Identification of an arginine carrier in the vacuolar membrane of Neurospora crassa

A number of arginine derivatives were tested for their ability to inhibit arginine uptake into vacuolar membrane vesicles of Neurospora crassa. The guanido side chain and L-configuration were found to be important for recognition by the arginine carrier. Based upon the specificity of recognition, a reactive arginine derivative (N alpha-p-nitrobenzyloxycarbonyl arginyl diazomethane) was synthesized which has an intact guanido side chain and a diazo group at the carboxyl end. The latter decomposes to a reactive carbene group. This derivative inhibited arginine uptake into vacuolar membrane vesicles at low concentrations. Radioactive N alpha-p-nitrobenzyloxycarbonyl arginyl diazomethane was covalently bound to vacuoles. Binding was specific for a single membrane protein with an approximate molecular weight of 40,000, saturable (2 pmol/mg vacuolar membrane protein), and inhibited by 100 mM L-arginine but not by 100 mM L-lysine. The results suggest that this protein is the arginine carrier.     

The enzymic conversion of linoleic acid into 9-(nona-1′,3′-dienoxy)non-8-enoic acid, a novel unsaturated ether derivative isolated from homogenates of Solanum tuberosum tubers

1. A major component of the lipids in aqueous (pH7.5) homogenates of tuber tissue from Solanum tuberosum was isolated and characterized as 9-(nona-1',3'-dienoxy)non-8-enoic acid. 2. This novel unsaturated ether fatty acid derivative, which contains a butadienylvinyl ether function, has the structure: [Formula: see text] and is formed from linoleic acid by a sequence of enzymic reactions. 3. A precursor of the unsaturated ether derivative is 9-d-hydroperoxyoctadeca-10,12-dienoic acid, formed by the action of S. tuberosum lipoxygenase on linoleic acid. 4. An enzyme that converts the fatty acid hydroperoxide into the unsaturated ether derivative was isolated from S. tuberosum. The pH optimum of this enzyme is approx. 9, although the overall conversion of linoleic acid into the ether derivative is maximal at pH7.5. 5. An unusual feature of this pathway is the insertion of an oxygen atom into the alkyl chain of a fatty acid. 6. This novel mechanism may play a role in the breakdown of polyunsaturated fatty acids to volatile products in plants.     

New bile alcohols--synthesis of 5beta-cholestane-3alpha, 7alpha, 25-triol and 5beta-cholestane-3alpha, 7alpha, 25-24 (14C)-triol.

5beta-Cholestane-3alpha, 7alpha, 25-triol and 5beta-cholestane-3alpha, 7alpha, 25-24(14-C)-triol were synthesized from 3alpha, 7alpha-dihydroxy-5beta-cholanoic acid (chenodeoxycholic acid). Chenodeoxycholic acid was converted to the diformoxy derivative (II) using formic acid. Reaction of II with thionyl chloride yielded the acid chloride which was treated with diazomethane (CH-2-N-2 or 14-CH-2-N-2) to produce 3alpha, 7alpha-diformoxy-24-oxo-25-diazo-25-homocholane (III, A or B). 25-Homochenodeoxycholic acid (IV, A or B) was formed from III by means of the Wolff rearrangement of the Arndt-Eistert synthesis. The methyl ester of V (A or B) was treated with methyl magnesium iodidi in ether to provide the desired triol, VI (A and B). The triol was identified by mass spectrometry and elemental analysis and was characterized by thin-layer and gas-liquid chromatography. The 3alpha, 7alpha, 25-triol is of possible significance as an intermediate in the pathway of bile acid formation from cholesterol.     

Synthesis and characterization of a tetracycline-platinum (II) complex active against resistant bacteria

A tetracycline-platinum(II) complex, [PtCl2(C22H24N2O8)], was synthesized and characterized by elemental analysis, conductivity and thermogravimetric analyses, and infrared spectroscopy. The interaction of tetracycline (Tc) with platinum(II) ions was also studied in aqueous solution by 1H NMR and circular dichroism spectroscopies. Tetracycline forms a 1:1 complex with platinum via the oxygen of the hydroxyl group at the A ring and that of the amide group. The complex is as efficient as tetracycline in inhibiting the growth of two Escherichia coli sensitive bacterial strains and six times more potent against E. coli HB101/pBR322, a bacterial strain resistant to tetracycline. This finding is very important because the use of tetracycline to treat bacterial infections has declined due to the emergence of resistant organisms.     

