A comparison of acylation, phosphorylation and binding in related substrates and inhibitors of serum cholinesterase

1. The K_m and catalytic-centre activities for human serum cholinesterase and methyl, ethyl, n-propyl and n-butyl butyrate substrates were determined and compared with the related inhibition constants of a similarly substituted organophosphate inhibitor series based on malaoxon. The results indicated that the catalytic-centre activities approximated to k_+2(a), the acylation rate constant, and that K_m approximated to the equilibrium binding constant. The inhibition constants measured were K_a, the equilibrium binding constant, and k_+2(p), the phosphorylation rate constant. 2. The effects of the alkyl substituents on k_+2(p) and k_+2(a) were closely parallel, and the decreasing order in each case was: n-butyl; methyl; n-propyl; ethyl. The Taft constants did not follow this order, suggesting that alkyl substituents did not primarily effect acylation or phosphorylation by electron induction. 3. For comparable homologues, the k_+2(a) values were on average 435 times the k_+2(p) values. The k_+2(p) values at 25° and pH7·6 ranged from 6·6min.⁻¹ for the diethyl member to 22·6min.⁻¹ for the di-n-butyl member. 4. The effect of the alkyl substituents on K_a and K_m were closely paralleled. The increasing order in each case was: n-butyl; n-propyl; ethyl; methyl. The K_a values were about 100 times less than the comparable K_m values. 5. Consideration of the binding energies suggested that only one of the two alkyl groups on the malaoxon homologues bound to the active site. 6. The possibility that malaoxon acted as a substrate as well as an inhibitor for cholinesterase was also investigated, but no evidence of a substrate reaction was found.     

The metabolism of urethane and related compounds

1. Urethane is metabolized in the rat, rabbit and man by a process of N-hydroxylation. This occurs to a smaller extent when methyl, n-propyl and n-butyl carbamates are administered to the rat and rabbit. 2. Other metabolites which have been detected in urine of animals dosed with urethane and N-hydroxyurethane are ethylmercapturic acid, ethylmercapturic acid sulphoxide and N-acetyl-S-carbethoxycysteine. 3. Substances which appear to be S-ethylglutathione and S-ethylglutathione sulphoxide have been detected in the bile of rats dosed with urethane or N-hydroxyurethane. 4. Methyl, ethyl, n-propyl and n-butyl N-hydroxycarbamates are excreted unchanged in the urine of rats dosed with these compounds to extents depending on the dose administered. 5. Animals dosed with methyl, ethyl, n-propyl or n-butyl carbamate or the corresponding N-hydroxycarbamate excrete the corresponding carbamate and N-hydroxycarbamate in the urine. 6. Methyl, n-propyl and n-butyl carbamates and N-hydroxycarbamates are excreted more slowly than are urethane and N-hydroxyurethane. 7. The probable role of N-hydroxyurethane and the processes of alkylation and carbethoxylation, and of hydroxylamine, nitroxyl and hyponitrous acid in carcinogenesis and chemotherapy with urethane, have been discussed.     

The interaction of carbon-14-labelled alkyl carbamates, labelled in the alkyl and carbonyl positions, with DNA in vivo

The binding of ethyl carbamate labelled with carbon-14 in the alkyl or carbonyl group, and of methyl, n-butyl and n-propyl carbamates labelled in the alkyl group, to the DNA of mouse liver, lung and kidney has been studied in male Crackenbush mice. Only ethyl carbamate bound to liver and kidney DNA to any significant extent.The binding of ethyl carbamate labelled with carbon-14 in the C₁, C₂ or the carbonyl position was examined and compared. The levels of binding of [1-¹⁴C]- and [2-¹⁴C]ethyl carbamate to liver DNA were not significantly different (328 ± 34 and 267 ± 24 dpm/mg DNA, respectively), but there was very little binding of the [carbonyl-¹⁴C]ethyl carbamate (26 ± 3 dpm/mg DNA). Furthermore, only 18% of the radioactivity was removed from the DNA labelled with the alkyl-labelled carbamates, whereas 65% of the radioactivity was removed from the DNA labelled with carbonyl-labelled ethyl carbamate on continuous ether extraction. It was concluded that the bound molecule does not contain the carbonyl carbon and is probably an ethyl group.     

