海洋链霉菌Streptomyces lusitanus SCSIO LR32中芳酰胺类代谢产物的研究

采用硅胶柱色谱和半制备反相高效液相色谱分离方法,对南海海洋放线菌Streptomyces lusitanus SCSIOLR32的次级代谢产物进行了研究,分离得到四个酰胺类化合物,经MS1、H和13 C NMR波谱分析鉴定为二甲基甲苯2,4-二氨基甲酸甲酯(1),甲苯2,4-二氨基甲酸乙酯(2),甲苯2,6-二氨基甲酸甲酯(3)和甲苯2,6-二氨基甲酸乙酯(4)。运用X-单晶衍射确定了1的结构式。其中化合物2和4是首次从自然界中分离得到。采用16S分子生物学方法鉴定该菌株为链霉菌属放线菌。     

A new approach to preparative enzymatic synthesis

A new approach to preparative organic synthesis in aqueous–organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system “water–water-immiscible organic solvent.” Thereby the enzyme is localized in the aqueous phase—this eliminates the traditional problem of stabilizing the enzyme against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations “water–water-miscible organic solvent,” in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important source for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L -tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L -tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.     

A new approach to preparative enzymatic synthesis. Reprinted from Biotechnology and Bioengineering, Vol. XIX, No. 9, Pages 1351-1361

A new approach to preparative organic synthesis in aqueous-organic systems is suggested. It is based on the idea that the enzymatic process is carried out in a biphasic system "water-water-immiscible organic solvent." Thereby the enzyme is localized in the aqueous phase-this eliminates the traditional problem of stabilizing the enzymes against inactivation by a nonaqueous solvent. Hence, in contrast to the commonly used combinations "water-water-miscible organic solvent," in the suggested system the content of water may be infinitely low. This allows one to dramatically shift the equilibrium of the reactions forming water as a reaction product (synthesis of esters and amides, polymerization of amino acids, sugars and nucleotides, dehydration reactions, etc.) toward the products. The fact that the system consists of two phases provides another very important sources for an equilibrium shift, i.e., free energies of the transfer of a reagent from one phase to the other. Equations are derived describing the dependence of the equilibrium constant in a biphasic system on the ratio of the volumes of the aqueous and nonaqueous phases and the partition coefficients of the reagents between the phases. The approach has been experimentally verified with the synthesis of N-acetyl-L-tryptophan ethyl ester from the respective alcohol and acid. Porous glass was impregnated with aqueous buffer solution of chymotrypsin and suspended in chloroform containing N-acetyl-L-tryptophan and ethanol. In water (no organic phase) the yield of the ester is about 0.01%, whereas in this biphasic system it is practically 100%. The idea is applicable to a great number of preparative enzymatic reactions.     

Products of estradiol/peroxidase interaction; their structural features and biopolymeric character

Loss of tritium from [2,4,6 alpha, 7 alpha-3H]estradiol and from [2-3H]estradiol during their conversion into polyestradiol (PEL) by horseradish peroxidase/H2O2 and the NMR spectrum of PEL permethyl ether suggest that PEL is composed of two or more different subunits, each formed by the joining of four molecules of estradiol with the loss of five hydrogen atoms from positions 2 and 4 and of three phenolic hydrogens leading to the formation of one C-C bond and three C-O bonds. At very low concentrations of estradiol the main reaction products were monomers; this is attributed to the initial formation of transient tetraestradiols which combine with water at high dilution and with themselves at low dilution. Association of the monomeric products to oligomers occurred on a Sephadex G-50 column and was readily reversed in phosphate buffer. In aqueous solution PEL underwent non-covalent changes induced by heat, time and electrolytes, and affecting its solubility, u.v. absorbance, extraction by organic solvents and ability to bind estradiol.     