1H nuclear magnetic resonance studies of the conformations of vitamin D compounds in various solvents

Using proton NMR, the solution conformation of the A ring of vitamin D3 and its analogs has been studied by application of the Karplus relation to the observed coupling constants. The A-ring conformations of vitamins D3, D2, and 25-hydroxyvitamin D3 were found to be solvent dependent, with a clear preference for an equatorial hydroxyl group in polar solvents such as methanol and dimethyl sulfoxide. Conversion of the hydroxyl group to an acetate did not affect solution conformation appreciably, but the corresponding t-butyl-dimethylsilyl ether derivative of vitamin D3 showed a strong preference for the 3 beta-equatorial conformer. The A-ring conformation of the active hormone, 1,25-dihydroxyvitamin D3, which has two hydroxyl groups competing for the equatorial position, was found not to be solvent-dependent.     

Isolation and identification of the urinary pigment uroerythrin.

The red pigment uroerythrin, a chromophore known to be adsorbed by the amorphous urate sediments (sedimentum lateritium), has been isolated from human urine and further purified as its trimethyl derivative. Urine was applied to a column of Amberlite XAD-2 resin on which uroerythrin and other pigments were adsorbed. The pigments were eluted with methanol and uroerythrin was further purified by extraction with ether at pH 4.0, repeated chromatography on lipophilic Sephadex LH-20 and thin-layer chromatography on silica gel. For optimal purification uroerythrin was converted into the trimethyl derivative and chromatographed on silical gel thin-layer plates. The structure of the pigment has been studied by chromate degradation followed by identification of the imide products by thin-layer chromatography. From these results and from infrared, mass spectral and nuclear magnetic resonance data a tripyrrole structure for uroerythrin is concluded. The proposed structure for the chromophore is related to that of the bile pigment biliverdin consisting, however, only of the rings A, B and C.     

Synthesis, characterization, and antibacterial activity of three palladium(II) complexes of tetracyclines

Pd(II) complexes with three antibiotics of the tetracycline family (tetracycline, doxycycline and chlortetracycline) were synthesized and characterized by elemental, thermogravimetric, and conductivity analyses, and infrared spectroscopy. The interactions between Pd(II) ions and tetracycline were investigated in aqueous solution by (1)H NMR. All the tetracyclines studied form 1:1 complexes with Pd(II) via the oxygen of the hydroxyl group at ring A and that of the amide group. The effect of the three complexes on the growth of bacterial strains sensitive and resistant to tetracycline was studied. The Pd(II) complex of tetracycline is practically as efficient as tetracycline in inhibiting the growth of two Escherichia coli (E. coli) sensitive bacterial strains and 16 times more potent against E. coli HB101/pBR322, a bacterial strain resistant to tetracycline. Pd(II) coordination to doxycycline also increased its activity in the resistant strain by a factor of 2.     

Role of oxygen radicals in the phototoxicity of tetracyclines toward Escherichia coli B.

Photoillumination of tetracycline derivatives with low-intensity (320- to 400-nm) light and visible light generated superoxide, observed as the reduction of ferricytochrome c. The rate of reduction was dependent on the tetracycline concentration and on the derivative being examined, with doxycycline greater than or equal to demeclocycline greater than tetracycline greater than oxytetracycline. Tetracycline-mediated cytochrome c reduction was oxygen dependent and inhibited up to 70% by superoxide dismutase. Illuminated tetracyclines were lethal to Escherichia coli B incubated in a glucose minimal medium containing chloramphenicol. This lethality was light dependent, oxygen dependent, and dependent on the concentration of tetracycline. Kill rates also varied according to the derivative under study, with doxycycline greater than or equal to demeclocycline greater than tetracycline greater than oxytetracycline. The addition of superoxide dismutase and catalase to the incubation medium partially protected E. coli B against the light-dependent lethality. Preinduction of intracellular superoxide dismutase and catalase substantially protected E. coli B against the phototoxicity of tetracyclines. Iron EDTA augmented the phototoxicity of tetracyclines, while diethylenetriaminepentaacetic acid protected against their lethality. Hydroxyl radical scavengers also conferred protection against tetracycline phototoxicity. The extent of protection was in order of the in vitro reactivity of the scavengers with the hydroxyl radical. These results indicate that superoxide, hydrogen peroxide, and the hydroxyl radical are generated by illuminated tetracyclines and are molecular agents of tetracycline phototoxicity in E. coli B.     