Effect of gem 2,2′-disubstitution and base in the formation of spiro- and ansa-1,3-propandioxy derivatives of cyclotriphosphazenes

The gem-dialkyl effect has been investigated in the reactions of cyclotriphosphazene, N₃P₃Cl₆1, with various 2,2′-derivatives of 1,3-propandiol, CXY(CH₂OH)₂, in either THF or DCM to form spiro (6-membered) and ansa (8-membered ring) derivatives. The reactions were made with a number of symmetrically-substituted (X = Y, methyl, ethyl, n-butyl and a malonate ester) and unsymmetrically-substituted (X ≠ Y, methyl/H, phenyl/H, methyl/n-propyl, ethyl/n-butyl and Br/NO₂) 1,3-propandiols. The products were analysed by ¹H and ³¹P NMR spectroscopy and some of the spiro and ansa derivatives were also characterized by X-ray crystallography. Reactions of 1 with unsymmetrically-substituted 1,3-propandiols results in the formation of two structural isomers of ansa-substituted compounds, both isomers (endo and exo) have been structurally-characterized by X-ray crystallography for the ethyl/n-butyl derivative. It is found that the regioselectivity of the reaction is changed when the base is changed. The relative proportions of spiro and ansa compounds formed under different reaction conditions were quantified by ³¹P NMR measurements of the reaction mixtures. The results were rationalised mainly in terms of the electronic effect of the substituents, whereas the steric effect has a secondary role in the formation of both spiro and ansa compounds.     

The differential effects of C-16,17-dihydro gibberellins and related compounds on stem elongation and flowering in Lolium temulentum

Plants of Lolium temulentum L. cv. Ceres grown under short days (SDs) can be induced to initiate inflorescences either by exposure to one long day (LD) or by single applications of some gibberellins (GAs), which also enhance the flowering response to one LD. Single doses of up to 25 μg per plant of C-16, 17-dihydro-GA₅ were about as effective as GA₅ for promoting flowering after one LD but inhibited stem elongation by up to 40% over three weeks. The promotion of flowering but not the inhibition of elongation by 16, 17-dihydro-GA₅ was reduced in SDs or in LDs low in far-red (FR) radiation. With shoot apices cultured in vitro, 16, 17-dihydro-GA₅ was more florigenic than GA₃ for apices excised after one LD of 14 h or more, but less florigenic for apices excised from plants in shorter days. 16, 17-Dihydro-GA₅ was ineffective compared with GA₁, GA₃ and GA₅ for α-amylase production by half-seeds of Lolium, a response concordant with its effect on stem elongation. As with GA₅, 16, 17-dihydro derivatives of GA₁, GA₃, GA₂₀ and several other GAs were more effective for flowering and less effective for stem elongation than the GAs from which they were derived. Hydroxylation at C-17 and/or C-16 generally reduced the effectiveness of 16, 17-dihydro-GA₅ for flowering. These results extend the known features of GA structure which favour flowering relative to stem elongation in L. temulentum. Moreover, C-16, 17-dihydro-GA₅ mimics, in its daylength- and wavelength-dependence and lack of stem elongation, characteristics of the LD stimulus in L. temulentum.     

Production and Purification of Thermostable Amylase and Protease of Thermomonospora viridis

Maximum yields of amylase were produced by the thermophilic actinomycete Thermomonospora viridis in a modified Simpson and McCoy medium containing 1.5% corn starch and 0.5% mycological peptone with an initial pH 7.0. Best yields of amylase were obtained after incubation for 48 h, when the pH of the medium had risen to 8.2. Amylase was purified 313-fold by precipitation with n-propyl alcohol, dialysis against tap water, adsorption on Ca₃(PO₄)₂, and fractionation on Sephadex G-100. Protease was produced in nutrient broth containing 0.5% starch and 1.0% corn steep liquor and at an initial pH 7.0. Maximum yields of protease were produced after 42 h. The protease was purified 54-fold by precipitation with n-propyl alcohol, dialysis against tap water, adsorption on Ca₃(PO₄)₂, and fractionation on Sephadex G-200.     

Synthesis of Compounds with Juvenile Hormone Activity

A new and convenient non-stereoselective synthetic route to the C₁₈-Cecropia juvenile hormone (as a stereoisomeric mixture) was developed. Employing this method, several juvenile hormone analogues with various alkyl groups at the terminal position were synthesized as stereoisomeric mixtures. Two analogues with two ethyl groups or n-propyl and methyl groups at the terminal position were more active than the C₁₈-Cecropia juvenile hormone on Tenebrio molitor and Tribolium castaneum.     

