Quantitative gas chromatography and single-ion detection of aliphatic α-keto acids from urine as their o-trimethylsilylquinoxalinol derivatives

A method is described for the quantitative determination of aliphatic α-keto acids in urine after derivatization with o-phenylenediamine and bis(trimethylsilyl)trifluoroacetamide, α-Ketovaleric acid and α-ketocaprylic acid are used as internal standards.The chemical yield is 80–100%. At physiological concentrations, the coefficient of variation after repeated derivatizations is 4% for pyruvic acid and 14% for α-ketoglutaric acid.With mass spectrometric single-ion detection at $\text{m}\text{c}$ = 217, 232 and 245, the biologically interesting aliphatic α-keto acids can be determined at very low levels in biological fluids.     

α-(15-Hydroxyhexadecyl)itaconic acid from Usnea aliphatica

The structure of a new aliphatic acid from Usnea aliphatica was established by spectroscopic and chemical methods as α- (15-hydroxyhexadecyl)itaconic acid.     

Molecular orbital calculations on the conformation of polypeptides and proteins. V. Conformational energy maps and stereochemical rotational states of aliphatic residues

The quantum-mechanical calculations by the PCILO method on the conformation of amino acid residues of proteins have been extended to the valyl, leucyl, and isoleucyl residues. In distinction to the earlier “empirical” computations, the quantum-mechanical results indicate very similar energy contours for the stable conformations of the three residues. Their general outline is also similar to that of the alanyl residue, although reduced by about 25%. Contrary to the “empirical” computations, the present results predict that the region corresponding to the α-helix should be one of great stability for the three residues and in particular for the valyl residue. The quantum-mechanical results are in excellent agreement with the experimental conformations of the aliphatic residues in lysozyme and myoglobin. Their prediction as to the ready availability of the valyl residue in the α-helical conformation agrees moreover with Ptitsyn's statistical evaluation of the participation of this residue in the inner turns of the helical regions in six globular proteins. The maximum conformational space allowed for the aliphatic residues is somewhat smaller than that allowed for the aromatic ones, while the minimum conformational space (region of stability common to all the residues) is similar in both groups.     

Cloning of a Nitrilase Gene from the Cyanobacterium Synechocystis sp. Strain PCC6803 and Heterologous Expression and Characterization of the Encoded Protein

The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector. The encoded protein was heterologously expressed in the native form and as a His-tagged protein in Escherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate. The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits. The purified nitrilase converted various aromatic and aliphatic nitriles. The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile. From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed. The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents.     

Reactive Site of API-2c,an Alkaline Protease Inhibitor,for Subtilisin BPN′

Differences between α- and β-lipovitellin were examined, especially in regard to the polypeptide and carbohydrate composition of apolipoprotein. Both lipoproteins were composed of at least eight polypeptides with similar molecular weights ranging from 35,000 to 140,000 daltons. Polypeptides with 110,000 daltons were common major constituents. The close similarity of component polypeptides in the two lipoproteins was also assumed from similar amino acid compositions and the identical immunological properties of the two lipoproteins. However, some notable differences were found in the composition of the polypeptides. α-Lipovitellin contained much more polypeptide with 85,000 daltons than β-lipovitellin. Both apolipovitellins were found to be glycoprotein containing mannose, galactose, glucosamine and sialic acid. The sialic acid in α-lipovitellin exceeded that in β-lipovitellin by six times, though only slight differences were found in the content of neutral and amino sugars. The relatively acidic nature of a-lipovitellin compared with β-lipovitellin is attributed not only to the relative predominance in protein phosphorus but also to the predominance in the sialic acid.     

