磷酰氨基酸的酯交换反应研究

本文利用^3^1P NMR , 对比研究了磷酰化组氨酸与其它磷酰化氨基酸和一级醇的磷上酯交换的反应速度,实验表明磷酰化组氨酸的反应速度最快. 由此提出了以下机理:由于咪唑环的参与,磷可以形成六配位过渡态,从而加快了反应速度.     

A general preparation of protected phosphoamino acids

Fmoc-O-benzyl-l-phosphoserine is an important building block in the synthesis of Forigerimod, a phosphopeptide being investigated for Systemic Lupus Erythematosus (SLE). An efficient one-pot process was developed using inexpensive, readily available starting materials. This general procedure was used to prepare a variety of protected phosphoamino acids.     

High-performance liquid chromatography of acid-stable and acid-labile phosphoamino acids

A high-performance liquid chromatographic system has been developed which permits the separation of both acid-stable and acid-labile phosphoamino acids. An anion-exchange resin and two buffers of different ionic strength and near neutral pH are used. A low-ionic-strength buffer is used for the separation of N-omega-phosphoarginine and N-epsilon-phospholysine, while the higher-ionic-strength buffer permits the clear separation of tau-phosphohistidine, omicron-phosphoserine and omicron-phosphothreonine. An in-stream fluorometric detection system using omicron-phthalaldehyde permits the rapid analysis of samples containing as little as 25 pmoles of phosphoamino acid. This method has been applied to the detection of tau-phosphohistidine from alkaline digests of chemically phosphorylated calf thymus histone 4 and bovine myelin basic protein.     

Non-radioactive determination of PTH and dabsyl phosphoamino acids by capillary electrophoresis

Summary Capillary electrophoresis is a novel technique in the non-radioactive determination of phosphoamino acids. The main advantage of the method presented is the high selectivity and the ability to separate all phosphoamino acid derivatives. Non-radioactive determination of PTH or dabsyl phosphoamino acids by capillary electrophoresis provides a fast and simple screening procedure for all O-phosphorylated amino acids in protein and peptides in the low picomolar range.     

Metal-specific allosteric activation and deactivation of a diamine

The reaction of a potentially tetradentate bis(pyridyl-thiazole) ligand with acetone is allosterically activated upon complexation with Cd(II) but deactivated by reaction with Cu(I), demonstrating metal-specific allosteric controlled reactivity.     

Determination of phosphoamino acids by micellar electrokinetic capillary chromatography with laser-induced fluorescence detection

A micellar electrokinetic capillary chromatography (MEKC) method with laser-induced fluorescence detection (LIF) was developed for analyzing three phosphoamino acids including phosphotyrosine (P-Tyr), phosphoserine (P-Ser), and phosphothreonine (P-Thr). 3-(2-Furoyl)quinoline-2-carboxaldehyde (FQ), a fluorogenic dye, was employed for derivatization of these phosphoamino acids. Results indicated that the complete baseline resolution of each phosphoamino acid was obtained within 10 min, using 20 mmol l⁻¹ sodium borate buffer (pH 9.35) containing 20 mmol l⁻¹sodium deoxycholate (SDC) and 10 mmol l⁻¹ Brij35. Other common amino acids, especially Glu and Asp, did not disturb the assay of these phosphoamino acids. There was a linear relationship between the peak area for analyte and its concentration, with correlation coefficients in the range of 0.9966-0.9996. The concentration detection limits (signal-to-noise = 3) for P-Tyr, P-Ser, and P-Thr were 10, 40, and 75 nmol l⁻¹, respectively. The developed method was successfully applied for determining phosphoamino acids in the hydrolysis sample of a phosphorylated protein kinase.     

On the activation and deactivation of patch-like carbon-mineral adsorbents

Some possibilities of activation and deactivation of carbon-mineral (silica) adsorbents are discussed. These can be achieved either by hydrothermal treatment of the comples adsorbents at different temperatures or by chemical modification of their mineral part.     

Thin-layer electrophoresis with PhastSystem facilitates analysis of phosphoamino acids from proteins bound to Immobilon.

A modification of thin-layer electrophoresis of phosphoamino acids on miniplates, using the PhastSystem (Pharmacia) is described. This method not only reduces the amount of radioactivity required but also the radioactive waste. In addition, it also decreases the handling of hazardous substances and takes only a fraction of the time required for the standard procedure.     

