Characterisation of rhodococci using peptide hydrolase substrates based on 7-amino-4-methylcoumarin

Enzyme activity profiles of 105 rhodococci and related actinomycetes were obtained using peptide hydrolase substrates based on 7-amino-4-methylcoumarin. All of the test strains produced l-alanyl-, l-lysyl-, l-methionyl, l-seryl-, l-tyrosyl, S-Bz-l-cysteinyl and l-alanyl-l-alanyl-l-phenyl-alanyl-peptidases but the distribution of the remaining enzymes gave results of differential value. Fluorogenic probes prepared from 7-amino-4-methylcoumarin provide a simple and rapid means of detecting specific exo- and endopeptidases in small amounts of whole rhodococci.     

A nanoprobe for nonprotein thiols based on assembling of QDs and 4-amino-2,2,6,6-tetramethylpiperidine oxide

A new fluorescent nanoprobe, 4-amino-2,2,6,6-tetramethylpiperidine oxide (AT)-functionalized CdTe quantum dots (QDs-AT), was synthesized, for selective detection of nonprotein thiols based on electron transfer (ET). In the presence of nonprotein thiols, the nitroxide radicals in QDs-AT were converted to hydroxylamines, resulting in the fluorescence recovery of the quenched QDs. The detection mechanism of the probe was investigated using Rh-Se-2 probe. The nanoprobe has high sensitivity toward glutathione (GSH) with a detection limit of 7.1 × 10?? M. The fluorescent imaging of living cells showed that QDs-AT could distinguish the concentration differences of GSH in HL-7702 and HepG2 cells.     

On the specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein as a probe for nitric oxide

The specificity of 4-amino-5-methylamino-2',7'-difluorofluorescein (DAF-FM) for nitric oxide was evaluated in in vitro systems. The probe was found fairly specific for nitric oxide. Potential sources of artifacts include the autoxidation of DAF-FM, potentiated by light, and its oxidation by sources of superoxide and peroxyl radicals, leading to fluorescence spectra indistinguishable from those of the nitric oxide adduct. Although DAF-FM reacts with peroxynitrite, this reaction seems to be of secondary importance under quasi-physiological conditions. On the other hand, a simultaneous presence of a nitric oxide source and a superoxide or hydrogen peroxide decreases or increases the fluorescence of DAF-FM, respectively, resulting in biased estimates of nitric oxide production.     

Conversion of pyridylamino sugar chains to 1-amino-1-deoxy derivatives, intermediates for tagging with fluorescein and biotin.

Pyridylamino (PA) derivatives of sugar chains were converted to 1-amino-1-deoxy derivatives. PA-lactose as a model compound was reduced with hydrogen, then treated with hydrazine. The product obtained was identified as 1-amino-1-deoxylactitol by mass spectrometry and chromatography with 1-amino-1-deoxylactitol as standard. PA-N-acetylglucosamine was converted to 1-amino-1-deoxy-N-acetylglucosaminitol under the same conditions. As an application, Man alpha 1-6(Man alpha 1-3)Man alpha 1- 6(Man alpha 1-2Man alpha 1-3)-Man beta 1-4GlcNAc beta 1-4GlcNAc-PA was converted to the 1-amino-1-deoxy derivative, which was further derivatized with fluorescein isothiocyanate or biotin sulfo-N-hydroxy-succinimide ester. Binding of these derivatives to concanavalin A dot-blotted on a nitrocellulose membrane was confirmed by fluorescence and by streptavidin-peroxidase conjugate. This conversion allowed replacement of the PA-group in PA-sugar chains which can be easily purified from glycoconjugates.     

9-amino-2-methoxy-6-chloroacridine monocation fluorescence analysis by phase-modulation fluorometry

The fluorescence of the 9-amino-2-methoxy-6-chloroacridinic monocation (ACMA) in various alcohol-water solutions was studied by phasemodulation fluorometry. Apparent phase and modulation lifetimes were determined at different observation wavelengths for three modulation frequencies. The results are explained by an orientational relaxation of the solvent cage subsequent to ACMA photoexcitation.     