黄连木叶挥发油化学成分研究

采用水蒸气蒸馏法提取中药黄连木叶挥发油。通过气相色谱／质谱联用技术测定了提取物的化学成分,从中鉴定出99个化合物,主要含有多种长链脂肪烃、棕榈酸及芳樟醇等多种萜类化合物。初步分析了所含成分的药效。结果显示,黄连木叶有一定的药用价值。     

Noncoded residues as building blocks in the design of specific secondary structures: Symmetrically disubstituted glycines and β-alanine

Structural changes can be induced in a peptide by selective substitution of coded α-amino acid residues by noncoded α-amino acid residues and the consequent production of analogues with modified structure and conformational preferences. In this review article we summarize the solid state structural results and the conformational preferences of two classes of “building blocks”: (a) the linear and cyclic symmetrically α, α-disubstituted glycines in which either two identical n-alkyl groups replace the hydrogen atoms of the glycine residue or a cyclic aliphatic side-chain system is formed by linking the two α-carbon side chains, respectively; and (b) the β-alanine residue. Examples, whenever possible, of the use of these residues for the elucidation of the bioactive conformation in the appropriate biological systems will be given. © 1993 John Wiley & Sons, Inc.     

Kinetics of the reactions of pentacyanonitrosylferrate(II) with mono- and diamino acids

The reaction of Fe(CN)₅NO^2? with glycine, α-alanine, β-alanine, γ-aminobutyric acid, ornithine and lysine were gas-volumetrically studied in a weakly-alkaline medium. The kinetic data show that the reactivity of the amino group depends on the basicity of the amine, on the behaviour of nucleophilic centers of the carbon chain, and on their steric positions.The kinetic results are compared to the data of the reactions of amino acids with nitrous acid and of the complex with aliphatic amines.     

A highly enantioselective rhodium catalyst for the hydrogenation of aliphatic α-keto esters

The enantioselective hydrogenation of several α-keto acid derivatives with rhodium diphosphine catalysts has been investigated using a random screening approach. The neutral rhodium catalyst prepared in situ from bis(2,5-norbornadiene rhodium chloride) and NORPHOS has been found to be an excellent catalyst for preparing aliphatic α-hydroxy esters in high optical purities. The reaction parameters for the hydrogenation of ethyl 2-oxo-4-phenyl-butyrate, an intermediate for the ACE inhibitor Benazepril, were optimized and the best optical yields obtained were 96%.     

红菜薹6个品种氨基酸含量的分析比较

对红菜薹6个品种的氨基酸种类、含量进行分析测定,结果表明,红菜薹含有17种氨基酸,其中7种为人体必需氨基酸,各品种间各类氨基酸含量差异不显著;相对于茎干而言,红菜薹的薹叶具有更高的营养价值和药用价值;不同的采收温度对各类氨基酸含量的影响不同。     

Formation of amino acids from aliphatic nitriles and aliphatic amines by contact glow discharge electrolysis

Contact glow discharge electrolyses (CGDE) were carried out relative to the prebiotic formation of amino acids by amination of aliphatic nitrile in aqueous ammoniacal solution, and by cyanization of aliphatic amine by sodium cyanide. The CGDE of propionitrile by amination followed by hydrolysis resulted in the formation of glycine, alanine and β-alanine. The reaction of ethylamine by cyanization, gave glycine, alanine, β-alanine, aspartic acid, and serine. In these reactions, a relatively high ratio of glycine was observed. This could be explained by the cleavage of the α,β-carbon bond, which was broken easily, due to the strong electron-attracting property of the nitrile group of propionitrile and the resulting α-aminopropionitrile.     

Cloning of a nitrilase gene from the cyanobacterium Synechocystis sp. strain PCC6803 and heterologous expression and characterization of the encoded protein

The gene encoding a putative nitrilase was identified in the genome sequence of the photosynthetic cyanobacterium Synechocystis sp. strain PCC6803. The gene was amplified by PCR and cloned into an expression vector. The encoded protein was heterologously expressed in the native form and as a His-tagged protein in Escherichia coli, and the recombinant strains were shown to convert benzonitrile to benzoate. The active enzyme was purified to homogeneity and shown by gel filtration to consist probably of 10 subunits. The purified nitrilase converted various aromatic and aliphatic nitriles. The highest enzyme activity was observed with fumarodinitrile, but also some rather hydrophobic aromatic (e.g., naphthalenecarbonitrile), heterocyclic (e.g., indole-3-acetonitrile), or long-chain aliphatic (di-)nitriles (e.g., octanoic acid dinitrile) were converted with higher specific activities than benzonitrile. From aliphatic dinitriles with less than six carbon atoms only 1 mol of ammonia was released per mol of dinitrile, and thus presumably the corresponding cyanocarboxylic acids formed. The purified enzyme was active in the presence of a wide range of organic solvents and the turnover rates of dodecanoic acid nitrile and naphthalenecarbonitrile were increased in the presence of water-soluble and water-immiscible organic solvents.     

