with ortho-trifluoromethoxyphenyl group have an essential role in inhibiting the LPS-stimulated ROS production in RAW 264.7 macrophages. The most significant inhibitory effect on LPS-stimulated ROS production in RAW 264.7 macrophages was observed in compound 30 that possessed para-hydroxyphenyl group along with ortho-trifluoromethoxyphenyl group.     

Comparative study of hemolymph phenoloxidase activity in Aedes aegypti and Anopheles quadrimaculatus and its role in encapsulation of Brugia malayi microfilariae

Hemolymph phenoloxidase activity of sugar-fed and blood-fed females of Anopheles quadrimaculatus and Aedes aegypti showed similar characteristics. Phenoloxidase was present as an inactive proenzyme in both mosquito species and was partially activated during collection of the hemolymph. In both mosquito species, phenoloxidase activity was modulated by different buffers and activated phenoloxidase did not need Ca²⁺. Enzymatic activity was higher in the hemocytes than in the plasma in both mosquito species. Trypsin, laminarin, and blood-feeding on uninfected and Brugia malayi-infected jirds enhanced hemolymph phenoloxidase activity in both mosquito species. The appearance of hemolymph phenoloxidase activity was inhibited by p-nitrophenyl p′-guanidinobenzoate HCl, soybean trypsin inhibitor, ethylenediaminetetraacetic acid, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea and reduced glutathione, but not by benzamidine in A. quadrimaculatus. The appearance of hemolymph phenoloxidase activity was inhibited by benzamidine, diethyldithiocarbamic acid, saturated 1-phenyl-2-thiourea, reduced glutathione, β-nitrophenyl p′-guanidinobenzoate and soybean trypsin inhibitor, but not by ethylenediaminetetraacetic acid in A. aegypti. It is suggested that in both mosquito species, blood-feeding and migration of sheathed microfilariae in the homocoel activated the prophenoloxidase in the hemolymph and caused the encapsulation and melanization of microfilarial sheaths and microfilariae of B. malayi.     

Effects of cationic micelles on the rate of reaction of p-nitrophenyl esters with dihydrolipoic acid and dihydrolipoylamido derivatives

Dihydrolipoic acid (DHLA) 2a and its surfactant derivatives, trialkyl(2-lipoylamidoethyl) ammonium salts 2b-c, have been investigated, mainly in micellar solutions of cetyltrimethyl-ammonium bromide (CTABr), as esterolytic reagents toward p-nitrophenyl esters. The origins of the observed kinetic effects are discussed, and the reactivity of these reagents are compared with that of other thiolytic systems. The results indicate that DHLA, although not a surfactant, is effectively comicellized by CTABr, and micelles of CTABr and DHLA are among the most effective esterolytic systems, at moderately alkaline pHs, so far reported.     

Discovery of a novel class of 2-mercaptohexanoic acid derivatives as highly active PPARα agonists

A novel and robust scaffold for highly active PPARα agonists based on the 2-mercaptohexanoic acid substructure is presented. Systematic structural variation of the substitution pattern of the phenolic backbone yielded detailed SAR especially of ortho and meta substituents. We corroborated the importance of the sulfur atom as well as of the n-butyl chain for PPARα activity in the 2-mercaptohexanoic acid head group by preparation of carbon analogs and α-unsubstituted derivatives. Compound 10 represents a low nano molar active PPARα activator with excellent selectivity towards PPARγ.     

