Fluorescence labeling method for estimation of soluble polymers in the living material

The highly fluorescent derivatives of fluorescein, bearing the aliphatic primary amino groups, N-(2-aminoethylcarbonyl)-5(6)-aminofluorescein and 5-[N′-(2-aminoethyl)thioureido]fluorescein, were prepared for labeling of soluble polymers. The absorption and emission properties of these labels and polymers labeled with them were compared with properties of fluorescein and fluorescein isothiocyanate (FITC)-bovine serum albumin conjugate. Effects of the chemical structure of the polymer on the relative fluorescence quantum yield of a covalently attached label were evaluated using ionogenic, olefinic, or phenolic groups in side chains. The fluorescence of labeled polymers was adequate for their tracing in all the cases studied. The most pronounced quenching of fluorescence in the presence of phenolic groups is comparable with the quenching of fluorescence of FITC observed in FITC-protein conjugates. The long-term stability of the polymer-fluorochrome bond was checked in solutions of pH 2.10, 7.46, and 11.84; a higher stability of simple amide over amide plus the thiourea bond was found. The quantitative method of measurement of the concentration of labeled polymers in the biological material in a range of about 10 ng was developed; factors affecting the reproducibility are discussed.     

TMEDA-derived biscationic amphiphiles: An economical preparation of potent antibacterial agents

Bis-alkylated derivatives of N,N,N′,N′-tetramethylethylenediamine (TMEDA) represent a well-known class of versatile biscationic amphiphiles, owing to their low cost and ease of preparation. Asymmetric TMEDA derivatives, however, have been studied significantly less, particularly in regards to their antimicrobial properties. We have thus prepared a series of 36 mono- and bis-alkylated TMEDA derivatives to evaluate their inhibition of bacterial growth. This series of compounds showed low micromolar activity against a panel of four bacteria. Optimal inhibition was observed when the biscationic amphiphiles possessed modest asymmetry and were composed of between 20 and 24 total carbon atoms in the side chains. These amphiphiles were prepared in a simple two-step procedure, utilizing inexpensive materials and atom-economical reactions, making them practical for further development.     

Aminopeptidase C of Aspergillus niger Is a Novel Phenylalanine Aminopeptidase

A novel enzyme with a specific phenylalanine aminopeptidase activity (ApsC) from Aspergillus niger (CBS 120.49) has been characterized. The derived amino acid sequence is not similar to any previously characterized aminopeptidase sequence but does share similarity with some mammalian acyl-peptide hydrolase sequences. ApsC was found to be most active towards phenylalanine β-naphthylamide (F-βNA) and phenylalanine para-nitroanilide (F-pNA), but it also displayed activity towards other amino acids with aromatic side chains coupled to βNA; other amino acids with nonaromatic side chains coupled to either pNA or βNA were not hydrolyzed or were poorly hydrolyzed. ApsC was not able to hydrolyze N-acetylalanine-pNA, a substrate for acyl-peptide hydrolases.     

Discovery of benzimidazole derivatives as orally active renin inhibitors: Optimization of 3,5-disubstituted piperidine to improve pharmacokinetic profile

We previously identified 2-tert-butyl-4-[(3-methoxypropyl)amino]-N-(2-methylpropyl)-N-[(3S,5R)-5-(morpholin-4-ylcarbonyl)piperidin-3-yl]pyrimidine-5-carboxamide 3 as a potent renin inhibitor. Since 3 showed unacceptably low bioavailability (BA) in rats, structural modification, using SBDD and focused on physicochemical properties was conducted to improve its PK profile while maintaining renin inhibitory activity. Conversion of the amino group attached at the 4-position of pyrimidine to methylene group improved PK profile and decreased renin inhibitory activity. New central cores with carbon side chains were explored to improve potency. We had designed a series of 5-membered azoles and fused heterocycles that interacted with the lipophilic S3 pocket. In the course of modification, renin inhibitory activity was enhanced by the formation of an additional hydrogen bonding with the hydroxyl group of Thr77. Consequently, a series of novel benzimidazole derivatives were discovered as potent and orally bioavailable renin inhibitors. Among those, compound 13 exhibited more than five-fold of plasma renin inhibition than aliskiren in cynomolgus monkeys at dose ratio.     

