氨基酸的消旋研究

在纯乙酸溶剂中,以乙酸酐酰化消旋酪氨酸,消旋温度为90℃,L-酪氨酸和乙酸酐的摩尔比为12,每消旋1gL-酪氨酸时,溶剂乙酸的用量为5mL,效果较好。同样的方法应用于L-脯氨酸,L-苏氨酸,L-缬氨酸,L-赖氨酸,L-半胱氨酸等氨基酸,消旋率均可达到100%。     

The preparation of amino bile acid derivatives

Bile acid derivatives have been prepared by reaction of their mixed anhydrides with diaminoethane. The new compounds have side chains modified by amide bond formation which extends the side chain by two carbon atoms and terminates in a primary amino group. Important properties of the new compounds are their solubility in acid, and their ability to act as nucleophiles in the carbodiimide reaction or mixed anhydride reaction. The derivatives have been used to prepare high molar ratio immunogens with bovine serum albumin and to prepare I labelled ligands for radioimmunoassay     

Comparison of two solid-phase peptide syntheses of a 32-amino acid carboxyl-terminal fragment of human parathyroid hormone,hPTH-(53–84)

Two approaches were employed in separate syntheses of the carboxyl-terminal third of human parathyroid hormone, hPTH-(53–84). The standard N,N′-dicychohexylcarbodiimide coupling method was compared to amino acid incorporation using in situ and preformed symmetrical anhydrides of tert-butoxycarbonyl-amino acids. Also, protection of the δ-carboxyl function of glutamic acid by the benzyl group was compared to protection by the chlorobenzyl group. The products of both synthetic strategies were chemically evaluated. The purified synthetic hormone fragment prepared by standard methods was found to be homogeneous and virtually identical to the native carboxyl-terminal region of the hormone by the analytical criteria employed. However, the synthetic product resulting from use of symmetrical anhydrides was heterogeneous. Also, several practical advantages were noted for amino acid incorporation by the standard method. Since the symmetrical anhydride method has been found to be extremely successful as an approach to the synthesis of other peptides, this report indicates that the choice of symmetrical anhydride versus standard coupling methods may in part be determined by the sequence selected for synthesis. In addition, both approaches produced a large number of side products. Hence, methods selected for purification of the desired product were essential to the successful preparation of this synthetic peptide.     

A convenient method for the synthesis of C-protected esters of BOC-/Z-alpha,alpha- dialkylamino acids by the mixed carboxylic carbonic anhydride method

The synthesis of C-protected esters of Boc-/Z-alpha,alpha-dialkylamino acids is accomplished by using alkyl/aryl chloroformate in presence of DMAP as a catalyst. The reaction proceeds through mixed carboxylic carbonic anhydride, which was monitored by IR. The reaction was clean and complete in about 2 hr. All the esters prepared have been obtained in good yield and are fully characterized.     

醋酸酐对太平洋白对虾(Penaeus vannamei)β-N-乙酰- D-氨基葡萄糖苷酶的抑制动力学

β-N-乙酰-D-氨基葡萄糖苷酶与南美白对虾的食物消化吸收、蜕壳生长有着密切关系. 海水里存在的有机污染物将影响酶生理功能, 从而进一步影响虾的正常蜕壳,严重将导致对虾的死亡. 醋酸酐是常用的有机溶剂, 故本文应用动力学方法研究醋酸酐对南美白对虾β-N-乙酰-D-氨基葡萄糖苷酶催化pNP-NAG水解时酶活力的变化规律. 表明在醋酸酐浓度低于20.0 mmol/L, 酶的抑制作用是可逆的, 测得醋酸酐对酶抑制的IC50为9.0 mmol/L. 用双倒数作图法测定醋酸酐与游离酶(E)和酶-底物络合物(ES)的结合平衡常数, 结果显示醋酸酐是酶的非竞争性抑制剂. 用底物反应动力学方法观测在不同底物浓度下酶在0.0、3.0、6.0、9.0、12.0 mmol/L的醋酸酐溶液中的失活过程,分别测定了酶的微观失活速度常数k+0及复活速度常数k-0, 结果表明醋酸酐对酶的影响是快速结合再缓慢失活的过程. 比较微观失活速度常数k+0及复活速度常数k-0, 结果暗示在高浓度的醋酸酐溶液中, 酶将完全失活.     

