Determination of asparagine and glutamine in polypeptides using bis(I,I-trifluoroacetoxy)iodobenzene

A simple yet accurate method is described by which the numbers of asparagine and glutamine residues in polypeptides can be determined. The method involves difference analysis of the aspartic acid and glutamic acid contents of the polypeptide after acid hydrolysis (in 6 n HCl), without and with prior treatment of the sample with bis(I,I-trifluoroacetoxy)iodobenzene. Under the conditions described, this reagent quantitatively converts the carboxamide residues to the corresponding amines, which are eluted near (and interfere with the estimation of) the lysine peak on conventional ion-exchange amino acid analysis. During the carboxamide conversion, certain amino acid residues sensitive to oxidation are partially or completely destroyed and cannot be accurately determined.     

Fluorometric quantitation of amino sugars in the picomole range.

A fluorometric method has been developed for the convenient and quantitative assay of amino sugars over the concentration range of 10 nm to 6 mm. Linear results are obtained for reaction mixtures containing 6 pmol to 60 nmol hexosamine. The procedure involves the condensation of amino sugars with the fluorogenic reagent o-phthalaldehyde, at alkaline pH in the presence of 2-mercaptoethanol. Relative fluorescence intensities are then determined using excitation and emission wavelengths of 340 and 455 nm, respectively. The presence of 2-mercaptoethanol in reaction mixtures not only enhanced sensitivity of the assay, but also defined the excitation/emission spectra. Under the conditions described, amino acids were also found to react with o-phthalaldehyde, yielding fluorescence intensities similar to those of amino sugars. These results suggest the applicability of fluorescence techniques in automated amino sugar analyses, as well as the potential interference of other compounds containing primary amines.     

Discovery of a class of diheteroaromatic amines as orally bioavailable CDK1/4/6 inhibitors

The discovery of a class of diheteroaromatic amines based on LY2835219 as cyclin-dependent kinase (CDK1/4/6) inhibitors was described. The series was found to have much more improved CDK1 inhibition and potent in vitro anti-proliferative effects against cancer cell lines. The synthesis and structure–activity relationship studies of these compounds were reported. One promising compound was selected to evaluate as a novel lead compound after in vitro and in vivo profiling.     

Spectrophotometric method for estimation of aliphatic primary amines in biological samples

A method based on Rimini test for aliphatic amines was studied and developed for quantitative estimation of aliphatic primary amines. The method involves action of the amine with acetone to form schiff base which complexes with sodium nitroprusside to give violet colour. The absorption maximum in the visible range of the spectrum, for the reaction mixture was found to be 550 nm. The pH (8–11) and reaction time scan for the assay were optimized. A linear relation of concentration (0.2–3 mg/mL) of amine against absorbance at 550 nm was established. Interference due to other reaction components was negligible (±0.02 mg/mL) as compared to the sample in buffer. 1, 3-dimethyl butylamine was used as the model amine and the method was applied to other amines; it was observed that when electron-withdrawing substituents are present in the molecule the reaction is retarded, as the incubation time was longer. This method is useful for estimation of aliphatic primary amine in biological samples.     

Chromatographic analysis of amino acids and primary amines with o-phthalaldehyde detection

The applicability of the o-phthalaldehyde reaction with fluorometric detection to the simultaneous chromatographic analysis of amines and nonprotein amino acids is demonstrated. The majority of the compounds tested could be quantitatively determined with high sensitivity. The method is particularly well suited for the analysis of β-amino acids. Amino compounds lacking an α-hydrogen atom are detected with lower sensitivity, due to a lower relative fluorescence. Secondary amino compounds are undetectable with this system.     

A chromatographic technique for the analysis of oxidized metabolites: Application to carcinogenic N-hydroxyarylamines in urine

A new chromatographic detection method for oxidized metabolites has been developed based on the reaction of eluted compounds with an Fe⁺³-bathophenanthroline colorimetric reagent in a postcolumn reactor. The method is sensitive to N-hydroxyarylamines, aryldiamines, phenolic amines, and ascorbic acid. It has been applied to the analysis of toxic N-oxidized metabolites in rhesus monkey urine after the animals were dosed with the bladder carcinogens, 1- and 2-napthylamine. These compounds are oxidized to the corresponding N-hydroxyarylamines in the liver, conjugated as the N-glucuronide, and excreted in the urine. The N-glucuronide has been shown to undergo acidic hydrolysis in the urine to release the free N-hydroxyarylamine, an ultimate carcinogen for the induction of bladder tumors. In this study, the N-hydroxy-N-glucuronide of 2-naphthylamine was found to be excreted at a rate that was 6.8 times that of the 1-naphthylamine isomer. This is consistent with the much higher carcinogenic potency of 2-naphthylamine in a variety of species.     