Aeromonas neutral protease: Specificity toward extended substrates

Kinetic measurements on the action of Aeromonas neutral protease toward blocked peptide substrates were made in order to determine the most favorable fit on the enzyme subsites that bind the residues flanking the scissile bond and to define the number of secondary sites involved in catalysis. Variations in the identity of P₁′,³ the residue furnishing the amino group to the scissile bond, produced significant changes in k_rmcat, whereas the identity of P₁′, the residue donating the carboxyl group, was of much less catalytic importance. Comparison of these results with those of previous investigators of other bacterial neutral proteases indicated distinct differences in specificity of the Aeromonas enzyme and revealed that phenylalanyl residues, rather than leucyl, were preferred in the P₁′ position. Additional binding sites on the carboxyl side of the scissile bond were shown to be important to catalytic efficiency and it is evident that at least three residues (P₁t$\text{,}\text{?}$ P₂′, P₃′) are involved while only two residues (P₂, P₁) on the amino terminal side of the sensitive bond are implicated.     

Enhancing the catalytic repertoire of nucleic acids. II. Simultaneous incorporation of amino and imidazolyl functionalities by two modified triphosphates during PCR

The incorporation of potentially catalytic groups into DNA is of interest for the in vitro selection of novel deoxyribozymes. We have devised synthetic routes to a series of three C7 modified 7-deaza-dATP derivatives with pendant aminopropyl, Z-aminopropenyl and aminopropynyl side chains. These modified triphosphates have been tested as substrates for Taq polymerase during PCR. All the modifications are tolerated by this enzyme, with the aminopropynyl side chain giving the best result. Most protein enzymes have more than one type of catalytic group located in their active site. By using C5-imidazolyl-modified dUTPs together with 3-(aminopropynyl)-7-deaza-dATP in place of the natural nucleotides dTTP and dATP, we have demonstrated the simultaneous incorporation of both amino and imidazolyl moieties into a DNA molecule during PCR. The PCR product containing the four natural bases was fully digested by XbaI, while PCR products containing the modified 7-deaza-dATP analogues were not cleaved. Direct evidence for the simultaneous incorporation during PCR of an imidazole-modified dUTP and an amino-modified 7-deaza-dATP has been obtained using mass spectrometry.     

Cytokinin structure-activity relationships and the metabolism of N-(delta-isopentenyl)adenosine-8-C in phaseolus callus tissues

The activities of the free base and ribonucleoside forms of cytokinins bearing saturated and unsaturated N⁶-isoprenoid side chains have been examined in callus cultures derived from Phaseolus vulgaris cv. Great Northern, P. lunatus cv. Kingston, and the interspecific hybrid Great Northern × Kingston. In callus of cv. Great Northern, cytokinins bearing saturated side chains (N⁶-isopentyladenine, N⁶-isopentyladenosine, dihydrozeatin, and ribosyldihydrozeatin) were always more active than the corresponding unsaturated analogs (N⁶-[Δ²-isopentenyl]adenine, N⁶-[Δ²-isopentenyl]adenosine, zeatin, and ribosylzeatin). In callus of cv. Kinston, the cytokinins bearing unsaturated side chains were either more active or equally as active as the saturated compounds. These differences in cytokinin structure-activity relationships were correlated with differences in the metabolism of ¹⁴C-N⁶-(Δ²-isopentenyl)adenosine. In Great Northern tissues, this cytokinin was rapidly degraded to adenosine; in Kingston tissues, the major metabolite was the corresponding nucleotide. The growth responses of callus of the interspecific hybrid were intermediate between the parental tissues, and the metabolism of ¹⁴C-N⁶-(Δ²-isopentenyl)adenosine by the hybrid callus exhibited characteristics of both parental tissues. The results are consistent with the hypothesis that the weak activity of cytokinins with unsaturated side chains in promoting the growth of Great Northern callus is due to the rapid conversion of these cytokinins to inactive metabolites.     