Solid-phase peptide synthesis of somatostatin using mild base cleavage of N alpha-9-fluorenylmethyloxycarbonylamino acids

Experimental details for the "Fmoc solid phase peptide synthesis" of somatostatin are described. The 9-fluorenylmethyloxycarbonyl group was rapidly and quantitatively cleaved by 55% piperidine in dimethylformamide and monitored (u.v.) manually. For a kinetic study, a centrifugal reactor with a photometric control system and reference cell was used at each stage. The symmetrical anhydride coupling reaction was rapid and either acetic anhydride or fluorescamine termination was incorporated to minimize formation of deletion peptides. Anchor-bond cleavage was effected with trifluoroacetic acid which simultaneously removed all the acid labile tert.-butyl side chain protecting groups. N alpha-9-fluorenylmethyloxycarbonyl peptides may be obtained by omitting the piperidine deprotection step after the last cycle of synthesis. From several syntheses, analytically pure di-S-protected somatostatin 14-peptide was obtained in 55-60% overall yield. The S-protecting groups were removed and the product was purified by gel filtration to give homogeneous dihydrosomatostatin (91%) yield. Oxidation of dihydrosomatostatin with potassium ferricyanide and purification by countercurrent distribution provided analytically pure homogeneous somatostatin.     

Adducts between nucleophilic amino acids and hexahydrophthalic anhydride, a structure inducing both types I and IV allergy.

Haptens causing type I allergy have been shown to predominantly form lysine adducts in the carrier protein, while many haptens giving rise to type IV allergy preferentially form adducts with cysteine residues. Hexahydrophthalic anhydride derivatives are strong sensitizers capable of inducing allergic rhinitis, asthma and urticaria (type I allergy) and allergic contact dermatitis (type IV allergy). The ability of hexahydrophthalic anhydride (HHPA) to form adducts with nucleophilic amino acids and a model peptide in vitro is presented. Adduct formation was monitored by high-performance liquid chromatography with ultraviolet light/vis detection (LC-UV/vis) and high-performance liquid chromatography with mass spectrometric detection (LC/MS). The characterization was obtained by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS and MS/MS). It was found that HHPA formed adducts with N(alpha)-acetylated lysine and cysteine and the non-acetylated alpha-amino group of proline and, to some extent, also with other nucleophilic amino acids. The adducts with lysine and proline were chemically stable. Addition of one HHPA to a model carrier peptide with all important nucleophilic amino acid residues showed N-terminal proline to be the major site of reaction. The addition of a second hapten gave a lysine adduct, but a minor cysteine adduct was also found. The cysteine-HHPA adducts were shown to be chemically unstable and participated in further reactions with lysine forming lysine-HHPA adducts. The results will be useful for understanding the formation of HHPA-protein adducts with the capability of being markers of exposure, and also to a deeper understanding of the chemical structures causing types I and IV allergy.     

Synthesis of glutathione analogues with modified ionizable groups for the study of enzyme:ligand interactions

The syntheses and analyses of GSSG analogues with modified ionizable groups are described. Using the mixed anhydride method, three symmetrical and three asymmetrical peptide analogues were obtained in moderate overall yields. The products were analyzed by HPLC, 1H-NMR, thin-layer electrophoresis, and amino acid analysis. They were used to study enzyme:ligand interactions in glutathione reductase.     

Determination of pyrrolidone carboxylate and gamma-glutamyl amino acids by gas chromatography.

Gas chromatographic methods for the quantitation of pyrrolidone carboxylate and γ-glutamyl amino acids are described. These intermediates of the γ-glutamyl cycle were separated by ion exchange chromatography and converted to their N-acyl-ester derivatives in a reaction with a mixture of 2,2,3,3,3-pentafluoro-1-propanol and pentafluoropropionic anhydride. The derivatives have excellent electron capture properties thus making possible their determination even in small amounts of material of biological origin. The method was applied for the determination of concentrations of pyrrolidone carboxylate in human urine and cerebrospinal fluid, and in the brain, liver, and kidney of the mouse. It was also used to demonstrate the formation in mouse tissues of several γ-glutamyl derivatives of amino acids after administration of the corresponding free amino acid.     