Coordination chemistry of vitamin C. Part II. Interaction of L-ascorbic acid with Zn(II), Cd(II), Hg(II), and Mn(II) ions in the solid state and in aqueous solution

The reaction of L-ascorbic acid with the zinc group and manganese ions has been investigated in aqueous solution at pH 6-7. The solid salts of the type M (L-ascorbate)2.2H2O, where M = Zn(II), Cd(II) and Mn(II) were isolated and characterized by 13C NMR and Fourier Transform infrared (FT-IR) spectroscopy. Spectroscopic evidence showed that in aqueous solution, the bindings of the Zn(II) and Mn(II) ions are through the ascorbate anion O-3 and O(2)-H groups (chelation), while the Cd(II) ion binding is via the O-3 atom only. In the solid state, the binding of these metal ions would be through two acid anions via O-3, O-2 of the first and O-1, O-3 of the second anion as well as to two H2O molecules, resulting in a six-coordinated metal ion. The Hg(II) ion interaction leads to the oxidation of the ascorbic acid in aqueous solution.     

Hydrogen turnover and subcellular compartmentation of hepatic [2-(13)C]glutamate and [3-(13)C]aspartate as detected by (13)C NMR.

(13)C NMR monitored the dynamics of exchange from specific hydrogens of hepatic [2-(13)C]glutamate and [3-(13)C]aspartate with deuterons from intracellular heavy water providing information on alpha-ketoglutarate/glutamate exchange and subcellular compartmentation. Mouse livers were perfused with [3-(13)C]alanine in buffer containing or not 50% (2)H(2)O for increasing periods of time (1 min < t < 30 min). Liver extracts prepared at the end of the perfusions were analyzed by high resolution (13)C NMR (150.13 MHz) with (1)H decoupling only and with simultaneous (1)H and (2)H decoupling. (13)C-(2)H couplings and (2)H-induced isotopic shifts observed in the glutamate C2 resonance, allowed to estimate the apparent rate constants (forward, reverse; min(-1)) for (i) the reversible exchange of [2-(13)C]glutamate H2 as catalyzed mainly by aspartate aminotransferase (0.32, 0.56), (ii) the reversible exchange of [2-(13)C]glutamate H3(proS) as catalyzed by NAD(P) isocitrate dehydrogenase (0.1, 0.05), and (iii) the irreversible exchanges of glutamate H3(proR) and H3(proS) as catalyzed by the sequential activities of mitochondrial aconitase and NAD isocitrate dehydrogenase of the tricarboxylic acid cycle (0.035), respectively. A similar approach allowed to determine the rates of (1)H-(2)H exchange for the H2 (0.4, 0.5) or H3(proR) (0.3, 0.2) or the H2 and H3(proS) hydrogens (0.20, 0.23) of [3-(13)C]aspartate isotopomers. The ubiquitous subcellular localization of (1)H-(2)H exchange enzymes and the exclusive mitochondrial localization of pyruvate carboxylase and the tricarboxylic acid cycle resulted in distinctive kinetics of deuteration in the H2 and either or both H3 hydrogens of [2-(13)C]glutamate and [3-(13)C]aspartate, allowing to follow glutamate and aspartate trafficking through cytosol and mitochondria.     

Ionization and phase behavior of fatty acids in water: application of the Gibbs phase rule