Mild hydrogenolysis of in-situ and isolated Pinus radiata lignins

The Pd/C-catalysed hydrogenolysis of in-situ and isolated lignins from Pinus radiata wood was investigated to gain a more complete understanding of the factors affecting yield and composition of the hydrogenolysis products. Such hydrogenolysis products could potentially be refined into aromatic feedstock chemicals providing sustainable alternatives to petroleum-derived phenols. Lignins were converted into solvent-soluble oils composed of monomeric, dimeric and oligomeric products in high yields, up to 89% of the original lignin. The main monomer products were dihydroconiferyl alcohol and 4-n-propyl guaiacol. Dimeric and oligomeric compounds constituted 75% of the hydrogenolysis oils and were mainly composed of dihydroconiferyl alcohol and 4-n-propyl guaiacol units linked by β-5, 5-5, 4-O-5 and β-1 linkages. Hydrogenolysis of steam exploded wood gave lower yields of lignin hydrogenolysis products compared to unmodified wood due to fewer reactive aryl-ether linkages in the lignin.     

Stereochemistry of some acetyl coenzyme a condensations

The absolute configuration of four naturally occurring α-alkylmalic acids (ethyl, n-propyl, isopentyl, and benzyl) has been established by synthesis, using the addition of alkyl lithium cuprates to the (R)-(+)-epoxide of dimethyl itaconate. It is concluded that α-alkylmalic acids biosynthesized from acetyl-CoA and α-ketoacids are, with few exceptions, formed by attack at the re face of the carbonyl group to generate (R)-(?) enantiomers.     

Thermal Decarboxylation of Sulfur-Containing Amino Acids in the Presence of Acetophenone,a Preparation of 3-Methylthiopropylamine Hydrochloride

3-Methylthiopropylamine hydrochloride was prepared from d,l-methionine and acetophenone in 90~92% yield by heating. Methionine sulfone was decarboxylated to γ-aminopropylmethyl sulfone, which migrated at the same rate as the authentic sample obtained from 3-methylthiopropylamine by hydrogen peroxide treatment. S-Alkylcysteines (R = methyl, ethyl, n-propyl, n-butyl and n-amyl) were also decarboxylated to give a product which showed new spots of 2-alkylthioethylamine with higher R_F values than those of the corresponding amino acids.     

Volatile constituents and fatty acid composition of lipids in Durio zibethinus

The predominating flavour compounds in the fruit pulp of Durio zibethinus were hydrogen sulfide, ethyl hydrodisulfide and several dialkyl polysulfides, particularly (C₂H₅)₂S_n, where n = 2 or 3. Ethyl acetate, 1,1-diethoxyethane and ethyl 2-methylbutanoate contribute to an additional fruity odour note. Hydrodisulfides are probably the precursors of the dialkyl sulfides. In the pericarp and seed no volatile sulfur compounds could be detected. The fatty acid composition of the lipids in pericarp, pulp and seed depended on the origin and/or harvest season of the fruit. The main components were oleic and palmitic acids or arachidic acid together with appreciable quantities of palmitoleic, stearic, linoleic and linolenic acids.     

Effects of Ring D-Modified Gibberellins on Gibberellin Levels and Development in Selected Sorghum bicolor Maturity Genotypes

Plants of early flowering mutant and wild type genotypes of Sorghum bicolor were treated with ring D-modified gibberellins (GAs), and the effects on endogenous GA levels were determined. The growth and timing of floral initiation in 58M plants grown under 18-h days (which significantly delays floral initiation in this short day plant) following treatment with these compounds, relative to GA₃ and GA₅ treatments, were also investigated. Application of the endo-isomer of C16,17-dihydro-GA₅ (endo-DiHGA₅), the exo-isomer of C16,17-dihydro-GA₅ (exo-DiHGA₅), and C16α,17-dichloromethanodihydro-GA₅ (DMDGA₅) altered GA levels in both genotypes. Each ring D-modified GA significantly inhibited shoot growth while significantly decreasing levels of GA₁ and increasing levels of its immediate precursor, GA₂₀. Gibberellin A₈ levels also decreased. Tillering was not affected by any treatment. For the early flowering genotype 58M, grown under noninductive long days, both dihydro-GA₅ isomers promoted floral initiation while shoot growth was strongly inhibited, and floral development was strongly advanced beyond floral stage 4. Gibberellin A₃ and GA₅, applied under the same conditions, promoted shoot growth slightly and gave ``floral-like' apical meristems that did not develop past floral stage 1. These results suggest that the reduced shoot growth of sorghum, which follows application of those ring D-modified GAs, is due to their inhibiting the 3β hydroxylation of GA₂₀ to GA₁, thereby reducing the GA₁ content. That floral initiation was hastened and floral development promoted in genotype 58M by application of both isomers of DiHGA₅ are in contrast to the effects of other GA biosynthesis inhibitors, which act earlier in the GA biosynthesis pathway, but are consistent with results seen for long day grasses. This suggests that endo-DiHGA₅ and exo-DiHGA₅ may be acting directly in promoting floral initiation and subsequent floral apex development of this short day plant under long day conditions. Received October 3, 1996; accepted January 22, 1997     