Photoprocesses of molecules with 2-nitrobenzyl protecting groups and caged organic acids

The 308 nm photoinduced formation of the nitroso product and the intermediacy of the aci-nitro form(s) were studied for a series of 2-nitrobenzyl alkyl and aryl esters (1a-4e) and bis-(nitrophenyl)methyl acetates (5a-6b) by time-resolved UV-vis spectroscopy. A triplet state appears as major transient, when 2-nitrobenzyl derivatives 1 are substituted by 4,5-dimethoxy (2) and 4,5-methylenedioxy (3/4) groups. This triplet of charge transfer character is, however, not part of the route via the aci-nitro into the 2-nitroso form. The activation energy and preexponential factor of the longest lifetime component (tau(aci)), i.e. the major part of the aci-nitro decay, were determined. The carboxylic acids as leaving groups have rather small effects on tau(aci). An additional nitrated phenyl ring in alpha-position (5) leads generally to shorter tau(aci) value. Otherwise, the photogeneration of nitroso products is similar. The quantum yield (Phi(d)) varies only moderately with structure, the yield of the aci-nitro form and Phi(d) are correlated and little affected by solvent properties.     

Simultaneous protection and activation of amino acids using propargyl pentafluorophenyl carbonate

[reaction: see text] A very efficient method for the simultaneous protection of the amino group and activation of the carboxyl group of amino acids is reported using propargyl pentafluorophenyl carbonate (PocOPfp). The amino group is protected as a propargyloxycarbonyl (Poc) derivative, and the carboxyl group is activated as a pentafluorophenyl ester. The yields obtained are good to excellent ranging from 60 to 87%.     

Single-step analysis of low abundance phosphoamino acids via on-line sample preconcentration with chemical derivatization by capillary electrophoresis

New strategies for rapid, sensitive and high-throughput analysis of low abundance metabolites in biological samples are required for future metabolomic research. In this report, a direct method for sub-micromolar analyses of phosphoamino acids was developed using on-line sample preconcentration with 9-fluorenylmethyloxycarbonyl chloride (FMOC) derivatization by capillary electrophoresis (CE) and UV detection. Analyte focusing by dynamic pH junction and FMOC labeling efficiency were influenced by several experimental factors including buffer pH, ionic strength, sample injection length and FMOC concentration. About a 200-fold enhancement in concentration sensitivity was achieved under optimal conditions relative to conventional off-line derivatization, as reflected by a detection limit (S/N approximately 3) of 0.1 microM. In-capillary sample preconcentration with chemical labeling by CE offers a unique single-step analytical platform for high-throughput screening of low abundance metabolites without intrinsic chromophores.     

Superelectrophilic activation of polyfunctional organic compounds using zeolites and other solid acids

Zeolites and other available solid acids have been successfully applied to initiate reactions, which were earlier recognised to involve superelectrophilic intermediates and thus required excess of superacids to be carried out.     

Rapid and sensitive determination of phosphoamino acids in phosvitin by N-hydroxysuccinimidyl fluorescein-O-acetate derivatization and capillary zone electrophoresis with laser-induced fluorescence detection

A method was developed for the determination of phosphoamino acids by capillary zone electrophoresis-laser-induced fluorescence detection (argon ion laser, excitation at 488 nm and emission at 520 nm) using derivatization with N-hydroxysuccinimidyl fluorescein-O-acetate (SIFA). Different variables affecting the derivatization (SIFA concentration, derivatization pH, reaction temperature and reaction time) and the separation (type, pH and concentration of buffer, applied voltage and injection mode) were investigated in detail. The optimized separation conditions were 40 mM boric acid buffer (pH 9.2) for background electrolyte, 25 kV for the separation voltage, 25 degrees C for the capillary temperature and 5 s at 0.5 psi for the sample injection. Under the optimal conditions, the SIFA-labeled phosphoamino acids were fully separated within 7 min. The detection limits ranged from 0.1 to 0.3 nM, which are the lowest values reported for capillary electrophoresis (CE) methods. The proposed methodology allowed the rapid, sensitive and selective determination of phosphoamino acids in hen egg yolk phosvitin by the standard addition method. The recovery of these compounds in real sample was 94.0-103.5%. The developed method surpasses previously published CE methods in terms of detection limit, separation time, stability and simplicity of the electrophoretic procedure.     

Cyclic caged morpholinos: conformationally gated probes of embryonic gene function

Feeling a bit cagey: morpholino-based antisense reagents have been caged through oligonucleotide cyclization, enabling photocontrol of gene expression in zebrafish embryos and larvae. Using these reagents, the timing of exocrine cell fate commitment in the developing pancreas has been examined.     