The phase behavior of several medium-chain (10- and 12-carbon) and long-chain (18-carbon) fatty acids in water was examined as a function of the ionization state of the carboxyl group. Equilibrium titration curves were generated above and below fatty acid and acid-soap chain melting temperatures and critical micelle concentrations, and the phases formed were characterized by X-ray diffraction, 13C NMR spectroscopy, and phase-contrast and polarized light microscopy. The resulting titration curves were divided into five regions: (i) at pH values less than 7, a two-phase region containing oil or fatty acid crystals and an aqueous phase; (ii) at pH approximately 7, a three-phase region containing oil, lamellar, and aqueous (or fatty acid crystals, 1:1 acid-soap crystals, and aqueous) phases; (iii) between pH 7 and 9, a two-phase region containing a lamellar fatty acid/soap (or crystalline 1:1 acid-soap) phase in an aqueous phase; (iv) at pH approximately 9, a three-phase region containing lamellar fatty acid-soap (or crystalline 1:1 acid-soap), micellar, and aqueous phases; and (v) at pH values greater than 9, a two-phase region containing micellar and aqueous phases. Interpretation of the results using the Gibbs phase rule indicated that, for oleic acid/potassium oleate, the composition of the lamellar fatty acid/soap phase varied from approximately 1:1 to 1:3 un-ionized to ionized fatty acid species. In addition, constant pH regions observed in titration curves were a result of thermodynamic invariance (zero degrees of freedom) rather than buffering capacity. The results provide insights into the physical states of fatty acids in biological systems.(ABSTRACT TRUNCATED AT 250 WORDS)     

Stereoselective synthesis of 2,3,7-trimethylcyclooctanone and related compounds and determination of their relative and absolute configurations by the MalphaNP acid method

The copper/chiral phosphoramidite (L(1))-catalyzed conjugate addition of dimethylzinc to cycloocta-2,7-dienone 4, followed by the methylation of the intermediate enolate, yielded a single isomer of 7,8-dimethylcyclooct-2-enone (+)-5. Compound (+)-5 was subjected to the second conjugate addition with ent-L(1) giving only one stereoisomer of 2,3,7-trimethylcyclooctanone (+)-6, which was converted to 2,3,7-trimethylcyclooctanol 7. To determine the relative and absolute configurations of these compounds, the (1)H NMR anisotropy method using (S)-(+)-2-methoxy-2-(1-naphthyl)propionic acid {(S)-(+)-MalphaNP acid} 1 was applied. Racemic alcohol (+/-)-7 was esterified with (S)-(+)-MalphaNP acid 1 yielding diastereomeric esters, which were efficiently separated by HPLC on silica gel affording the first-eluted MalphaNP ester (-)-10a and the second-eluted one (-)-10b. The relative and absolute configurations of ester (-)-10a were determined to be (S;1R,2S,3R,7S) by analyzing the (1)H and (13)C NMR spectra of (-)-10a and (-)-10b, especially their HSQC-TOCSY and NOESY spectra, and by applying the MalphaNP anisotropy method. The alcohol 7 formed from (+)-6 was similarly esterified with (S)-(+)-MalphaNP acid 1 yielding an MalphaNP ester, which was identical with (-)-10a, and the relative and absolute configurations of 2,3,7-trimethylcyclooctanone (+)-6 were determined to be (2S,3R,7S).     

Enzymatic esterification in organic media: role of water and organic solvent in kinetics and yield of butyl butyrate synthesis

Summary The respective roles of organic solvent and of water in butyl butyrate synthesis from n-butanol and n-butyric acid in n-hexane by Mucor miehei lipase have been investigated by analysis of the kinetics and the reaction balances. Esterificaton was found to take place in both low water systems containing solid enzyme in hexane and in biphasic aqueous enzyme solution/hexane systems. In the solid enzyme system, the enzyme adsorbed the water produced, thus delaying the appearance of a discrete aqueous phase. As expected, the presence of some water was indispensable for this system, as its removal or exclusion by various means (adsorption, distillation) affected enzyme activity. However, water removal had little effect on the final yield of esterification. Reaction velocities were quite similar for the solid enzyme/hexane system and for the biphasic aqueous enzyme solution/hexane system. In the latter case, the butyl butyrate formed was almost exclusively found in the organic phase. Ethyl butyrate, a more polar compound, was synthesized with a lower yield. These results allow the conclusion that the reaction took place in a phase consisting of either solid hydrated enzyme with no discrete aqueous phase or of an aqueous enzyme solution by basically similar mechanisms according to the amount of water available to the system, the esterification being driven to completion by transfer of the ester into the organic phase because of a favourable partition coefficient. Offprint requests to: F. Monot     