Nitrogen-containing non-steroidal secondary metabolites of Solanum, Cyphomandra, Lycianthes and Margaranthus

Of the 49 species of Solanum studied, cuscohygrine has been detected in 25, solamine and related amines in 17 and solamine-derived amides in 16. Five species of Cyphomandra examined all contained both amines and amides. From roots of Margaranthus solanaceus cuscohygrine has been isolated which probably occurs, too, in roots of Lycianthes rantonnettii. The distribution of these compounds throughout the taxa could be of chemotaxonomic value.     

Staining for protease activity on polyacrylamide gels

A high-performance liquid chromatographic procedure has been developed for the detailed analysis of amino acids and related compounds in 10-μl samples of perilymph from the guinea pig cochlea (inner ear). The procedure employs an Aminco amino acid analyzer and combines the use of a single chromatographic column, lithium citrate buffers for elution, a change of column temperature, and fluorometric detection of o-phthaldialdehyde/2-mercaptoethanol adducts of primary amines. Sensitivity is about 0.2 pmol referenced to leucine. Fifty-four primary amine components are detectable in perilymph collected in relative silence. Twentynine compounds have been identified, and six are putative amino acid neurotransmitters. The present method provides new information about the chemical composition of perilymph and is suitable for the analysis of physiological fluids available only in volumes of several microliters.     

Mutagenicity of benzidine and 4-aminobiphenyl after metabolic activation with isolated hepatocytes and liver 9000 × g supernatant from rat,hamster and guinea pig

The mutagenicity of benzidine and 4-aminobiphenyl towards Salmonella typhimurium strain TA1538 was measured in the presence of isolated hepatocytes from rat, hamster and guinea pig. The mutagenic potency of these compounds was also assayed with S9 (9000 × g supernatant) prepared from disrupted hepatocytes of these aryl amines was investigated.For all 3 animal species it was found that the mutagenicity of benzidine is higher with intact hepatocytes than with S9 prepared from disrupted hepatocytes. Addition of acetyl coenzyme A to the S9 fraction increased the mutagenicity of benzidine. In contrast to benzidine, the mutagenicity of 4-aminobiphenyl appeared to be lower with hepatocytes than with S9. Addition of acetyl coenzyme A to the S9 fraction decreased the mutagenicity of 4-aminobiphenyl.The mutagenic potency of 4-aminobiphenyl was almost equal in the presence of the liver preparations from the 3 different species, whereas obvious species differences were seen with benzidine.     

Amine substitution of quinazolinones leads to selective nanomolar AChE inhibitors with ‘inverted’ binding mode

Selective and nanomolar acetylcholinesterase inhibitors were obtained by connecting tri- and tetracyclic quinazolinones—previously described as moderately active and unselective cholinesterase (ChE) inhibitors—via a hydroxyl group in para position to an anilinic nitrogen with different amines linked via a three carbon atom spacer. These tri- and tetracyclic quinazolinones containing different alicyclic ring sizes and connected to tertiary amines were docked to a high-resolution hAChE crystal structure to investigate the preferred binding mode in relation to results obtained by experimental structure–activity relationships. While the ‘classical orientation’ locating the heterocycle in the active site was rarely found, an alternative binding mode with the basic aliphatic amine in the active center (‘inverted’ orientation) was obtained for most compounds. Analyses of extended SARs based on this inverted binding mode are able to explain the compounds’ binding affinities at AChE.     

Synthesis, multinuclear magnetic resonance and crystal structures of Pt(II) complexes containing amines and bidentate carboxylate ligands

A novel synthetic method for the synthesis of the complexes cis-Pt(amine)₂R(COO)₂ is compared to two other methods involving the use of either barium dicarboxylate or sodium carboxylate. Pt(II) compounds with monodentate and bidentate amines were studied. The reaction involves the use of a silver dicarboxylato complex, which is the intermediate in the new synthetic procedure. The crystal structure of the silver intermediate with the ligand 1,1-cyclobutanedicarboxylate (1,1-CBDCA) was determined by X-ray diffraction. The crystal Ag₂(1,1-CBDCA) has a very interesting 3-D extended structure. The complexes cis-Pt(amine)₂R(COO)₂ were studied in solution by multinuclear (¹H, ¹³C and ¹⁹⁵Pt) magnetic resonance spectroscopy, but the solubilities are very low. D₂O was found to be the best solvent. In ¹⁹⁵Pt NMR, the complexes containing bidentate amines forming five-membered chelates were observed at higher fields than those containing monodentate amines. The resonances of the NH₃ compounds were also found at lower fields than the primary amine complexes. All the dicarboxylato ligands form six-membered chelates except 1,2-CBDCA, whose Pt(II) compounds were observed at lower fields than the others. The crystal structures of Pt(en)(1,1-CBDCA), Pt(Meen)(1,1-CBDCA) and Pt(en)(benzylmalonato) were confirmed by X-ray diffraction methods. Several compounds are disordered. The crystals are stabilized by intermolecular hydrogen bonds between the -NH₂ groups and the carboxylato O atoms.     