Novel interleukin-5 inhibitors based on hydroxyethylaminomethyl-4H-chromen-4-one scaffold

Hydroxyethylaminomethyl-4H-chromenones were previously discovered as fairly strong IL-5 inhibitor. For determination of detail structure activity relationship, N-substituted hydroxyethylaminomethylchromenones 4a–n were prepared and evaluated for their IL-5 inhibitory activity. Shifting the hydrophobic group to nitrogen from 1-position of hydroxyethylamino moiety of hydroxyethylaminomethyl-4H-chromenones enhances the activity. The increment in bulkiness or hydrophobicity of alkyl side chain at amino group increases the activity. The same level of activity of 5-(cyclohexylmethoxy)-3-(N-benzyl-2-hydroxyethylaminomethyl)-4H-chromenone analogs regardless of hydrophobic or hydrophilic substituents at 4th position of phenyl ring might infer the existence of tunnel structure in the putative receptor for accepting these side chains.     

Function of Mannan Chains of Yeast Repressible Acid Phosphatase on Its Enzymatic Properties

To find the function of the mannan chains covalently attached to yeast repressible acid phosphatase, the N-glycosidic carbohydrate chains were removed by endo-β-N-acetyl-glucosaminidase H under native conditions. Almost all of the N-glycosidic mannan chains were cleaved off by the glycosidase. The deglycosylated enzyme was shown to be a dimer structure as is the native enzyme. The deglycosylated enzyme retained enzyme activity, the same Km, and the same circular dichroism spectra as the native enzyme. These results indicate that the carbohydrate chains are not essential for maintaining the active enzyme structure, but the deglycosylated enzyme was shown to be more sensitive to acidic pH and high temperature.     

Block copolymerization of α-amino acid N-carboxyanhydride initiated by vinyl polymers having a terminal amino group and the characterization of the block copolymers

Poly(methyl methacrylate) and polystyrene having terminal amino groups were synthesized by the radical polymerization of those monomers in the presence of 2-mercaptoethylammonium chloride as a chain-transfer agent. By the terminal group analysis and the molecular weight determination of the polymers, 0.5–1.3 amino groups were found in a chain of poly(methyl methacrylate) and 0.5–2.5 amino groups in a chain of polystyrene. Using these polymers having a terminal amino group as an initiator, the block polymerization of α-amino acid N-carboxyanhydride (NCA) was carried out. In the polymerizations of Glu(OBzl) NCA and Lys(Z) NCA by the poly(methyl methacrylate) initiator, the terminal amino group underwent a nucleophilic addition reaction to NCA and initiated the polymerization, yielding A-B-type block copolymers in a high yield. The same was observed in the polymerizations of Gly(OBzl) NCA and Lys(Z) NCA by the polystyrene initiator. By eliminating the protecting groups of the side chains of the polypeptide segment, the block copolymers poly(methyl methacrylate)-poly(Glu), poly(methyl methacrylate)-poly(Lys), polystyrene-poly(Glu) and polystyrene-poly(Lys) were synthesized with little side reactions. The side chain amino groups of poly(Lys) segment in the poly(methyl methacrylate)-poly(Lys) block copolymers were sulphonated or stearoylated successfully.     

Crystal structures of complexes of bacterial DD-peptidases with peptidoglycan-mimetic ligands: the substrate specificity puzzle