Hollow-Fibre Enzyme Reactor Integrated with Solvent Extraction for Synthesis of Aspartame Precursor

A new process for the simultaneous enzymic synthesis and purification of N-(benzyloxycarbonyl)- -aspartyl- -phenylalanine methyl ester (ZAPM), a precursor of aspartame, has been developed. The enzymic reaction between N-(benzyloxycarbonyl)- -aspartic acid (ZA) and -phenylalanine methyl ester (PM) was carried out in a biphasic hollow-fibre rector with an aqueous phase an a butyl acetate phase. The reaction took place in the aqueous phase and by maintaining the pH at 5, the product (ZAPM) was extracted into the organic phase. Product purity was greater than 90% and reasonable productivity could be achieved with this system.     

Curtius Rearrangement Using Ultrasonication: Isolation of Isocyanates of Fmoc-Amino Acids and Their Utility for the Synthesis of Dipeptidyl Ureas

An efficient conversion of N^α-[(9–fluorenylmethyl)oxy]carbonyl (Fmoc) amino acid azides to the corresponding isocyanates using ultrasound is described. The Curtius rearrangement was accomplished using acid azides in toluene solution as well as solid powder at room temperature. All isocyanates synthesized have been obtained as crystalline solids and were characterized. Coupling of isocyanates with amino acid methyl ester hydrochloride salts in presence of N-methylmorpholine (NMM) resulted in Fmoc-protected dipeptidyl urea esters, which have been well characterized by ¹H NMR, ¹³C NMR and mass spectrometry.     

Study by NMR and circular dichroism of synthetic "zinc finger" peptide of viral Gag protein from HIV-2

We report the synthesis in solid phase of an 18-amino acid peptide that contains the cysteine-rich region of the structural Gag protein of HIV-2. The characterization of this fragment and of its interaction with Zn2+ has been made by one- (1 D) and two-dimensional (2D) NMR techniques and by circular dichroism. Our results suggest that in aqueous solution the complexation produces a significant perturbation in the conformation of this peptide.     

Coordination chemistry of vitamin C. Part I. Interaction of L-ascorbic acid with alkaline earth metal ions in the crystalline solid and aqueous solution

The interaction of L-ascorbic acid with alkaline earth metal ions has been investigated in aqueous solution at pH 6-7. The solid salts of the type Mg(L-ascorbate)2.4H2O, Ca(L-ascorbate)2.2H2O, Sr(L-ascorbate)2.2H2O and Ba(L-ascorbate)2.2H2O were isolated and characterized by means of 13C NMR and FT-IR spectroscopy. Spectroscopic and other evidence suggested that in aqueous solution, the binding of the alkaline earth metal ions is through the O-3 atom of the ascorbate anion, while in the solid state the binding of the Mg(II) is different from those of the other alkaline earth metal ion salts. The Mg(II) ion binds to the O-3, O-1 atom of the two ascorbate anions and to two H2O molecules, while the eight-coordination around the Ca(II), Sr(II), and Ba(II) ions would be completed by the coordination of three acid anions, through O-5, O-6 of the first, O-3, O-5, O-6 of the second and O-1 of the third anion as well as to two H2O molecules. The structural properties of the alkaline earth metal-ascorbate salts are different in the solid and aqueous solution.     