N,N-Bis(glycityl)amines as anti-cancer drugs

A series of N,N-bis(glycityl)amines with promising anti-cancer activity were prepared via the reductive amination of pentoses and hexoses, and subsequently screened for their ability to selectively inhibit the growth of cancerous versus non-cancerous cells. For the first time, we show that this class of compounds possesses anti-proliferative activity, and, while the selective killing of brain cancer (LN18) cells versus matched (SVG-P12) cells was modest, several of the amines, including d-arabinitylamine 1a and d-fucitylamine 1g, exhibited low micromolar IC₅₀ values for HL60 cells. Moreover, these two amines showed good selectivity towards HL60 cells when compared to non-cancerous HEK-293 cells. The compounds also showed low micromolar inhibition of the leukaemic cell line, THP-1. The modes of action of amines 1a and 1g were then determined using yeast chemical genetics, whereby it was established that both compounds affect similar but distinct sets of biochemical pathways. Notably purine nucleoside monophosphate biosynthesis was identified as an enriched mechanism. The rapid synthesis of the amines and their unique mode of action thus make them attractive targets for further development as anti-cancer drugs.     

Development of a peroxidase-coupled fluorometric assay for lysyl oxidase

Lysyl oxidase catalyzes the oxidation of peptidyl lysine in elastin and collagen and also acts upon nonpeptidyl amines, although the enzyme becomes slowly inactivated while processing nonpeptidyl substrates. In spite of this complexity, it has been possible to devise a continuously monitored peroxidase-coupled fluorometric assay for the oxidation of simple amines by lysyl oxidase. In the present study, optimal assay conditions have been explored and found to include assay temperatures of 50 to 60°C, the presence of urea in the assay, and the use of diaminopentane as substrate. Although the assay is subject to interference by contaminating macromolecules in enzyme fractions, a linear assay response to enzyme concentration is obtained with highly purified lysyl oxidase with a limiting sensitivity of 0.3 μg of enzyme per assay.     

Chemical induction of poly-cis carotenoid biosynthesis

A new class of synthetic bioregulators is reported which cause the accumulation of poly-cis carotenoids in the flavedo of Marsh white seedless grapefruit. The compounds tested were all secondary amines: dibenzylamine, substituted dibenzylamines (4-F; 4-Cl; 4-Br; 2-, 3-and 4-Me; 4-NO₂; 4-CN; 4-Cl, 4′-Me; 4-Me, 4′-NO₂), N-benzyl phenethylamine and N-benzyl 2-naphthalenemethylamine. The most effective, 4-chlorodibenzylamine, caused the accumulation of 74μgg/g dry wt of poly-cis carotenoids. Prolycopene was the predominant pigment but substantial amounts of proneurosporene, poly-cis-γ-carotenes and other cis carotenes were also present. The mode of action of these new bioregulators is probably gene derepression, the same as that of the lycopene inducers. However, the secondary amines probably derepress a recessive gene governing the biosynthesis of poly-cis carotenoids; whereas, the lycopene inducers derepress the dominant gene that gives rise to the normal all-trans carotenoids. The new compounds did not seem to inhibit the cyclase(s), as the lycopene inducers do.     

The purification and properties of a thiaminase I enzyme from nardoo (Marsilea drummondii)

Fronds of the fern nardoo (Marsilea drummondii) contain a thiaminase I enzyme at very high levels of activity. Highest levels of enzyme activity were found in vigorously growing plant material. The thiaminase I has been purified to a final sp act value of 2.07 μkat/mg protein at 30° and pH 6.5. It was shown to have similar properties to thiaminase I enzymes purified from bracken fern, rock fern and freshwater mussels. These enzymes have MW values in the range 93 000–115 000, energies of activation of 14 000 cal mol, pH optima of 8–9 and are quite stable in the pH range 3 to 12 and to extended incubation at 55°. The temperature for 50 % denaturation is 60–65°. p-CMB, mersalyl acid and HgCl₂ (10^t-6 M) are potent inhibitors, but monoiodacetic acid (10^?4 M) has no effect. A wide range of heterocyclic bases, sulphydryl compounds, and amines, including the non-aromatic amines 6-aminohexanoic acid and ethanolamine, act as co-substrates in the thiaminase I reaction; however, their effectiveness is dependent on both their degrees of basicity and to some extent, their stereochemistry. When the co-substrate activity of a range of substituted anilines were compared, no correlation was found between the degree to which the base activates the reaction and the pK_b (or Hammett's sigma constant) of the base.     