The X-ray crystal structures of covalent complexes of the Actinomadura R39 dd-peptidase and Escherichia coli penicillin-binding protein (PBP) 5 with β-lactams bearing peptidoglycan-mimetic side chains have been determined. The structure of the hydrolysis product of an analogous peptide bound noncovalently to the former enzyme has also been obtained. The R39 dd-peptidase structures reveal the presence of a specific binding site for the d-α-aminopimelyl side chain, characteristic of the stem peptide of Actinomadura R39. This binding site features a hydrophobic cleft for the pimelyl methylene groups and strong hydrogen bonding to the polar terminus. Both of these active site elements are provided by amino acid side chains from two separate domains of the protein. In contrast, no clear electron density corresponding to the terminus of the peptidoglycan-mimetic side chains is present when these β-lactams are covalently bound to PBP5. There is, therefore, no indication of a specific side-chain binding site in this enzyme. These results are in agreement with those from kinetics studies published earlier and support the general prediction made at the time of a direct correlation between kinetics and structural evidence. The essential high-molecular-mass PBPs have demonstrated, to date, no specific reactivity with peptidoglycan-mimetic peptide substrates and β-lactam inhibitors and, thus, probably do not possess a specific substrate-binding site of the type demonstrated here with the R39 dd-peptidase. This striking deficiency may represent a sophisticated defense mechanism against low-molecular-mass substrate-analogue inhibitors/antibiotics; its discovery should focus new inhibitor design.     

Specificity and stereospecificity of α-chymotrypsin

1. The optically pure p-nitrophenyl esters of the d and l enantiomers of N-acetyl-tryptophan, N-acetylphenylalanine and N-acetyl-leucine, and the p-nitrophenyl ester of N-acetylglycine, have been prepared. 2. These materials are all substrates of α-chymotrypsin, and the rates of deacylation of the corresponding acyl-α-chymotrypsins have been determined. 3. As the size of the amino acid side chain increases, the l series deacylate progressively faster than the N-acetylglycyl-enzyme, and the d series progressively more slowly. 4. The results are interpreted in terms of a three-locus model of the enzyme's active site, which accounts for the interrelationship between substrate specificity and stereospecificity observed. 5. The concepts of negative specificity and of specificity saturation are introduced.     

Side chain interactions in aromatic dipeptides

A study of the 100-MHZ nuclear magnetic resonace spectra in D₂O solution was made of a series of linear dipeptides of the types L -phenylalanine-L -and-D -X, and L -phenylalanine-L -and-D -Y, where X comprised a group of amino acid residues with polar side chains (X = glutamine, glutamic acid, arginine, and N^ε-acetyllysine) and Y comprised amino acid residues with purely aliphatic side chains (Y = α-aminobutyric acid and norvaline). It was found that regardless of the side chain length, resonances due to the α-methylene protons in the X and Y side chains of the L -Phe-D -Y series consistently exhibited upfield shifts greater than any other protons in these side chains, when compared to the corresponding side chain resonances of the nonaromatic dipeptide series L -Ala-L -X and L -Ala-L -Y. The magnitudes of these shielding effects were consistently and considerably greater for the L -Phe-D -X series than for the L -Phe-D -Y series. An intramolecular complex–formed by association of armatic π-electrons with the positive end of the dipole in the polar side chains—was proposed as one plausible interpretation of the enhanced shielding effects. An increase in temperature from 32 to 70–80° was sufficient to overcome the enhanced shielding attributable to the suggested complex.     

Glycoproteines sulfates des membranes de l'oeuf de poule et de l'oviducte Isolement et caracterisation de glycopeptides sulfatesSulfated glycoproteins form egg shell membranes and hen oviduct. Isolation and characterization of sulfated glycopeptides