Enzymatic resolution of (S)-(+)-naproxen in a trapped aqueous-organic solvent biphase continuous reactor

A trapped aqueous-organic biphase system for the continuous production of (S)-(+)-2-(6-methoxy-2-naphthyl) propionic acid (Naproxen) has been developed. The process consists of a stereoselective hydrolysis of the racemic Naproxen methyl ester by Candida rugosa lipase in a trapped aqueous-organic biphase system. The reaction has been carried out in a laboratory-scale continuous-flow stirred tank reactor (CSTR). The staring material has been supplied in and remaining substrate recovered by organic phase. YWG-C(6)H(5), a poorly polar synthetic support, has been employed to immobilize the lipase and to restrict the aqueous phase. Lipase immobilized on YWG-C(6)H(5) containing aqueous phase has been added into the CSTR to catalyze the hydrolysis. A dialysis membrane tube containing a continuous flow closed-loop buffer has been applied in the CSTR for the extraction of product and recruiting of the aqueous part consumed. Various reaction conditions have been studied. The activity of immobilized enzyme was effected by the polarity of support, the substrate concentration, logP value of organic phase and the product inhibition. At steady-state operating conditions, an initial conversion of 35% has been obtained. The CSTR was allowed to operate continuously for 60 days at 30 degrees C with a 30% loss of activity. The hydrolysis reaction yielded (S)-(+)-Naproxen with >90% enantiomeric excess and overall conversion of 30%.     

Molecular mobility and phase structure of biodegradable poly(butylene succinate) and poly(butylene succinate-co-butylene adipate)

Molecular mobility and phase structure of biodegradable poly(butylene succinate) (PBS) and poly(butylene succinate-co-20 mol % butylene adipate) [P(BS-co-20 mol % BA)] have been investigated by high-resolution solid-state (13)C NMR. For both samples, two components with different (13)C spin-lattice relaxation time (T(1C)) values have been observed in the crystalline region. The crystalline component with shorter T(1C) value is assignable to the interface near amorphous phase. The crystalline component with longer T(1C) value is ascribed to the inside of the crystalline region. On the basis of T(1C), it has been concluded that the BA units are not included in the crystalline region of P(BS-co-20 mol % BA). Molecular mobility and higher-ordered structure of amorphous phase have been also compared between the melt and solid state. Variable-temperature high-resolution (13)C NMR measurements for the amorphous phase have revealed the remarkable difference in dynamics and structure between the melt and solid state.     

Enzymatic synthesis of arginine-based cationic surfactants

A novel enzymatic approach for the synthesis of arginine N-alkyl amide and ester derivatives is reported. Papain deposited onto solid support materials was used as catalyst for the amide and ester bond formation between Z-Arg-OMe and various long-chain alkyl amines and alcohols (H2N-Cn2, HO-Cn; n = 8-16) in organic media. Changes in enzymatic activity and product yield were studied for the following variables: organic solvent, aqueous buffer content, support for the enzyme deposition, presence of additives, enzyme loading, substrate concentration, and reaction temperature. The best yields (81-89%) of arginine N-alkyl amide derivatives were obtained at 25 degrees C in acetonitrile with an aqueous buffer content ranging from 0 to 1% (v/v) depending on the substrate concentration. The synthesis of arginine alkyl ester derivatives was carried out in solvent-free systems at 50 or 65 degrees C depending on the fatty alcohol chain length. In this case, product yields ranging from 86 to 89% were obtained with a molar ratio Z-Arg-OMe/fatty alcohol of 0.01. Papain deposited onto polyamide gave, in all cases, both the highest enzymatic activities and yields. Under the best reaction conditions the syntheses were scaled up to the production of 2 g of final product. The overall yields, which include reaction, Nalpha-benzyloxycarbonyl group (Z) deprotection and purification, varied from 53 to 77% of pure (99.9% by HPLC) product.     

Synthesis and solid state 13C and 1H NMR analysis of new oxamide derivatives of methyl 2-amino-2-deoxy-alpha-D-glucopyranoside and ester of amino acids or dipeptides

The syntheses of new oxamide derivatives of methyl 2-amino-2-deoxy-alpha-D-glucopyranoside and amino acid or peptide esters are presented. The reaction of methyl 3,4,6-tri-O-acetyl-2-acetamido-2-deoxy-alpha-D-glucopyranoside and oxalyl chloride gave N-(methyl 3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranosid-2-yl) oxamic acid chloride which on reaction with the ester of Gly, L-Ala, L-Phe, GlyGly, Gly-L-Phe and Gly-L-Ala afforded N-(methyl 3,4,6-tri-O-acetyl-2-deoxy-alpha-D-glucopyranosid-2-yl), N'-oxalyl-amino acid or dipeptide esters. The structure of the oxamides was studied using 1H, 13C NMR in solution and solid state.     