Non-covalent DNA groove-binding by 2-amino-1-methyl-6-phenylimidazo[4,5-b]pyridine.

The cooked meat mutagen 2-amino-1-methyl-6-phenyl-imidazo[4,5-b]pyridine (PhIP) is metabolized in vivo to electrophilic intermediates that covalently bind to DNA guanines. Here we address the mechanism of PhIP's non-covalent interaction with DNA by using spectroscopic and computational methodologies. NMR methodologies indicated that upon addition of DNA, PhIP aromatic protons underwent a small, 0.11-0.12 p.p.m. upfield shift. DNA phosphorus resonances of non-covalent PhIP-DNA complexes broadened and slightly shifted upfield, while DNA base imino proton resonances shifted slightly downfield relative to DNA alone. UV and fluorescence spectra of PhIP titrated with DNA showed no detectable shifting and hypochromism of absorbance or fluorescence bands. In the presence of DNA, PhIP fluorescence was efficiently quenched by acrylamide, but not by silver ion. Further, the NMR spectra suggest that PhIP is in fast exchange with the DNA, and is slightly specific for adenine-thymine (A-T) sequences. Finally, structural arguments based on quantum chemistry calculations suggested that PhIP and its metabolites are unlikely to intercalate into DNA. These data collectively indicate that PhIP non-covalently binds in a groove of DNA.     

Synthesis and characterization of N-(2,4-diphosphobenzyl)-1-amino-5-naphthalenesulfonic acid, a new fluorescent analogue of diphosphoglyceric acid

The synthesis of N-(2,4-diphosphobenzyl)-1-amino-5-naphthalenesulfonic acid (DIPANS) is described. It entails the synthesis of 2,4-diphosphobenzaldehyde from the action of POCl3 on 2,4-dihydroxybenzaldehyde. This is followed by coupling of the 2,4-diphosphobenzaldehyde to 1-amino-5-naphthalenesulfonic acid. Subsequent reduction with NaBH4 yields the desired product. The DIPANS exhibits an excitation maximum at 337 nm and a fluorescence emission maximum at 504 nm. This dye is quantitatively displaced by inositol hexaphosphate and is an effective analogus of diphosphoglyceric acid (DPG), possessing a KD at pH 7.0 in 0.05 M [bis(2-hydroxyethyl)amino]tris(hydroxymethyl)methane (bis-Tris) plus 0.1 M chloride of 6.88 microgram, with 1.0 molecule bound/hemoglobin tetramer. Like DPG its binding to deoxyhemoglobin decreases with increasing pH; in the presence of 0.1 M chloride it binds 0.031 times as tightly to CO hemoglobin and it yields a value for free energy coupling of 2.0 kcal/mol. The presence of 1 mM DIPANS decreases the affinity of hemoglobin for oxygen in the absence of salt from p1/2 of 0.8 mm Hg to 12.4 mm Hg. Using DPG as a competitor of DIPANS binding, a dissociation constant of 11.4 micrometer was calculated for DPG binding to deoxy-Hb at pH 7.0 in the presence of 0.05 M bis-Tris and 0.1 M chloride.     

2-amino-2-desoxy-6-O-(D-glykofuranosylurono-6,3-lacton)-D-galaktopyranosen

Condensation of 2-amino-2-deoxy-D-galactopyranose with D-glucuronic acid or D-mannurono-6,3-lactone in the presence of hydrochloric acid gave the corresponding 2-amino-2-deoxy-6-O-(D-glycofuranosylurono-6,3-lactone)-D-galactopyranoses. The α-D configuration of the disaccharide derived from D-glucuronic acid was determined by its resistance towards β-D-glucuronidase.     