Lipase-catalyzed acetylation of N-acetylneuraminic acid derivative

A facile preparation of triacetylated derivative of 2-phenylthioglycoside of N-acetylneuraminic acid (4) was achieved by treatment with lipase PS in vinyl acetate. The major product 4 has a free hydroxyl group at C-7. Results of time-course HPLC analysis indicate that the reactivity of the hydroxyl groups under this condition is in the following order; C-9 > C-4 > C-8 > C-7.     

Internally repeated sequences in ras gene products.

The preparation of peptides terminating in -Arg-CHN2 has been attempted because of their potential value as proteinase inactivators. We have succeeded in one case, converting Cbz-Phe-ArgOH to the diazomethane without blocking the guanidino group. As expected from previous results with such reagents, the new derivative was extremely effective in inactivating a cysteine proteinase specific for cleaving at arginyl bonds, that is, clostripain. However, in contrast with the inertness of serine proteinases to reagents of this type in the cases examined previously, plasma kallikrein was inactivated by Cbz-Phe-Arg-CHN2, although with a considerably lower rate constant than with clostripain. Trypsin, however, was not inactivated, but gradually destroyed the reagent, as had been observed previously with chymotrypsin and Cbz-Phe-CHN2. This has now been re-examined with rho-nitro-Cbz-Ala-Phe-CHN2 and shown to involve a cleavage to rho-nitro-Cbz-Ala-PheOH, probably with liberation of diazomethane.     

Improved high-performance liquid chromatographic method for the combined analysis of phospholipase, lipoxygenase and cyclooxygenase activities

We report herein an improved method for the high-performance liquid chromatographic separation and analysis of eicosanoids formed during the stimulation of human platelets in vitro with collagen. Since the products of interest, excepting arachidonic acid, contain hydroxyl groups (one to several), our method involves the conversion of the hydroxyl groups into acetates (pyridine/acetic anhydride) after derivatization with anthryl diazomethane (ADAM) rendering the compounds with much decreased polarity for separation on a reversed-phase column. This procedure is superior to that involving ADAM esters only, i.e. with free hydroxyl groups, as it leads to the excellent separation of the desired compounds from each other and from extraneous peaks observed due to the ADAM reagent and sharpens the peak of thromboxane. We have successfully applied the method to investigate the formation of thromboxane B₂ and 12-hydroxyheptadecatrienoic acid (HHT) (products of cyclooxygenase and thromboxane A₂ synthase), 12-hydroxyeicosatetraenoic acid (12-HETE, a 12-lipoxygenase product) and arachidonic acid (AA, product of phospholipase A₂) formed during the in vitro aggregation of human platelets induced by collagen. A correlation between the inhibition of aggregation by aspirin and thromboxane/HHT formation was observed. All four compounds can be chromatographed in a single run. We employed prostaglandin B₁ (PGB₁) as internal reference standard to quantify the products. The method is useful to investigate selectivity of drugs which may affect either or all of these enzyme pathways at the same time.     

Tin(II) chloride catalyzed reactions of diazodiphenylmethane with vicinal diols in an aprotic solvent. The reactions with cis- and trans-1,2-cyclohexanediols and 1,2-propanediol

The paper reports the tin(II) chloride catalyzed reactions of diazodiphenylmethane with the cis- and trans-1,2-cyclohexanediols and R,S-1,2-propanediol in 1,2-dimethoxyethane and the identification of the monodiphenylmethyl ethers formed. The catalyst is shown to work for both the cis- and trans-cyclohexanediols, but the catalyst is unstable at high reagent concentrations, especially in the case of the trans-isomer. Conditions where catalyst destruction is negligible show that the rate of the reaction with the trans-isomer is larger than with the cis-isomer. The reactions with 1,2-propanediol show small difference between the selectivity for the primary and secondary hydroxyl groups. This is in contrast with the tin(II) chloride catalyzed reactions of diazomethane and diazophenylmethane in methanol with carbohydrates, glycerol and ribonucleosides, where the primary hydroxyl group does not react.     