Lack of effect of vaginal lavages and aliphatic acids on ejaculatory responses in rhesus monkeys: Behavioral and chemical analyses

Four behavioral experiments involving a total of 19 adult male and 27 adult spayed female rhesus monkeys failed to reveal significant sexual stimulatory properties of vaginal lavages obtained from estrogen-treated donor females when the material was applied to spayed nonestrogenized recipient females. However, all but one of the males copulated to ejaculation when paired with estrogenized females. Two of three males showed moderate increases in sexual behavior with recipients when the vaginal lavage tested was contaminated with 24-hr-old ejaculate. When purified aliphatic acids were applied to spayed nonestrogenized recepients, one of two males showed increased frequencies of mounting behavior, but intromissions and ejaculations were not displayed.Quantitative analyses of short chain aliphatic acids in vaginal lavages, the hypothesized active component, revealed that (a) spayed females had nondetectable levels of aliphatic acids; (b) daily estradiol benzoate treatment for 6–10 days (25 μg/day i.m.) induced detectable levels of acetic, propionic, and butyric acids; (c) exposure to estradiol for six months resulted in a fairly constant plateau of aliphatic acid concentrations; and (d) ejaculate from the male caused up to fivefold elevations in the aliphatic acid concentrations.Three intact females were studied throughout a menstrual cycle, and the peak values of aliphatic acids occurred in the luteal phase, several days after presumed ovulation. Three spayed females treated chronically with estradiol were given four daily injections of 5 mg progesterone, and the mean concentrations of aliphatic acids increased from 199.7 to 801.0 μg/ml. However, the endocrine conditions associated with maximum concentrations of aliphatic acids in either intact or spayed females are known from other studies to be associated with decreased likelihood of copulation.We conclude that for the majority of males studied, the application of vaginal lavages obtained from estrogenized donors did not significantly increase copulatory behavior with spayed, nonestrogenized recipient females. Moreover, the data from aliphatic acid determinations suggest that increases in concentrations of these substances are not always associated with facilitation of copulation, since the largest increases were found either (1) following copulation to ejaculation, (2) during the luteal phase in cycles free of copulation, or (3) following progesterone treatment of spayed, estrogenized females. Finally, comparison of our results with those from other laboratories suggests that the mechanism involved in positive effects may depend upon associative learning or upon extinction and disinhibition of sexual interest.     

Conversion of aliphatic nitriles by the arylacetonitrilase from <Emphasis Type="Italic">Pseudomonas fluorescens</Emphasis> EBC191

The conversion of aliphatic nitriles by the arylacetonitrilase from Pseudomonas fluorescens EBC191 (NitA) was analyzed. The nitrilase hydrolysed a wide range of aliphatic mono- and dinitriles and showed a preference for unsaturated aliphatic substrates containing 5–6 carbon atoms. In addition, increased reaction rates were also found for aliphatic nitriles carrying electron withdrawing substituents (e.g. chloro- or hydroxy-groups) close to the nitrile group. Aliphatic dinitriles were attacked only at one of the nitrile groups and with most of the tested dinitriles the monocarboxylates were detected as major products. In contrast, fumarodinitrile was converted to the monocarboxylate and the monocarboxamide in a ratio of about 65:35. Significantly different relative amounts of the two products were observed with two nitrilase variants with altered reaction specifities. NitA converted some aliphatic substrates with higher rates than 2-phenylpropionitrile, which is one of the standard substrates for arylacetonitrilases. This indicated that the traditional classification of nitrilases as “arylacetonitrilases”, “aromatic” or “aliphatic” nitrilases might require some corrections. This was also suggested by the construction of some variants of NitA which were modified in an amino acid residue which was previously suggested to be essential for the conversion of aliphatic substrates by a homologous nitrilase.     