Facile synthesis of 7-amino-4-carbamoylmethylcoumarin (ACC)-containing solid supports and their corresponding fluorogenic protease substrates

The bifunctional fluorophore, 7-amino-4-carbamoylmethylcoumarin (ACC) without any protection groups, was regioselectively attached to different solid supports functionalized with a primary amino group. The resulting resins were used to synthesize fluorogenic protease substrates with high yield and purity.     

Synthesis of ethyl 2-acetamido-6-S-(5-amino-5-deoxy-beta-D- arabinopyranosyl)-2-deoxy-1,6-dithio-beta-D-glucopyranoside: a sulfur-linked 5-amino-5-deoxyglycopyranosyl disaccharide

A novel pseudo-disaccharide having an imino sugar residue at the non-reducing end, namely, a sulfur-linked 5-amino-5-deoxyglycopyranosyl disaccharide, which is a potential specific inhibitor for glycosidases that recognize not only the glycosidic linkage but also the aglycone moiety, was synthesized. Glycosidation of N-Boc-5-amino-5-deoxy-D-arabinose with ethyl 2-acetamido-3,4-di-O-acetyl-2-deoxy-1,6-dithio-beta-D- glucopyranoside in the presence of TsOH gave exclusively the corresponding 1,2-cis-linked thioglycoside. The interglycosidic linkage proved stable enough under conditions for the deprotection of the N-Boc group with TFA. This pseudodisaccharide was unstable at pH > 5, but stable at lower pH. The sulfur-linked 5-amino-5-deoxyglycopyranosyl disaccharide was shown to be formed from 5-amino-5-deoxy-D-arabinose and ethyl 2-acetamido-2-deoxy-1,6-dithio-beta-D-glucopyranoside in an acidic buffer solution.     

Asymmetric biocatalysis of S-3-amino-3-phenylpropionic acid with new isolated Methylobacterium Y1-6

β-amino acids are widely used in drug research, and S-3-amino-3-phenylpropionic acid (S-APA) is an important pharmaceutical intermediate of S-dapoxetine, which has been approved for the treatment of premature ejaculation. Chiral catalysis is an excellent method for the preparation of enantiopure compounds. In this study, we used (±)-ethyl-3-amino-3-phenylpropanoate (EAP) as the sole carbon source. Three hundred thirty one microorganisms were isolated from 30 soil samples, and 17 strains could produce S-APA. After three rounds of cultivation and identification, the strain Y1-6 exhibiting the highest enantioselective activity of S-APA was identified as Methylobacterium oryzae. The optimal medium composition contained methanol (2.5 g/L), 1,2-propanediol (7.5 g/L), soluble starch (2.5 g/L), and peptone (10 g/L); it was shaken at 220 rpm for 4–5 days at 30 °C. The optimum condition for biotransformation of EAP involved cultivation at 37 °C for 48 h with 120 mg of wet cells and 0.64 mg of EAP in 1 ml of transfer solution. Under this condition, substrate ee was 92.1% and yield was 48.6%. We then attempted to use Methylobacterium Y1-6 to catalyze the hydrolytic reaction with substrates containing 3-amino-3-phenyl-propanoate ester, N-substituted-β-ethyl-3-amino-3-phenyl-propanoate, and γ-lactam. It was found that 5 compounds with ester bonds could be stereoselectively hydrolyzed to S-acid, and 2 compounds with γ-lactam bonds could be stereoselectively hydrolyzed to (-)-γ-lactam.     