Metabolism of lysophosphatidylserine, a potentiator of histamine release in rat mast cells

Lysophosphatidylserine (lysoPS) strongly enhances degranulation of rat mast cells induced by concanavalin A (Con A). In the present paper, the metabolism of exogenous lysoPS in intact mast cells was investigated. Incubation of mast cells with 1-stearoyl-sn-glycero-3-phospho-[3-3H]serine resulted in the rapid binding of lysoPS to mast cells and the time-dependent formation of a considerable amount of [3H]phosphatidylserine. No other radiolabeled lipid metabolites were detected. These results suggest that phosphatidylserine (PS) is synthesized through acylation of lysoPS incorporated into mast cells. Most of the lysoPS associated with mast cells was removed by washing with bovine serum albumin, whereas PS newly formed from lysoPS was not. The cells washed with albumin showed no appreciable histamine release upon subsequent addition of Con A. A different set of experiments was performed using lysoPS analogs which were modified at the hydroxyl group at position 2 of glycerol to avoid acylation. 1-Stearoyl-2-O-methyl-glycero-3-phosphoserine showed almost the same potentiating activity as 1-stearoyl-lysoPS, although the former does not have the free hydroxyl moiety at position 2 of the glycerol residue. The enhancing activity of another lysoPS analog, 1-stearyl-propanediol-3-phosphoserine, which lacks the hydroxyl group altogether, was quite similar to that of 1-stearyl-lysoPS. From these results we conclude that the acylation of lysoPS bears no relation to its potentiating activity and that lysoPS acts toward mast cells as lysoPS itself without any conversion to PS. The effect of replacement of an ester bond at position 1 of glycerol in lysoPS with an ether bond, and the phospholipid composition of rat mast cells are also discussed.     

Selective phenolic acylation of 10-hydroxycamptothecin using poly (ethylene glycol) carboxylic acid

Selective acylation of the phenolic hydroxyl group of 10-hydroxycamptothecin has been accomplished using phenyl dichlorophosphate. Additional modification of the 10-OH as an ether permits a 20-acyl derivative to be synthesized. This result along with data from a 6-hydroxyquinoline model strongly suggests that powerful intermolecular hydrogen bonding exists in the parent molecule.     

Gas-chromatographic resolution of the enantiomers of 3-(3,4-dihydroxyphenyl)alanine and its α-methyl analog

Methylation of (R,S)-DOPA with diazomethane gave the trimethyl derivative in which the phenolic hydroxy groups and the carboxy group were methylated. N-Methylated side products were also formed. N-Acylation of the racemic trimethyl derivative with (S)-α-methoxy-α-trifluoromethylphenylacetyl chloride gave two diastereomeric amides which were resolved by gas chromatography, the diastereomer derived from (S)-(−)-DOPA cluting first. The procedure was also applied to α-methyl-DOPA.     

Synthesis of polyhydroxy amino acids based on D- and L-alanine from D-glycero-D-gulo-heptono-1,4-lactone

2-Amino-2,3-dideoxy-D-manno-heptonic acid (7) has been synthesized from 2,5,6,7-tetra-O-acetyl-3-deoxy-D-gluco-heptono-1,4-lactone (1), which was readily prepared from D-glycero-D-gulo-heptono-1,4-lactone. O-Deacetylation of 1 followed by treatment with 13:1 (v/v) 2,2-dimethoxypropane/acetone in the presence of p-toluenesulfonic acid gave methyl 3-deoxy-4,5:6,7-di-O-isopropylidene-D-gluco-heptonate (3) as a crystalline product (80% yield). The free hydroxyl group (OH-2) of 3 was mesylated and substituted by azide to give the corresponding azide derivative 5. Hydrogenolysis and further hydrolysis of the ester function of 5 afforded alpha-amino acid 7 (43% overall yield from 1). Compound 7 is an analog of L-alanine having a polyhydroxy chain attached to C-3. The diastereoisomer of 7 at C-2, 2-amino-2,3-dideoxy-D-gluco-heptonic acid (12) was also prepared from 3, by a route that involved 2,3-dideoxy-2-iodo derivative 8 as a key intermediate.     