Analysis of a method for testing azo dyes for mutagenic activity in Salmonella typhimurium in the presence of flavin mononucleotide and hamster liver S9

A protocol for assessing the mutagenic activity of azo dyes derived from mutagenic or potentially mutagenic aromatic amines was evaluated, using 4 model compounds. This protocol is based upon one developed in Sugimura's laboratory with modifications, including the use of flavin mononucleotide (FMN) rather than riboflavin to reduce the azo compounds to free amines, and hamster liver S9 rather then rat liver S9 for metabolic activation. The protocol developed differs from the standard Ames Salmonella plate incorporation assay in 5 ways: (1) uninduced hamster liver S9 rather than Aroclor 1254-induced rat liver S9 is used; (2) 150 μl of S9 is used rather than the maximum of 50 μl of S9 used in the standard assay; (3) FMN is added to the cofactor mix; (4) the cofactor mix is modified to include exogenous glucose 6-phosphate dehydrogenase, NADH, and 4 times the standard amount of glucose 6-phosphate; and (5) a 30-min “pre-incubation” step is used before addition of top agar. We found that each of these 5 changes is necessary for optimal mutagenic activity of azo dyes derived from the mutagenic aromatic amines benzidine, o-tolidine or o-dianisidine. The use of hamster liver S9 rather than rat liver S9 was also required for optimal mutagenic activity of benzidine itself. Rat liver S9 inhibited the ability of hamster S9 to activate benzidine to a mutagen. The presence in rat liver S9 of an inhibitor of the metabolic activation of benzidine may account for the failure of benzidine and a benzidine dye (Congo red) to be strongly mutagenic when tested with this type of S9.     

9-Dihydroerythromycin ethers as motilin agonists—Developing structure–activity relationships for potency and safety

A series of derivatives of the amine of 9-dihydro-9-O-ethylamino-N-desmethyl-N-isopropyl erythromycin A derivatives were synthesized as motilin agonists. The compounds were developed for potency without showing antibacterial activity and inhibition of the hERG potassium channel. The formamide of the amide series was found to show the optimal combination of properties relative to carbamates, ureas, thioureas, and amines. This prompted an investigation of heterocyclic isosteres for the amide. In this series the triazole had the optimal combination of properties. From the study, two compounds met the criteria for detailed pharmacokinetic studies.     

Syntheses,properties, and use of fluorescent N-(5′-phospho-4′-pyridoxyl)amines in assay of pyridoxamine (pyridoxine) 5′-phosphate oxidase

A sensitive and simple fluorometric assay has been developed for detection of pyridoxamine (pyridoxine) 5′-phosphate oxidase. This technique utilizes fluorescent N-(5′-phospho-4′-pyridoxyl)amines as substrates that, upon incubation with the oxidase, release the free fluorescent amine. The substrates were prepared by condensation of pyridoxal 5′-phosphate with fluorescent amines and subsequent hydrogenation of the Schiff bases. Since N-(1-naphthyl)ethylenediamine is 15 times less fluorescent in the intramolecularly quenched substrate than the product amine, the direct increase of fluorescence, as well as selective extraction of more fluorescent product, can be utilized for assay. The apparent K_m value for this substrate is 8 μm, which is slightly less than that of pyridoxamine 5′-phosphate; V is larger than the natural substrate value. The greater sensitivity gained by this fluorimetric method allows detection of the oxidase in smaller quantities than can be determined by the conventional colorimetric assay.     

4-Hydroxybenzoylcholine: A natural product present in Sinapis alba

The total fractions of choline esters have been isolated from different parts of Sinapis alba. 4-Hydroxybenzoylcholine has been identified and found to be one of the quantitatively dominating choline esters in seed extracts of S. alba. The identity of the new natural product has been confirmed by comparison with synthetic reference compounds. Several different choline esters are present in this plant but in extracts of seedlings, leaves, and inflorescences they are not so quantitatively dominating as in seeds. The modified ion-exchange technique applied appeared to be an efficient tool in the isolation and separation of choline esters and amines from other types of phenolic plant constituents.     

Synthesis and antitubercular activity of substituted phenylmethyl- and pyridylmethyl amines

A total of 42 benzyl- and pyridylmethyl amines were synthesized either by reductive amination of aromatic/heteroaromatic aldehydes with amines or by conjugate addition of amines to the cinnamates followed by reduction of the ester group with lithium aluminium hydride to the respective propanolamines. All the synthesized compounds were evaluated against both avirulent and virulent strains of Mycobacterium tuberculosis. Many of the compounds exhibited MIC as low as 1.56 μg/mL. Few of potent compounds were also evaluated against clinical isolates of MDR TB and found to be active at one or other concentrations with MIC as low as 3.12 μg/mL.