Differential induction of polar and non‐polar metabolism during wound‐induced suberization in potato (Solanum tuberosum L.) tubers

Wound‐induced suberin deposition involves the temporal and spatial coordination of phenolic and fatty acid metabolism. Phenolic metabolism leads to both soluble metabolites that accumulate as defense compounds as well as hydroxycinnamoyl derivatives that form the basis of the poly(phenolic) domain found in suberized tissue. Fatty acid metabolism involves the biosynthesis of very‐long‐chain fatty acids, 1‐alkanols, ω‐hydroxy fatty acids and α,ω‐dioic acids that form a poly(aliphatic) domain, commonly referred to as suberin. Using the abscisic acid (ABA) biosynthesis inhibitor fluridone (FD), we reduced wound‐induced de novo biosynthesis of ABA in potato tubers, and measured the impact on the expression of genes involved in phenolic metabolism (StPAL1, StC4H, StCCR, StTHT), aliphatic metabolism (StCYP86A33, StCYP86B12, StFAR3, StKCS6), metabolism linking phenolics and aliphatics (StFHT) or acyl chains and glycerol (StGPAT5, StGPAT6), and in the delivery of aliphatic monomers to the site of suberization (StABCG1). In FD‐treated tissue, both aliphatic gene expression and accumulation of aliphatic suberin monomers were delayed. Exogenous ABA restored normal aliphatic suberin deposition in FD‐treated tissue, and enhanced aliphatic gene expression and poly(aliphatic) domain deposition when applied alone. By contrast, phenolic metabolism genes were not affected by FD treatment, while FD + ABA and ABA treatments slightly enhanced the accumulation of polar metabolites. These data support a role for ABA in the differential induction of phenolic and aliphatic metabolism during wound‐induced suberization in potato.     

Mutational analysis of type IV collagen alpha5 chain, with respect to heterotrimer formation

Alport syndrome (AS) is caused by mutations in type IV collagen α3, α4, and α5 chains. The three chains form a heterotrimer. In this study, we introduced 12 kinds of missense and three kinds of nonsense mutations, corresponding to AS mutations, into the NC1 domain of α5(IV) and characterized the mutant chains. Nine α5(IV) chains with amino acid substitutions and all three truncated α5(IV) chains did not form a heterotrimer and were not secreted from cells. Three α5(IV) chains with amino acid substitutions did, however, form heterotrimers in cells, but these were not secreted from cells. These findings indicate that a defect in heterotrimer formation is the main molecular mechanism underlying the pathogenesis of AS caused by mutation in the NC1 domain. We also showed that even a single amino acid deletion in the carboxyl-terminal region markedly affected the heterotrimerization, indicating that the carboxyl-terminal end is indispensable for heterotrimer formation.     

A general method for preparation of N‐Boc‐protected or N‐Fmoc‐protected α,β‐didehydropeptide building blocks and their use in the solid‐phase peptide synthesis