Syntheses of p-aminophenyl α-d-talopyranoside and 1-thio α-d-talopyranoside as analogs of glycosidase substrates

p-Nitrophenyl and p-aminophenyl α-d-talopyranoside and 1-thio-α-d-talopyranosides were prepared for studies on specificity of glycosidases. Reaction of α-d-talopyranose pentaacetate with p-nitrophenol gave exclusively p-nitrophenyl 2,3,4,6-tetra-O-acetyl-α-d-talopyranoside (2) in 63% yield. A similar reaction with p-nitrobenzenethiol afforded the 1-thio analog (3) of 2 in 41.8% yield; the p-nitrophenyl 2,3,4,6-tetra-O-acetyl-1-thio-β-d-talopyranoside (6) was also obtained in low yield (6.7%). The two α-d-talosides 2 and 3 were catalytically deacetylated in near-quantitative yields by methanolic sodium methoxide. The p-nitrophenyl α-d-talopyranoside (4) and 1-thio-α-d-talopyranoside (5) were reduced with palladium on barium sulfate catalyst to the corresponding p-aminophenyl talosides. The acetylated p-nitrophenyl d-talosides 2, 3, and 6 were determined, from their 250-MHz n.m.r. spectra, to exist in the ⁴C₁ (d) conformation in chloroform solution.     

Synthesis and biological evaluation of 2-phenol-4-chlorophenyl-6-aryl pyridines as topoisomerase II inhibitors and cytotoxic agents

A new series of 2-phenol-4-chlorophenyl-6-aryl pyridines were designed, synthesized, and evaluated for topoisomerase (topo) I and II inhibitory activities as well as cytotoxic activity against four different human cancer cell lines such as HCT15, T47D, DU145, and Hela. Most of the tested compounds exhibited stronger topo II inhibitory activity at 100 μM as compared to etoposide. All the compounds, except 39, did not show topo I inhibitory activity. Interestingly, compounds that showed better topo II inhibition than etoposide have ortho- or para-chlorophenyl at 4-position of central pyridine, and none of the compounds possess meta-chlorophenyl. SAR study revealed the importance of ortho- or para-chlorophenyl at 4-position of the central pyridine for selective topo II inhibitory activity. Similarly, all compounds possessing meta- or para-hydroxyphenyl moieties showed moderate to significant cytotoxic effects. Particularly, compounds 27–37, and 39 which showed excellent cytotoxicity (IC₅₀ = 0.68–1.25 μM) against T47D breast cancer cells suggest the importance of meta- or para-hydroxyphenyl moiety at 2-position of the central pyridine for the design of anticancer agents with related scaffolds.     

Thermodynamics of complexation of lanthanides by methoxybenzoates

The thermodynamic parameters of complexation of lanthanide cations by ortho-, meta- and para- methoxybenzoates have been measured using potentiometric and calorimetric techniques at 25 °C and an ionic strength of 0.10 M (NaClO₄). The values of logβ₁₀₁ correlate well with the ligand acid values of pK_a, reflecting the strongly ionic nature of the metal-ligand interaction. No evidence is found for extra charge polarization in these aromatic ligands due to the lanthanide complexation.     

Stereoselectivity in deacylation of nitrophenyl acylates by poly(ethylenimine) derivatives

Deacylation of nitrophenyl acetates containing carboxyl substituents [4-acetoxy-3-nitrobenzoic acid (1), 3-acetoxy-4-nitrobenzoic acid (2), and 2-acetoxy-5-nitrobenzoic acid (3)] was studied in the presence of poly(ethylenimine) derivatives. The polymers examined contained lauryl groups (Lau₁₂PEI) or both lauryl and imidazolyl groups (Lau₁₂Im₁₀PEI). The reaction with active ester proceeds through the attack of primary amino groups of the polymers at the acyl carbons of the substrates. The reaction of Lau₁₂Im₁₀PEI with a hydrophobic ester, p-nitrophenyl caproate (NPC), however, has been reported to involve the attack by the imidazolyl group of the polymer. Thus, the anionic (carboxyl-containing) and the hydrophobic esters bind to different domains on Lau₁₂Im₁₀PEI. Among the anionic substrates, 3 has uniquely large k_cat values compared with 1 or 2. This is explained in terms of closer proximity between a nucleophile amino group of the polymer and the scissile bond of the substrate in the polymer-substrate complex.     