A kinetic study of isoamyl acetate synthesis by immobilized lipase-catalyzed acetylation in n-hexane

The objective of this work was to propose a reaction mechanism and to develop a rate equation for the synthesis of isoamyl acetate by acylation of the corresponding alcohol with acetic anhydride using the lipase Novozym 435 in n-hexane. The reaction between isoamyl alcohol and acetic anhydride occurred at high rate in first place. Then, if excess alcohol was used, produced acetic acid further reacted with remaining alcohol, leading to yields higher than 100% (based on initial acetic anhydride content). This reaction was much slower and took place only when acetic anhydride had been totally consumed. Optimal pH for Novozym 435 was 7.7. Acetic acid strongly inactivated the enzyme but it was partially caused by the pH drop in the biocatalyst aqueous microenvironment. Acetic anhydride also showed an important inhibition effect. On the contrary, isoamyl alcohol and isoamyl acetate had no negative effect on the lipase. The analysis of the initial rate data showed that reaction followed a Ping-Pong Bi-Bi mechanism with inhibition by acetic anhydride. The kinetic constants were obtained by multiple regression analysis of experimental findings. Equation predictions and experimental reaction rate values matched very well at conditions where acetic acid concentration in the medium was low.     

Reactivity and ionization constants of the lysine residues in apovitellenin i of emu egg yolk low-density lipoprotein by competitive labelling.

Competitive labelling with[14C]acetic anhydride over a range of pH values has been used to explore the surface topography of the apovitellenin I moiety in emu egg yolk low-density lipoprotein. The reaction of the lysine xi-amino groups with acetic anhydride has been related to pH in a set of titration curves; from these, the reactivities relative to alanine and the ionization constants of all but the amino terminal lysines have been determined. All lysines have near normal pKa values around 10, and lower than normal reactivities (except the amino terminal lysine). At pH values above 10, the titration curves show breaks where the epsilon-amino groups become much more reactive, except for lysine 71 which in this regard behaves like a normally ionizing lysine in not showing a discontinuity. Most of the basic residues in this apoprotein may occur clustered at the surface of the molecule. This accounts best for the observed low reactivities and pKa values. The amino terminal lysine residue is presumably completely exposed to the aqueous environment.     

Diketopiperazine-mediated peptide formation in aqueous solution

Though diketopiperazines (DKP) are formed in most experiments concerning the prebiotic peptide formation, the molecules have not been paid attention in the studies of chemical evolution. We have found that triglycine, tetraglycine or pentaglycine are formed in aqueous solution of glycine anhydride (DKP) and glycine, diglycine or triglycine, respectively. A reaction of alanine with DKP resulted in the formation of glycylglycylalanine under the same conditions. These results indicate that the formation of the peptide bonds proceeds through the nucleophilic attack of an amino group of the amino acids or the oligoglycines on the DKP accompanied by the ring-opening.The formation of glycine anhydride, di-, tri- and tetraglycine was also observed in a mixed aqueous solution of urea and glycine in an open system to allow the evaporation of ammonia. A probable pathway is proposed for prebiotic peptide formation through diketopiperazine on the primitive Earth.     

Adducts between nucleophilic amino acids and hexahydrophthalic anhydride,a structure inducing both types I and IV allergy

Haptens causing type I allergy have been shown to predominantly form lysine adducts in the carrier protein, while many haptens giving rise to type IV allergy preferentially form adducts with cysteine residues. Hexahydrophthalic anhydride derivatives are strong sensitizers capable of inducing allergic rhinitis, asthma and urticaria (type I allergy) and allergic contact dermatitis (type IV allergy). The ability of hexahydrophthalic anhydride (HHPA) to form adducts with nucleophilic amino acids and a model peptide in vitro is presented. Adduct formation was monitored by high-performance liquid chromatography with ultraviolet light/vis detection (LC-UV/vis) and high-performance liquid chromatography with mass spectrometric detection (LC/MS). The characterization was obtained by nuclear magnetic resonance spectroscopy (NMR) and mass spectrometry (MS and MS/MS). It was found that HHPA formed adducts with N^α-acetylated lysine and cysteine and the non-acetylated α-amino group of proline and, to some extent, also with other nucleophilic amino acids. The adducts with lysine and proline were chemically stable. Addition of one HHPA to a model carrier peptide with all important nucleophilic amino acid residues showed N-terminal proline to be the major site of reaction. The addition of a second hapten gave a lysine adduct, but a minor cysteine adduct was also found. The cysteine–HHPA adducts were shown to be chemically unstable and participated in further reactions with lysine forming lysine–HHPA adducts. The results will be useful for understanding the formation of HHPA–protein adducts with the capability of being markers of exposure, and also to a deeper understanding of the chemical structures causing types I and IV allergy.     