Stereospecific ligands and their complexes. V. Synthesis, characterization and antimicrobial activity of palladium(II) complexes with some alkyl esters of (S,S)-ethylenediamine-N,N′-di-2-propanoic acid

Three new ligands and their palladium(II) complexes of general formula [PdCl₂(R₂-S,S-eddp)] (R = n-propyl, n-butyl and n-pentyl) have been synthesized and characterized by microanalysis, infrared and ¹H and ¹³C NMR spectroscopy. Antimicrobial activity of these ligands and complexes was tested by microdilution method and both minimal inhibitory and microbicidal concentration were determined. These tested complexes demonstrated the significant antifungal activity against pathogenic fungi Aspergillus flavus and Aspergillus fumigatus. On the other hand, these complexes demonstrated moderate antibacterial activity.     

Synthesis and Biological Activity of Analogs of the Cecropia Juvenile Hormone with Different Alkyl Substituents at C-7 and C-11

Sixteen new Cecropia juvenile hormone (JH) analogs with different alkyl substituents at C–7 and C–11 were synthesized as stereoisomeric mixtures. The epoxides with n-propyl or n-butyl and methyl groups at C-11 and methyl or ethyl group at C-7 showed high JH activity on Bombyx mori L. Structure-activity relationship of the JH analogs was discussed.     

Occurrence of fatty acid lower-alkyl esters in euonymus fruits

Small amounts of a mixture of fatty acid lower-alkyl esters (FALAEs) were obtained from chloroform extracts of the fruit arils and seeds of four euonymus species (Euonymus sp., Celastraceae). The FALAEs were the products of biosyntheses in the cell rather than experimental artifacts. By using GC-MS, this mixture was shown to contain a total of 19 individual FALAE species comprising four separate fractions, viz. methyl (FAMEs), ethyl (FAEEs), n-propyl, and n-butyl FA esters. Fruit FALAEs included mainly FAEEs and, to a lesser extent, FAMEs, while the n-propyl and n-butyl FA esters, which occurred less frequently, were found here for the first time as the plant products. The FALAE acid components included C₁₄-C₁₈ saturated, mono-, di-, and trienoic FAs with the predominance of ubiquitous linoleic, oleic, palmitic, and, in some cases, also α-linolenic acid. The indices of qualitative and quantitative composition of separate FALAE fractions varied considerably depending on the plant species, fruit part (aril or seed), and the extent of fruit maturity. It can be supposed that, in some euonymus species, FAMEs and FAEEs are formed at the expense of the same FA pool characteristic for a given species. As a whole, euonymus FALAEs and triacylglycerols seem to be synthesized from different FA pools. Discussed is the physiological significance of FALAE biosynthesis in plant metabolism, possible pathways of this biosynthesis, as well as the perspectives of further investigations of FALAEs of plant origin.     

A Study on Low-boiling Chemical Constituents of Cryptotaenia japonica Hassk

Low-boiling compounds escaping during steam distillation of Cryptotaenia japonica Hassk were collected and examined with chromatographies and by the preparation of their derivatives.

The following compounds were identified. Six paraffin hydrocarbons: n-pentane, n-hexane, n-heptane, n-octane, isooctane, n-nonane; seven carbonyl compounds: formaldehyde, acetaldehyde, propionaldehyde, n-butyraldehyde, isovaleraldehyde, acetone, 3-methyl-2-pentanone; three esters: n-propyl and isopropyl formates, n-propyl acetate; eight alcohols: methanol, ethanol, n-propanol, isopropanol, isobutanol, sec-butanol, isopentanol, n-octanol; two sulfides: dimethyl and methyl ethyl sulfides; six monoterpene hydrocarbons: α-pinene, camphene, β-pinene, sabinene, myrcene, limonene.     