Fluorogenic substrates for chymotrypsin with 2-quinolinone derivatives as leaving groups

Sensitive and convenient fluorometric assays for the determination of chymotrypsin have been developed by using the substrates Glt-Leu-Phe-NH-Meq, Glt-Phe-NH-FMeq and Glt-Leu-Phe-NH-FMeq, which have been synthesized utilizing the mixed anhydride method. The amidolytic activity of chymotrypsin was measured by the release of the highly fluorescent amine 7-amino-4-methyl-2-quinolinone (AMeq) or 7-amino-4-trifluoromethyl-2-quinolinone (AFMeq). The fluorescence properties of the synthesized substrates and the new fluorescent marker AFMeq were examined. Kinetic constants, as well as the maximum sensitivity for the hydrolysis of the new substrates, were determined. All new substrates permit a rapid and sensitive determination of chymotrypsin in a continuous assay system. As little as 0.7 ng of enzyme can be detected using the substrate Glt-Leu-Phe-NH-Meq, which is the most sensitive fluorogenic substrate thus far reported.     

Synthesis and structure of 6-amino-2,3,6-trideoxy-D-erythro-hexono-1,6-lactam and 6-amino-3,6-dideoxy-D-xylo-hexono-1,6-lactam

Solid-state conformations of 6-amino-2,3,6-trideoxy-D-erythro-hexono-1,6-lactam (3a) and 6-amino-3,6-dideoxy-D-xylo-hexono-1,6-lactam (7a) were determined using X-ray diffraction. Conformations of the compounds 3a, 7a, and their per-O-acetyl derivatives 4,5-di-O-acetyl-6-amino-2,3,6-trideoxy-D-erythro-hexono-1,6-lactam (3b) and 2,4,5-tri-O-acetyl-6-amino-3,6-dideoxy-D-xylo-hexono-1,6-lactam (7b) in solutions were deduced from the analysis of NMR spectra using a modified Karplus equation and compared with the results of circular dichroism measurement of lactams 3a and 7a. Conformation 4C(1,N) was revealed for solid lactams 3a and 7a and for lactams 7a and 7b in solution, while lactams 3a and 3b in solution exist in the approximately 1:1 equilibrium of the conformers 4C(1,N) and (1,N)C4.     

Formation of D-1-amino-2-propanol from L-threonine by enzymes from Escherichia coli K-12

Escherichia coli K-12 cells contain two dehydrogenases which in sequence catalyze the net conversion of L-threonine to the D-isomer of 1-amino-2-propanol. These two enzymes are L-threonine dehydrogenase (L-threonine + NAD⁺ → aminoacetone + CO₂ + NADH + H⁺) and D-1-amino-2-propanol dehydrogenase (aminoacetone + NADH + H⁺D-1-amino-2-propanol + NAD⁺). Each enzyme has been obtained in purified form free of the other; the nature of the reaction catalyzed by the latter dehydrogenase alone and in a coupled system with the former enzyme has been studied. The results provide an explanation on the enzymological level for the utilization of L-threonine by cell suspensions of certain microorganisms for the biosynthesis of the D-1-amino-2-propanol moiety of Vitamin B₁₂.     

Fluorescent 7- and 8-methyl etheno derivatives of adenosine and 6-amino-9-ethylpurine: syntheses and fluorescence properties

Two fluorescent adenosine derivatives (5 and 7) (Sch. 1) and two 6-amino-9-ethylpurine derivatives (6 and 8) (Sch. 1), were synthesised using 2-chloropropanal and 3-chloropropyne as reagents. The structures of the products were determined by spectroscopic and spectrometric methods (1H-, 13C- and 2D NMR, MS, UV and fluorescence spectrometry). Their fluorescence properties were determined and found to be similar to those of ethenoadenosine. Also, the stabilites of 5 and 7 in aqueous solutions were determined and found to be higher than that of the etheno derivative of adenosine.     

Site of inhibition by 2-amino-1,1,3-tricyanopropene of photosynthetic oxygen evolution by spinach leaf chloroplasts (Short Communication)

2-Amino-1,1,3-tricyanopropene inhibits photosynthetic O₂ evolution, but, unlike 3-(3,4-dichlorophenyl)-1,1-dimethylurea, has little effect on the steady-state fluorescence of chlorophyll. In chloroplasts prepared from spinach leaves and inhibited by 2-amino-1,1,3-tricyanopropene, a 3-(3,4-dichlorophenyl)-1,1-dimethylurea-sensitive photoreduction of ferricyanide may be restored by addition of semicarbazide. It is concluded that 2-amino-1,1,3-tricyanopropene acts at a point close to the photo-oxidation of water.     