Large fragments of nef-protein and gp110 envelope glycoprotein from HIV-1. Synthesis, CD analysis and immunoreactivity

Chemical synthesis of large peptide fragments (from 18 to 66 amino acid residues long) of the gp110 envelope glycoprotein and of nef-protein from HIV-1 was achieved by the solid phase method. Stepwise assembling of the peptide chains was carried out automatically on 4-(oxymethyl)-phenylacetamidomethyl resin using the N-alpha-butyloxycarbonyl amino acids with benzyl-based side chain protecting groups. Two elongation protocols were used depending on the peptide chain length: a standard cycle, mainly characterized by a single coupling step (Boc-amino acid symmetrical anhydride in dimethylformamide), and an optimized one for large peptides, based on a double coupling strategy (Boc-amino acid symmetrical anhydride first in dimethylformamide, then in dichloromethane). Final cleavage of the peptide from the solid support was carried out by anhydrous hydrogen fluoride and crude peptides were purified by C18 reverse phase medium pressure liquid chromatography after molecular filtration. Characterization of the purified peptides was done by analytical HPLC, amino acid content determination, and circular dichroism analysis both in polar (H2O) and in non-polar (TFE) environments. Immunoreactivity of anti-nef positive sera from HIV-1 infected patients by ELISA with the synthetic peptides was investigated. The results showed four major antigenic regions of nef-protein and mainly the immunodominance of the N- and C-termini of the molecule. Several of these peptides should prove to be useful for both diagnosis and vaccination purposes.     

A procedure for the rapid, quantitative N-acetylation of amino sugar methyl glycosides.

A rapid and quantitative procedure is described for the re-N-acetylation of amino sugar methyl glycosides prior to their analysis by gas-liquid chromatography. Two equivalents of pyridine are added to acidic methanolysates containing amino sugars, serving both to neutralize the acid and to act as a catalyst for the subsequent N-acetylation reaction with acetic anhydride. The N-acetylation is quantitative and complete within 10 min at ambient temperature. Excess acetic anhydride is destroyed by solvolysis with the methanolic solvent. The procedure has been used effectively for methanolysates containing 0.01–2.0 mg/ml glucosamine. The procedures utilizing ion-exchange columns and insoluble salts are thus circumvented and all reaction byproducts are volatile. The procedure is therefore ideally suited for the simultaneous workup of numerous samples for analytical procedures such as gas-liquid chromatography.     

Ion-exchange chromatography in lunar organic analysis

Although ion exchange chromatography has been used in separating amino acids from mineral salts, quantitative recovery has not been possible for the basic amino acids or for subnanomole concentrations of amino acids.As an analytical tool for amino acid analysis, ion-exchange chromatography has made it possible to resolve a relatively complex mixture of amino acids in less than an hour with detection limits of less than 10^–12 moles of amino acids. Reasonable specificity for amino acids is achieved by multiple wavelength detection of the reaction product found with ninhydrin. Unequivocal specificity must be obtained in conjunction with other methods such as mass spectrometry.In the analysis of subnanomole levels of amino acids, it is necessary to carry both reagent blanks and low-level amino acid standards through the entire sample preparation step since both contamination and selective losses occur and must be monitored.     

Mycobacterial ubiquitin-like protein ligase PafA follows a two-step reaction pathway with a phosphorylated pup intermediate

In Mycobacterium tuberculosis, the enzyme PafA is responsible for the activation and conjugation of the proteasome-targeting molecule Pup to protein substrates. As the proteasomal pathway has been shown to be vital to the persistence of M. tuberculosis, understanding the reaction mechanism of PafA is critical to the design of antituberculous agents. In this study, we have developed novel techniques to study the activity of PafA and have characterized fundamental features of the reaction mechanism. We show that PafA catalyzes a two-step reaction mechanism proceeding through a γ-glutamyl phosphate-mixed anhydride intermediate that is formed on the C-terminal glutamate of Pup before transfer of Pup to the substrate acceptor lysine. SDS-PAGE analysis of formation of the phosphorylated intermediate revealed that the rate of Pup activation matched the maximal steady-state rate of product formation in the overall reaction and suggested that Pup activation was rate-limiting when all substrates were present at saturating concentrations. Following activation, both ADP and the phosphorylated intermediate remained associated with the enzyme awaiting nucleophilic attack by a lysine residue of the target protein. The PafA reaction mechanism appeared to be noticeably biased toward the stable activation of Pup in the absence of additional substrate and required very low concentrations of ATP and Pup relative to other carboxylate-amine/ammonia ligase family members. The bona fide nucleophilic substrate PanB showed a 3 orders of magnitude stronger affinity than free lysine, promoting Pup conjugation to occur close to the rate limit of activation with physiologically relevant concentrations of substrate.     