Catalytic subunit of protein kinase A caged at the activating phosphothreonine

Caged reagents are photoactivatable molecules with applications in biological research. While a great deal of work has been carried out on small caged molecules, less has been done on caged macromolecules, such as proteins. Caged proteins would be especially useful in signal transduction research. Since most proteins involved in cell signaling are regulated by phosphorylation, a means to cage phosphorylated proteins would be generally applicable. Here we show that the catalytic subunit of protein kinase A can be activated by thiophosphorylation at Thr-197. The modified protein can then be caged with 4-hydroxyphenacyl bromide to yield a derivative with a specific catalytic activity that is reduced by approximately 17-fold. Upon photolysis at near UV wavelengths, an approximately 15-fold increase in activity is observed, representing an approximately 85-90% yield of uncaged product with a quantum yield phi(P) = 0.21. Because protein kinases belong to a superfamily with structurally related catalytic domains, the protein chemistry demonstrated here should be applicable to a wide range of signaling proteins.     

Hydrolysis of nucleic acids in single-cell protein concentrates using immobilized benzonase

Hydrolysis of nucleic acids for single-cell protein concentrates has been carried out in one step using immobilized benzonase on corn cob. The immobilization is carried out by tosylation of primary alcohols of cellulose of corn cob. The immobilized benzonase is more stable vs pH changes than native benzonase, but the same optimum values of [Mg(II)] and temperature are obtained. The DNase activity is greater than the RNase activity. The percentage of DNA is reduced to 3-6% and that of RNA to 50%. The protein loss is negligible (1%). The enzymatic activity per weight unit of enzyme is greater in the case of benzonase that in reported data for other nucleases insolubilized on corn cob by the same procedure.     

Resonant electron capture mass spectrometry of free fatty acids: examination of ion structures using deuterium-labeled fatty acids and collisional activation

The structures of [M-H](-) ions generated from free fatty acids in resonant electron capture at energies of 1.2 and 7.2 eV were investigated using deuterium-labeled isotopomers and collision-induced dissociation. The [M-H](- small middle dot) ions occur in both a carboxylate anion and a carbanion form. While the formation of the carboxylate anion at 1.2 eV involves the loss of a carboxylic hydrogen, that at 7.2 eV involves the loss of a hydrogen from different positions in the aliphatic chain followed by a rearrangement of a carboxylic hydrogen on to the radical site in the chain. The [M-H-H(2)O](-) ion which is a minor ion in the resonant electron capture spectrum at 7.2 eV is shown to be a precursor for the charge-remote fragment ions corresponding to formal losses of a hydrogen and elements of alkanes. The [M-H-H(2)O](-) ion corresponding to the second major ion in the resonant electron capture spectrum at 7.2 eV is demonstrated to be consistent with a cyclopentanone anion structure. On the basis of new insights obtained in the present study and taking into account previous results, an updated proposal is presented for the mechanism of charge-remote fragmentation which operates in resonant electron capture of free fatty acids at 7.2 eV.     

Selective functionalization of independently addressed microelectrodes by electrochemical activation and deactivation of a coupling catalyst

We demonstrate selective functionalization of independently addressed microelectrodes by electrochemical activation and deactivation of a coupling catalyst. 1,2,3-Triazole formation between terminal acetylenes and organic azides is efficiently catalyzed by copper(I) complexes (a Sharpless "click" reaction), while the oxidized copper(II) complexes are inactive. By electrochemically activating or deactivating the catalyst by switching its redox state, we demonstrate control over triazole formation between surface-immobilized azides and ethynylferrocene. The reaction proceeds on the time scale of minutes using submicromolar concentration of reactants and catalyst, requires mild potentials for catalyst activation and deactivation, and works in aqueous and mixed aqueous-organic solvents. By appropriate biasing of each electrode, we selectively modify one of two chemically identical 10-mum-wide electrodes separated by 10 mum in an interdigitated array. The ability to switch on or off the reaction by electrical addressing together with the chemoselectivity of this reaction makes Cu(I)-catalyzed triazole formation an ideal method for the chemical modification of multielectrode arrays.     

Enzyme treatment-free and ligation-independent cloning using caged primers in polymerase chain reactions

A new simple scheme for constructing recombinant vectors that does not require any restriction enzyme, ligase, or any other special enzyme treatment has been developed. By using caged primers in PCR, unnatural sticky-ends of any sequence, which are sufficiently long for ligation-independent cloning (LIC), are directly prepared on the product after a brief UVA irradiation. Target genes and vectors amplified by this light-assisted cohesive-ending (LACE) PCR join together in the desired arrangement in a simple mixture of them, tightly enough to be repaired and ligated in competent cells.