N‐(tert‐butyloxycarbonyl) or N‐(9‐fluorenylmethoxycarbonyl) dipeptides with C‐terminal (Z)‐α,β‐didehydrophenylalanine (?^ZPhe), (Z)‐α,β‐didehydrotyrosine (?^ZTyr), (Z)‐α,β‐didehydrotryptophan (?^ZTrp), (Z)‐α,β‐didehydromethionine (?^ZMet), (Z)‐α,β‐didehydroleucine (?^ZLeu), and (Z/E)‐α,β‐didehydroisoleucine (?^Z/EIle) were synthesised from their saturated analogues via oxidation of intermediate 2,5‐disubstituted‐oxazol‐5‐(4H)‐ones (also known as azlactones) with pyridinium tribromide followed by opening of the produced unsaturated oxazol‐5‐(4H)‐one derivatives in organic‐aqueous solution with a catalytic amount of trifluoroacetic acid or by a basic hydrolysis. In all cases, a very strong preference for Z isomers of α,β‐didehydro‐α‐amino acid residues was observed except of the ΔIle, which was obtained as the equimolar mixture of Z and E isomers. Reasons for the (Z)‐stereoselectivity and the increased stability of the aromatic α,β‐didehydro‐α‐amino acid residue oxazol‐5‐(4H)‐ones over the corresponding aliphatic ones are also discussed. It is the first use of such a procedure to synthesise peptides with the C‐terminal unsaturated residues and a peptide with 2 consecutive ΔPhe residues. This approach is very effective especially in the synthesis of peptides with aliphatic α,β‐didehydro‐α‐amino acid residues that are difficult to obtain by other methods. It allowed the first synthesis of the ?Met residue. It is also more cost‐effective and less laborious than other synthesis protocols. The dipeptide building blocks obtained were used in the solid‐phase synthesis of model peptides on a polystyrene‐based solid support. Peptides containing aromatic α,β‐didehydro‐α‐amino acid residues were obtained with PyBOP or TBTU as a coupling agent with good yields and purities. In the case of aliphatic α,β‐didehydro‐α‐amino acid residues, a good efficiency was achieved only with DPPA as a coupling agent.     

Synthesis and assay of structural intermediates of crustacean pigment-dispersing hormones (α and β-PDH)

Summary

The two characterized crustacean pigment-dispersing hormones (α-PDH; β-PDH) are octadecapeptides which differ in primary structure at six positions. Assays for melanophore pigment-dispersing activity showed β-PDH to be 21-fold more potent than α-PDH. In an effort to explain the difference in potencies between the two PDHs, we synthesized and purified six analogs of α-PDH (Leu^4?, Leu^11?, Lys^13?, Asn^16?, Asp^17?, and Glu³, Leu^4? α-PDH) in which the amino acid residues of α-PDH were substituted with those of β-PDH. Four analogs (Leu^11?, Lys^13?, Asn^16?, and Asp^17? α-PDH) possessed melanophore-dispersing activity equivalent to α-PDH. Leu^4? α-PDH and Glu³, Leu^4? α-PDH were 2.4? and 4-fold more potent than α-PDH, respectively. Glu^3-α-PDH was 3.3-fold more potent than α-PDH (Jorenby et al., 1987). These results suggest that the 21-fold increase in activity of β-PDH over α-PDH is due to an interactive effect of two or more substitutions rather than from the product of the effects brought about by individual substitutions.     

Quantitative Study of Anomeric Forms of Glucose Produced by α-Glucosidases and Glucoamylases

A gas-liquid chromatographic method was applied to the determination of anomeric forms of sugar produced by carbohydrases. Anomeric forms of glucose released from maltotriose, phenyl α-maltoside and phenyl α-glucoside were determined quantitatively. Thirteen α-glucosidases tested, including α-glucosidase from honey bee and acid α-glucosidase from pig′s liver, were found to produce α-glucose exclusively, and two kinds of glucoamylases, only β-glucose. This method proved very useful for the determination of the anomeric forms of sugar produced. It was confirmed that mammalian acid α-glucosidase does not belong to the category of exo-1,4-α-glucosidase (EC 3.2.1.3).     

Optical Resolution and Determination of Absolute Configurations of α-Isopropyl-4-substituted Phenylacetic Acids and Insecticidal Activities of Their 5-Benzyl-3-furylmethyl Esters

In connection with disclosure of a new class of insecticides, the modified phenylacetates, six new optically active α-isopropy-4-substituted phenylacetic acids whose substituents are respectively 4-methyl,4-methoxy, 4-fluoro, 4-chloro, 4-bromo and 3,4-methylenedioxy group were prepared by optical resolution and their absolute configurations were determined by comparative ORD with α-isopropylphenylacetic acid derivatives whose absolute configuration is known as (S)-(²). Esters of the (S)-(²)-acids with 5-benzyl-3-furylmethanol were nearly twice toxic to Musca domestica than those of the racemic esters. Optical purities of the resolved acids were determined by GLC and NMR (with Eu-FOD) as (?)-methyl esters.