Effect of ppGpp on the accuracy of protein biosynthesis

The maintenance of accuracy in protein biosynthesis in amino acid-starved rel+ strains of Escherichia coli has been attributed to an effect of ppGpp on the accuracy of aa-tRNA selection by the ribosome. It has been determined that concentrations of ppGpp characteristic of those found in amino acid-starved cells have no effect on the rate of reaction of poly(U)-programmed ribosomes with either the cognate (Phe) or the near-cognate (Leu2) ternary complexes. Neither the rate of GTP hydrolysis, which signals selection of the ternary complex, nor the rate of peptide formation, which signals the acceptance of the aa-tRNA after proofreading, is affected by the nucleotide. The results indicate that the effect of ppGpp in maintaining the accuracy of protein biosynthesis in cells starved for an amino acid is not due to a direct effect on the rate constants for substrate selection by the ribosome.     

Stopped-flow kinetic analysis of the bacterial luciferase reaction.

The kinetics of the reaction catalyzed by bacterial luciferase have been measured by stopped-flow spectrophotometry at pH 7 and 25 degrees C. Luciferase catalyzes the formation of visible light, FMN, and a carboxylic acid from FMNH2, O2, and the corresponding aldehyde. The time courses for the formation and decay of the various intermediates have been followed by monitoring the absorbance changes at 380 and 445 nm along with the emission of visible light using n-decanal as the alkyl aldehyde. The synthesis of the 4a-hydroperoxyflavin intermediate (FMNOOH) was monitored at 380 nm after various concentrations of luciferase, O2, and FMNH2 were mixed. The second-order rate constant for the formation of FMNOOH from the luciferase-FMNH2 complex was found to be 2.4 x 10(6) M-1 s-1. In the absence of n-decanal, this complex decays to FMN and H2O2 with a rate constant of 0.10 s-1. The enzyme-FMNH2 complex was found to isomerize prior to reaction with oxygen. The production of visible light reaches a maximum intensity within 1 s and then decays exponentially over the next 10 s. The formation of FMN from the intermediate pseudobase (FMNOH) was monitored at 445 nm. This step of the reaction mechanism was inhibited by high levels of n-decanal which indicated that a dead-end luciferase-FMNOH-decanal could form. The time courses for these optical changes have been incorporated into a comprehensive kinetic model. Estimates for 15 individual rate constants have been obtained for this model by numeric simulations of the various time courses.     

Chemical modification of a cerebral sodium-plus-potassium ion-stimulated adenosine triphosphatase preparation

1. A particulate Na(+)+K(+)-stimulated adenosine triphosphatase preparation obtained by treatment of bovine cerebral microsomes with a sodium iodide reagent has been further treated with acid anhydrides likely to convert amino groups into acidic derivatives. 2. The extent of acylation of amino groups was determined by reaction of the remaining amino groups with 2,4,6-trinitrobenzenesulphonic acid. The unmodified preparation contains about 1.2 muequiv. of amino groups/mg of protein of which only about 0.5 muequiv. are accounted for by protein amino groups. Kinetics of the trinitrobenzenesulphonic acid reaction with the unmodified preparation are complex and are altered by ATP or ouabain. 3. The compounds examined cause loss of Na(+)+K(+)-stimulated adenosine triphosphatase activity when relatively few amino groups are modified but ATP was found to afford partial protection against inactivation by methylmaleic anhydride. Na(+)+K(+)-stimulated adenosine triphosphatase activity is partly restored to the dimethylmaleylated preparation by hydrolysis of the dimethylmaleyl-amide bonds but not if more than about 20% of the amino groups have been acylated. 4. Supernatants obtained by high-speed centrifugation of the dimethylmaleylated preparation contained up to 45% of the total protein with less than 10% of the total phospholipid. Methylmaleyl and benzenetricarboxylyl derivatives of the enzyme preparation behaved similarly but tetrafluorosuccinylated material was almost entirely deposited by centrifugation.