Enzymatic synthesis of glycerides from DHA-enriched PUFA ethyl ester by glycerolysis under vacuum

Pseudomonas lipase immobilized on CaCO₃ powder was used for the glycerolysis of n−3 polyunsaturated fatty acid ethyl esters to prepare nutritionally valuable glycerides. The initial ethyl ester contained 65 or 99% docosahexaenoic acid ethyl ester (DHAEE) which is very unstable and readily oxidized. The process performance was intensified by: (1) using Pseudomonas lipase which has good specificity for docosahexaenoic acid (DHA), (2) shifting the reaction equilibrium by evaporation of the resulted ethanol under vacuum and (3) enhancing the enzyme operational stability by immobilization on CaCO₃ powder. Under these conditions, over 90% conversion of DHAEE was achieved in 5 h and the oxidative deterioration of DHA was avoided. The final product contained 53% partial glyceride and, thus, had good emulsifying power. The catalyst was reused 5 times showing a very good stability in this system. Other lipases were tried for this reaction and different glyceride compositions were obtained depending on the enzyme specificity for the 1(3)-position of glycerol.     

Purification and characterization of alkane solubilizing factor produced by Pseudomonas PG-1

The normal hexadecane emulsifying and solubilizing factor (PG-1 ESF C₁₆) produced by Pseudomonas PG-1 during growth on n-hexadecane was isolated and purified. The factor was composed of protein, carbohydrate and lipid, which were largely undialyzable. Ca²⁺ was necessary for activation and heat stability of the factor. Particle size of the factor was less than 10 nm. All the protein along with 68–74% of the carbohydrate in the factor was obtained in a single protein peak by gel filtration chromatography using Biogel P-30. The isolated protein fraction showed a 1–5 fold increase in n-hexadecane solubilizing activity. The isolated protein was shown to be a homogeneous, monomeric protein with a molecular weight of approximately 11,000 daltons by SDS-PAGE. The protein and carbohydrate moieties in the isolate were separated by DEAE-cellulose chromatography. Neither purified protein nor carbohydrate showed n-hexadecane solubilizing activity separately, but when these were mixed full activity was restored. Hydrocarbon emulsifying activity was confined to the lipid fraction, which was isolated to the extent of 85% from the Biogel P-30 column by ethyl ether extraction.     

Synthesis of hybrid analogues of caffeine and eudistomin D and its affinity for adenosine receptors

Four bis-N-n-propyl analogues (3–6) in the uracil ring of two hybrid molecules (1 and 2) of caffeine and eudistomin D, a β-carboline alkaloid from a marine tunicate, were synthesized, and their affinity and selectivity for adenosine receptors A₁, A_2A, and A₃ were examined. All the compounds (3–6) showed better potency as adenosine receptor ligands than caffeine. Bis-N-n-propylation (3 and 4, respectively) of the uracil ring in 1 and 2 resulted in higher affinity for A₁ and A_2A adenosine receptors. Furthermore, it was found that a compound (5) possessing a n-propyloxy group at C-7 in compound 3 with a nitrogen at the β-position of the pyridine ring (β-N type) enhanced remarkably affinity for adenosine receptor A₃ subtype, while n-propyloxy substitution (compound 6) at C-5 in compound 4 with a nitrogen at the δ-position of the pyridine ring (δ-N type) reduced affinity for all the adenosine receptor, A₁, A_2A, and A₃. Among all the compounds (1–6) examined, compound 5 showed the most potent affinity for adenosine receptor A₃ subtype (K_i value, 0.00382 μM).     

Changes in Flavor Components of Onion by γ-Irradiation

The effect of γ-irradiation on the flavor of Onions (Senshu Yellow) associated with sprout-inhibition has been investigated. The relative amounts of propionaldehyde, n-propyl mercaptan and di-n-propyl disulfide in onions stored for 0, 5, 10, 20 and 30 days, respectively, after irradiation were estimated by measurement of peak areas of gas chromatograms. It was observed that the lachrymatory character and the pungent flavor had been decreased by γ-irradiation (remarkably at 70 and 700 Krads). In this connection, the amounts of propionaldehyde and di-n-propyl disulfide were decreased with increasing radiation dose and storage period at room temperature (20 to 25°C) and at low temperature (4°C). Moreover, it was observed that the sweetness had been decreased by γ-irradiation, but the amount of n-propyl mercaptan was increased with radiation dose and storage period. Therefore it seems that there is no correlation between the sweetness of onion and the amount of n-propyl mercaptan.