Sulfated glycopeptides were isolated from pronaisc and tryptic digests of egg shell membranes and hen oviduct. They were precipitated by cationic detergents and separated by preparative electrophoresis, after removal of small quantities of glucuronoglycosaminoglycans detected only in the oviduct (isthmus and magnum). The principal isolated sulfated glycopeptides were divided according to increasing electrophoretic mobilities into two groups A and B. The homogeneity of the purified glycopeptides was confirmed by gel filtration and polyacrylamide gel electrophoresis.Glycopeptides from pool preparation of tissue are analysed and carbohydrate and amino acids average values are estimated. Hexosamines (mainly N-acetylglucosamine), hexoses (galactose, glucose, mannose) and fucose were found in Glycopeptides A. The molar ratio of hexose/hexosamine was 0.4. N-Acetylneuraminic acid and sulfate were also present in Glycopeptides A. The molar ratio of sulfate/hexosamine ranged from 0.1 to 0.25. The Glycopeptides A composition indicated the presence of chains with many glycosyl groups and a few of amino acids residues. The carbohydrate components of Glycopeptides B from egg shell membranes and magnum were found to be hexosamines (N-acetylgalactosamine and N-acetylglucosamine in equimolar proportions), hexoses (galactose mainly and glucose), N-acetylneuraminic acid, and fucose. The molar ratio of hexose/hexosamine was 1. Sulfate was also present and the molar ratio of N-acetylneuraminic acid and sulfate to hexosamine was ranged from 0.8 to 1. The main amino acid residues in these glycopeptides were serine and threonine with destruction of these hydroxyamino acids during alkali treatment. Glycopeptides B probably consist of short carbohydrate chains, linked to the polypeptide through O-glycosidic bonds involving N-acetylgalactosamine and serine and threonine. Approximately 40% of the amino acid residues were linked to carbohydrate chains.Glycopeptides B from egg shell membranes magnum and egg white were very similar in their carbohydrate and amino acid composition and in their properties.Gylcopeptides A from egg shell membranes, isthmus and magnum showed similarities and divergences especially in the amino acid composition. These results suggest that magnum and isthmus in oviduct are both concerned with the synthesis of egg shell membrane glycoproteins.     

A new method for degradation of the protein part of glycoproteins: isolation of the carbohydrate chains of asialofetuin

A new method has been developed for degrading the protein part of several glycoproteins, whilst leaving the carbohydrate portion virtually intact apart from partial degradation at the reducing end. The method is based upon stabilization of the glycosidic linkages of the sugar residues by trifluoroacetyl groups and subsequent cleavage of the peptide bonds by transamidation. The two reactions are carried out in a mixture of trifluoroacetic anhydride and trifluoroacetic acid. After O- and N-detrifluoroacetylation, the carbohydrate portion can be isolated and re-N-acetylated. The applicability of the method is demonstrated by the isolation from asialofetuin of the carbohydrate chains that are attached by N- and O-glycosyl links.     

An allosteric circuit in caspase-1

Structural studies of caspase-1 reveal that the dimeric thiol protease can exist in two states: in an on-state, when the active site is occupied, or in an off-state, when the active site is empty or when the enzyme is bound by a synthetic allosteric ligand at the dimer interface ∼ 15 Å from the active site. A network of 21 hydrogen bonds from nine side chains connecting the active and allosteric sites change partners when going between the on-state and the off-state. Alanine-scanning mutagenesis of these nine side chains shows that only two of them—Arg286 and Glu390, which form a salt bridge—have major effects, causing 100- to 200-fold reductions in catalytic efficiency (k_cat/K_m). Two neighbors, Ser332 and Ser339, have minor effects, causing 4- to 7-fold reductions. A more detailed mutational analysis reveals that the enzyme is especially sensitive to substitutions of the salt bridge: even a homologous R286K substitution causes a 150-fold reduction in k_cat/K_m. X-ray crystal structures of these variants suggest the importance of both the salt bridge interaction and the coordination of solvent water molecules near the allosteric binding pocket. Thus, only a small subset of side chains from the larger hydrogen bonding network is critical for activity. These form a contiguous set of interactions that run from one active site through the allosteric site at the dimer interface and onto the second active site. This subset constitutes a functional allosteric circuit or “hot wire” that promotes site-to-site coupling.     

Structural insights by molecular dynamics simulations into differential repair efficiency for ethano-A versus etheno-A adducts by the human alkylpurine-DNA N-glycosylase