Competitive lipase-catalyzed ester hydrolysis and ammoniolysis in organic solvents; equilibrium model of a solid-liquid-vapor system.

Enzymatic ester hydrolysis and ammoniolysis were performed as competitive reactions in methyl isobutyl ketone without a separate aqueous phase. The reaction system contained solid ammonium bicarbonate, which dissolved as water, ammonia, and carbon dioxide. During the reaction an organic liquid phase, a vapor phase, and at least one solid phase are present. The overall equilibrium composition of this multiphase system is a complex function of the reaction equilibria and several phase equilibria. To gain a quantitative understanding of this system a mathematical model was developed and evaluated. The model is based on the mass balances for a closed batch system and straightforward relations for the reaction equilibria and the solubility equilibria of ammonium bicarbonate, the fatty acid ammonium salt, water, ammonia, and carbon dioxide. For butyl butyrate as a model ester and Candida antarctica lipase B as the biocatalyst this equilibrium model describes the experiments satisfactorily. The model predicts that high equilibrium yields of butyric acid can be achieved only in the absence of ammoniolysis or in the presence of a separate water phase. However, high yields of butyramide should be possible if the water concentration is fixed at a low level and a more suited source of ammonia is applied.     

Synthesis and conformational investigation of cyclic dipeptides: 7-membered rings containing alpha- and beta-amino acids.

The synthesis of heterocyclic compounds containing the 7-membered ring system [1,4]diazepane-2,5-dione is described. The aim of this study was to elaborate the solid phase and solution synthesis of eight representatives of the cyclic scaffold and to investigate their chemical stability and their conformational properties. The solid phase synthesis was performed on aminomethyl polystyrene resin using 5-(4-formyl-3,5-dimethoxyphenoxy)valeric acid as a backbone linker system (BAL-linker). After attachment of the alpha- and beta-amino acid and deprotection of the amino function, the dipeptide ester was obtained. The molecule was cyclized on the solid support by treatment with NaOMe in MeOH/NMP. The product was cleaved from the resin by TFA. For the solution pathway the linear dipeptides were synthesized by coupling of the BOC-protected L-alpha-amino acid with the beta2-amino acid ester (EDC/HOBT). After N- and C-terminal deprotection of the dipeptide, the linear species was cyclized with EDC/HOBT at a concentration of 3 mM in DMF. The products showed high chemical stability after storage in DMSO at room temperature for weeks. The x-ray and two dimensional NMR investigations were performed to investigate the conformation of the molecules. Three types of configuration could be distinguished by NMR, depending on the substitution pattern of the cyclic compounds. The x-ray results confirmed the NMR observations. In general the 7-membered rings showed rigidity, thus they could represent optimal scaffolds for new receptor ligands.     

Advances towards resonance assignments for uniformly—13C, 15N enriched bacteriorhodopsin at 18.8 T in purple membranes

Solid state NMR spectra from uniformly (13)C, (15)N enriched bacteriorhodospin (bR) purified from H. salinarium were acquired at 18.8 T using magic angle spinning methods. Isolated resonances of 2D (13)C-(13)C spectra exhibited 0.50-0.55 ppm line-widths. Several amino acid types could be assigned, and at least 12 out of 15 Ile peaks could be resolved clearly and identified based on their characteristic chemical shifts and connectivities. This study confirms that high resolution solid state NMR spectra can be obtained for a 248 amino acid uniformly labeled membrane protein in its native membrane environment and indicates that site-specific assignments are likely to be feasible with heteronuclear multidimensional spectra.