Selective synthesis of 4-methoxyestrogen from 4-hydroxyestrogen.

The introduction of an oxygen atom into the C-6 position of 4-hydroxyestrogen allowed for the selective methylation of the two phenolic hydroxyl groups. When the 6-oxo derivative of 4-hydroxyestrone was benzylated in ethanol, only the 3-monobenzyl ether was obtained without formation of the 4-monobenzyl ether. Moreover, the 6-carbonyl group was further reduced to methylene almost quantitatively in the reaction of 4-acetoxy-6-oxoestrone 3-benzyl ether derivative with sodium borohydride. Therefore, 4-methoxyestrogen was synthesized by essentially combining these two reactions.     

Designer DNA-binding drugs: the crystal structure of a meta-hydroxy analogue of Hoechst 33258 bound to d(CGCGAATTCGCG)2.

An analogue of the DNA binding compound Hoechst 33258, which has the para hydroxyl group altered to be at the meta position, together with the replacement of one benzimidazole group by pyridylimidazole, has been cocrystallized with the dodecanucleotide sequence d(CGCGAATTCGCG)2. The X-ray structure has been determined at 2.2 A resolution and refined to an R factor of 20.1%. The ligand binds in the minor groove at the sequence 5'-AATTC with the bulky piperazine group extending over the CxG base pair. This binding is stabilised by hydrogen bonding and numerous close van der Waals contacts to the surface of the groove walls. The meta-hydroxyl group was found in two distinct orientations, neither of which participates in direct hydrogen bonds to the exocyclic amino group of a guanine base. The conformation of the drug differs from that found previously in other X-ray structures of Hoechst 33258-DNA complexes. There is significant variation between the minor groove widths in the complexes of Hoechst 33258 and the meta-hydroxyl derivative as a result of these conformational differences. Reasons are discussed for the inability of this derivative to actively recognise guanine.     

Chemical synthesis, biological evaluation and structure-activity relationship analysis of azaisoindolinones, a novel class of direct enoyl-ACP reductase inhibitors as potential antimycobacterial agents

The synthesis and biological evaluation of azaisoindolinone compounds embedding a lipophilic chain on the framework were performed. These compounds were designed as InhA inhibitors and as anti-Mycobacterium tuberculosis agents. Structure-activity relationships concerning the length and the location of the lipophilic chain around the azaisoindolinone framework, the suppression of the phenyl group, the bioisosteric substitution of ether link and alkylating of the tertiary hydroxyl and the hemiamidal nitrogen were also investigated, revealing insightful information and thereby enabling further diversification of the azaisoindolinone scaffold for new antitubercular agents.     

Distal Cleavage of 3-Chlorocatechol by an Extradiol Dioxygenase to 3-Chloro-2-Hydroxymuconic Semialdehyde

A 2,3-dihydroxybiphenyl 1,2-dioxygenase from the naphthalenesulfonate-degrading bacterium Sphingomonas sp. strain BN6 oxidized 3-chlorocatechol to a yellow product with a strongly pH-dependent absorption maximum at 378 nm. A titration curve suggested (de)protonation of an ionizable group with a pK_a of 4.4. The product was isolated, purified, and converted, by treatment with diazomethane, to a dimethyl derivative and, by incubation with ammonium chloride, to a picolinic acid derivative. Mass spectra and ¹H and ¹³C nuclear magnetic resonance (NMR) data for these two derivatives prove a 3-chloro-2-hydroxymuconic semialdehyde structure for the metabolite, resulting from distal (1,6) cleavage of 3-chlorocatechol. 3-Methylcatechol and 2,3-dihydroxybiphenyl are oxidized by this enzyme, in contrast, via proximal (2,3) cleavage.