A novel aryl-hydrazide from the marine lichen Lichina pygmaea: Isolation,synthesis of derivatives,and cytotoxicity assays

A new aryl-hydrazide l-glutamic acid derivative, pygmeine (3), was isolated from a methanolic extract of Lichina pygmaea, a marine lichen. Synthetic derivatives obtained via a two-step coupling of l-glutamic acid with phenylhydrazine moieties were useful to elucidate the structure of 3 and to carry out biological assays. Thus, the cytotoxicity of the ortho-, meta-, and para-hydroxyl isomers along with their respective benzyl intermediates, and a natural methoxylated analog, were evaluated on murine and human melanoma cells (B16, A375). The para-hydroxyl isomer 6 was found to be the most active (IC₅₀ = 1.6 μM) on B16 cells.     

Aryl 4-thioxylobioside and 1,4-dithioxylobiosides as effectors of the enzymic activity for fungal d-xylanases

Reaction of penta-O-acyl-4-thio-β-xylobiosyl bromide with either o-nitrophenol, p-nitrobenzenethiol, or p-aminobenzenethiol in acetone in the presence of potassium carbonate, followed by subsequent O-deacylation, led in excellent yield to o-nitrophenyl 4-thio-β-xylobioside (8), and p-nitrophenyl and p-aminophenyl 1,4-dithio-β-xylobiosides (10). Compound 8 behaved as a good chromogenic substrate for xylanases A and B from Sporotrichum dimorphosphorum with respective K_m of 2.4 and 6mm. The N-acetyl derivative of 10, as well as the free disaccharide 4-thioxylobiose, were competitive inhibitors for xylanase B (respective K_i of 2.5 and 0.72mm) but distinctly enhanced the rate of hydrolysis of d-xylan by xylanase A when incubated simultaneously with this substrate (respective K_A of 0.19 and 0.18mm); the rate of hydrolysis of the synthetic substrate 8 was not affected under similar conditions. These results suggest the possibility of a regulation mechanism for d-xylan hydrolysis by xylanase which may involve extracellular regulation by small oligosaccharides. The aminodithiodisaccharide 10 was coupled to CH-Sepharose 4B to give an affinity gel which retained xylanase activities.     

Sequence of a new Bowman-Birk inhibitor fromTorresea acreana seeds and comparison withTorresea cearensis trypsin inhibitor (TcTI2)

TaTI (Torresea acreana trypsin inhibitor), a new member of the Bowman-Birk trypsin inhibitor family, was purified from seeds ofTorresea acreana, one of the two known species ofTorresea, a Brazilian native Leguminosae of the Papilionoideae subfamily. Purification was performed by acetone fractionation, anion-exchange chromatography, and gel filtration. The TaTI appears asM _r 7000 in SDS-PAGE under reducing conditions. There are 63 amino acid residues present in the TaTI sequence, which was confirmed by mass spectrometry (8388 daltons). The putative reactive sites residues were Lys-15 and Arg-42 at the first and second site, respectively. The antibodies raised against TcTI2,Torresea cearensis trypsin inhibitor 2, showed a cross-reaction with TaTI, but not with other Bowman-Birk inhibitors purified from Leguminosae. The inhibition constants of TaTI and TcTI2 were comparable when measured against trypsin, chymotrypsin, and factor XIIa, but not on plasmin. The latter was tenfold more effectively inhibited by TcTI2 then by TaTI. Neither TaTI nor TcTI2 affects thrombin, plasma kallikrein, or factor Xa.     