1,N⁶-ethenoadenine adducts (εA) are formed by known environmental carcinogens and found to be removed by human alkylpurine-DNA N-glycosylase (APNG). 1,N⁶-ethanoadenine (ΕA) adducts differ from εA by change of a double bond to a single bond in the 5-member exocyclic ring and are formed by chloroethyl nitrosoureas, which are used in cancer therapy. In this work, using purified recombinant human APNG, we show that ΕA is a substrate for the enzyme. However, the excision efficiency of ΕA was 65-fold lower than that of εA. Molecular dynamics simulation produced similar structural motifs for εA and ΕA when incorporated into a DNA duplex, suggesting that there are no specific conformational features in the DNA duplex which can account for the differences in repair efficiency. However, when ΕA was modeled into the APNG active site, based on the APNG/εA-DNA crystallographic coordinates, in structures produced by 2 ns molecular dynamics simulation, we observed weakening in the stacking interaction between ΕA and aromatic side chains of the key amino acids in the active site. In contrast, the planar εA is better stacked at the enzyme active site. We propose that the observed destabilization of the ΕA adduct at the active site, such as reduced stacking interactions, could account for the biochemically observed weaker recognition of ΕA by APNG as compared to εA.     

Construction of chiral polyesters from polycondensation of multifunctional monomer containing both flexible amino acid and rigid pendant groups with aromatic diols

A number of chiral wholly aromatic polyesters (PEs) with phthalimido and flexible chiral unit in the backbone were prepared from a chiral synthesized diacid monomer, 5-(3-methyl-2-phthalimidylpentanoylamino)isophthalic acid (1), and various aromatic diols via the polyesterification reaction. The tosyl chloride/pyridine/N,N-dimethylformamide (DMF) system was used as a condensing agent. All of the these polymers having bulky phthalimido and amino acid functionalities in the side chain showed excellent solubility and readily dissolved in various solvents such as N-methyl-2-pyrrolidinone, N,N-dimethylacetamide and DMF. Since, these chiral polymers have natural amino acids in the polymer architecture, they are expected to be biodegradable and therefore may be classified under eco-friendly polymers. They had useful levels of thermal stability associated with excellent solubility. Thermogravimetric analysis (TGA) showed that the obtained PEs are rather thermally stable, 10% weight loss temperatures in excess of 317°C, and char yields at 700°C in the nitrogen atmosphere higher than 24%. The resulting polymers were obtained in good yields with inherent viscosities ranging between 0.22 and 0.56 dL/g and were characterized with FT-IR, ¹H-NMR, elemental and TGA techniques.     

A Remote Palm Domain Residue of RB69 DNA Polymerase Is Critical for Enzyme Activity and Influences the Conformation of the Active Site

Non-conserved amino acids that are far removed from the active site can sometimes have an unexpected effect on enzyme catalysis. We have investigated the effects of alanine replacement of residues distant from the active site of the replicative RB69 DNA polymerase, and identified a substitution in a weakly conserved palm residue (D714A), that renders the enzyme incapable of sustaining phage replication in vivo. D714, located several angstroms away from the active site, does not contact the DNA or the incoming dNTP, and our apoenzyme and ternary crystal structures of the Pol^D714A mutant demonstrate that D714A does not affect the overall structure of the protein. The structures reveal a conformational change of several amino acid side chains, which cascade out from the site of the substitution towards the catalytic center, substantially perturbing the geometry of the active site. Consistent with these structural observations, the mutant has a significantly reduced k_pol for correct incorporation. We propose that the observed structural changes underlie the severe polymerization defect and thus D714 is a remote, non-catalytic residue that is nevertheless critical for maintaining an optimal active site conformation. This represents a striking example of an action-at-a-distance interaction.     

1,2,3-Triazole analogs of combretastatin A-4 as potential microtubule-binding agents

A series of cis-restricted 1,4- and 1,5-disubstituted 1,2,3-triazole analogs of combretastatin A-4 (1) have been prepared. Cytotoxicity and tubulin inhibition studies showed that 2-methoxy-5-((5-(3,4,5-trimethoxyphenyl)-1H-1,2,3-triazol-1-yl)methyl)aniline (5e) and 2-methoxy-5-(1-(3,4,5-trimethoxybenzyl)-1H-1,2,3-triazol-5-yl)aniline (6e) were two of the most active compounds. Molecular modeling studies revealed that the N-2 and N-3 atoms in the triazole rings in 5e and 6e did not form hydrogen bonds with the amino acids in the anticipated pharmacophore.