Enzymatic preparation of cis and trans-3-amino-4-hydroxytetrahydrofurans and cis-3-amino-4-hydroxypyrrolidines

The lipase catalyzed resolution of cis and trans-3-amino-4-hydroxytetrahydrofurans and cis-3-amino-4-hydroxypyrrolidines have been studied. For all the heterocycles, the best enantioselectivity was obtained using Candida antarctica lipases A and B as catalysts in hydrolytic processes. The absolute configuration of the optically pure obtained heterocycles has been assigned.     

Vitamin B6-catalyzed reactions of α-amino- and α-amino-α-methyldiethylmalonate

The reactions of pyridoxal phosphate with α-amino- and α-amino-α-methyldiethylmalonate have been investigated at pH 6.25 (30°C, N₂ atmosphere). The data indicate that the parent α-amino compound is converted to glycine ethyl ester by a reaction sequence involving rate-limiting, B₆-independent formation of the half-ester of α-aminomalonic acid, and then B₆-dependent decarboxylation. The reaction of α-amino-α-methyldiethylmalonate with pyridoxal phosphate follows a similar pathway except that, in contrast to the parent compound, the vitamin enhances the rate of formation of the half-ester from the α-methyl derivative. Likely mechanisms for these reactions are discussed.     

Structural studies on bioactive compounds. Part 29: palladium catalysed arylations and alkynylations of sterically hindered immunomodulatory 2-amino-5-halo-4,6-(disubstituted)pyrimidines

The immunological agent bropirimine 5 is a tetra-substituted pyrimidine with anticancer and interferon-inducing properties. Synthetic routes to novel 5-aryl analogues of bropirimine have been developed and their potential molecular recognition properties analysed by molecular modelling methods. Sterically challenged 2-amino-5-halo-6-phenylpyrimidin-4-ones (halo = Br or I) are poor substrates for palladium catalysed Suzuki cross-coupling reactions with benzeneboronic acid because the basic conditions of the reaction converts the amphoteric pyrimidinones to their unreactive enolic forms. Palladium-mediated reductive dehalogenation of the pyrimidinone substrates effectively competes with cross-coupling. 2-Amino-5-halo-4-methoxy-6-phenylpyrimidines can be converted to a range of 5-aryl derivatives with the 5-iodopyrimidines being the most efficient substrates. Hydrolysis of the 2-amino-5-aryl-4-methoxy-6-phenylpyrimidines affords the required pyrimidin-4-ones in high yields. Semi-empirical quantum mechanical calculations show how the nature of the 5-substituent influences the equilibrium between the 1H- and 3H-tautomeric forms, and the rotational freedom about the bond connecting the 6-phenyl group and the pyrimidine ring. Both of these factors may influence the biological properties of these compounds.     

Interaction of N-phenyl-1-amino-8-sulfonaphthalene (ANS) with albumin in blood in hyperbilirubinemia

The fluorescent intensity of the N-phenyl-1-amino-8-sulfonaphthalene (ANS) probe significantly decreases in hyperbilirubinemic serum. A decrease of the albumin concentration and absorption of ANS fluorescence by bilirubin cannot explain such a considerable reduction of the probe fluorescence intensity. Measurements of the fluorescence decay kinetics has shown two types of sites occupied by ANS in albumin. ANS quantum yields in hyperbilirubinemic and normal serum are practically identical. The coupling parameters for ANS decrease, but the coupling constant increases under hyperbilirubinemia. As a result the coupling of organic anions with serum albumin significantly decreases if there is high anion concentration, and it does not decrease at low anion concentration. Bilirubin is not a main cause of a decrease of the albumin binding capacity.