Active site mapping of trypsin,thrombin and matriptase-2 by sulfamoyl benzamidines

The benzamidine moiety, a well-known arginine mimetic, has been introduced in a variety of ligands, including peptidomimetic inhibitors of trypsin-like serine proteases. According to their primary substrate specificity, the benzamidine residue interacts with the negatively charged aspartate at the bottom of the S1 pocket of such enzymes. Six series of benzamidine derivatives (1–73) were synthesized and evaluated as inhibitors of two prototype serine proteases, that is, bovine trypsin and human thrombin. As a further target, human matriptase-2, a recently discovered type II transmembrane serine protease, was investigated. Matriptase-2 represents an important regulatory protease in iron homeostasis by down-regulation of the hepcidin expression. Compounds 1–73 were designed to contain a fixed sulfamoyl benzamidine moiety as arginine mimetic and a linker-connected additional substructure, such as a tert-butyl ester, carboxylate or second benzamidine functionality. A systematic mapping approach was performed with these inhibitors to scan the active site of the three target proteases. In particular, bisbenzamidines, able to interact with both the S1 and S3/S4 binding sites, showed notable affinity. In branched bisbenzamidines 66–73 containing a third hydrophobic residue, opposite effects of the stereochemistry on trypsin and thrombin inhibition were observed.     

Potassium-p-nitrophenyl phosphate interactions with (Na+ + K+)-ATPase their relevance to phosphatase activity

The K⁺-dependent p-nitrophenylphosphatase activity catalyzed by purified (Na⁺ + K⁺)-ATPase from pig kidney shows substrate inhibition (K_i about 9.5 mM at 2.1 mM Mg²⁺). Potassium antagonizes and sodium favours this inhibition. In addition, K⁺ reduces the apparent affinity for substrate activation, whereas p-nitrophenyl phosphate reduces the apparent affinity for K⁺ activation. In the absence of Mg²⁺, p-nitrophenyl phosphate, as well as ATP, accelerates the release of Rb⁺ from the Rb⁺ occluded unphosphorylated enzyme. With no Mg²⁺ and with 0.5 mM KCl, trypsin inactivation of (Na⁺ + K⁺)-ATPase as a function of time follows a single exponential but is transformed into a double exponential when 1 mM ATP or 5 mM p-nitrophenyl phosphate are also present. In the presence of 3 mM MgCl₂, 5 mM p-nitrophenyl phosphate and without KCl the trypsin inactivation pattern is that described for the E₁ enzyme form; the addition of 10 mM KCl changes the pattern which, after about 6 min delay, follows a single exponential. These results suggest that (i) the shifting of the enzyme toward the E₁ state is the basis for substrate inhibition of the p-nitrophenulphosphatase acitivy of (Na⁺ + K⁺)-ATPase, and (ii) the substrate site during phosphatase activity is distinct from the low-affinity ATP site.     

Alkenyl substituted bicyclic nucleoside analogues retain nanomolar potency against Varicella Zoster Virus

Novel alkenyl substituted aryl bicyclic furano pyrimidines have been prepared and evaluated in vitro against Varicella Zoster Virus (VZV). The para-substituted analogues retain the nanomolar potency we have reported for p-alkyl analogues, while the ortho- and meta-alkenyl systems lose 3–4 orders of potency.     

Correlation of hydrogen-bonding propensity and anticancer profile of tetrazole-tethered combretastatin analogues

A series of 1,5-disubstituted tetrazole-tethered combretastatin analogues with extended hydrogen-bond donors at the ortho-positions of the aryl A and B rings were developed and evaluated for their antitubulin and antiproliferative activity. We wanted to test whether intramolecular hydrogen-bonding used as a conformational locking element in these analogues would improve their activity. The correlation of crystal structures with the antitubulin and antiproliferative profiles of the modified analogues suggested that hydrogen-bond-mediated conformational control of the A ring is deleterious to the bioactivity. In contrast, although there was no clear evidence that intramolecular hydrogen bonding to the B ring enhanced activity, we found that increased substitution on the B ring had a positive effect on antitubulin and antiproliferative activity. Among the various analogues synthesized, compounds 5d and 5e, having hydrogen-bonding donor groups at the ortho and meta-positions on the 4-methoxy phenyl B ring, are strong inhibitors of tubulin polymerization and antiproliferative agents having IC₅₀ value in micromolar concentrations.