Synthesis of stereochemical probes for new fluorogenic assays for yeast transketolase variants

For the screening of yeast transketolase (TK) variants with improved or new properties acquired by random mutagenesis, we report on the stereoselective synthesis of fluorogenic substrates as probes for measuring TK activity. Compound 1 (7-(2′,3′,5′-trihydroxy-4′-oxo-pentyl)oxycoumarine), prepared as previously described, [Tetrahedron Lett.2003, 44, 827-830] enabled us to evaluate wild type TK velocity in a simple, specific and reproducible way. To select TK mutants able to produce d-threo aldoses, we prepared compound 2 (dihydroxy-4-O-(2′-oxo-benzopyran-7′-yl-D-threose) from dimethyl tartrate. Starting from d-ribose, we successfully obtained compound 3 (7′-(2,3,5-trihydroxy-4-oxo-pentyl)oxycoumarine) as a probe for TK mutants able to produce l-erythro ketoses.     

A novel chromogenic and fluorogenic scaffold for detection of oxidative radicals

Radicals have profound biorelevance and their robust detections are warranted. We have devised a novel “covalent-assembly” type scaffold for sensitive detection of oxidative radicals.     

Comparative study of some synthesised and commercial fluorogenic substrates for horseradish peroxidase and its mimetic enzyme hemin by a flow injection method

Four 3,4-dihydroquinoxalin-2(1H)-one derivatives, i.e., 3,4-dihydroquinoxalin-2(lH)-one (DHQ), 3-methyl-3,4-dihydroquinoxalin-2(1H)-one (MDHQ), 3,4-dihydroquinoxalin-2(1H)-one-6-acid (DHQ-6-A) and 3-methyl-3,4-dihydroquinoxalin-2(1H)-one-6-acid (MDHQ-6-A), and N,N′-dicyanomethyl-o-phenylenediamine (DCM-OPA) were synthesised as potential substrates for horseradish peroxidase (HRP). Of these compounds DCM-OPA, DHQ and MDHQ can be prepared by very simple methods in a pure form in large quantities. Their properties for use as fluorogenic substrates for HRP and its mimetic enzyme hemin were compared with commercially available substrates, i.e., p-hydroxyphenylacetic acid (p-HPA), p-hydroxyphenylpropionic acid (p-HPPA), homovanillic acid (HVA) and tyramine, by a flow injection method. The results showed that DCM-OPA and MDHQ were the best among the five synthesised substrates and p-HPPA and p-HPA are better than HVA and tyramine. Substrates p-HPPA, p-HPA, DCM-OPA and MDHQ showed comparable ability for H₂O₂ detection in HRP and hemin catalysed reaction systems, with detection limits in the nmol l⁻¹ region. The stability of DCM-OPA is better than MDHQ, but both are stable for at least a month in a refrigerator.     

Solid-phase synthesis of arginine-containing peptides and fluorogenic substrates using a side-chain anchoring approach

Attachment of an amino acid to a solid support by its side chain is sometimes necessary to take advantage of an alpha-carboxylic group available for diverse modifications, including the incorporation of a fluorophore for the preparation of fluorogenic substrates. In contrast to most other amino acids, anchoring the guanidinium group of an arginine to a resin requires the use of a supplementary linker. To avoid the usually multistep synthesis of such a linker as well as its difficult attachment to the guanidine group, we developed a simple method where the guanidine group is built on a Rink amide resin. Our strategy followed the steps of guanidine formation: (i) addition of an isothiocyanate derivative of ornithine to the amino group of a solid support, yielding Nomega-linked thiocitrulline; (ii) S-methylation of thiourea; (iii) guanidinylation using ammonium acetate. Cleavage of the resin generated the arginine-containing compound, the amine group of the resin becoming part of the guanidine. We have demonstrated the usefulness of this method by the synthesis of a series of fluorogenic substrates for trypsin-like serine proteases, which were obtained in high yield and purity. Then, our strategy also allowed generation from the same precursor differentially substituted arginine derivatives, including Nomega-methyl- and Nomega-ethylarginines. The ability to prepare such analogues together with the intermediates thiocitrulline and S-methylisothiocitrulline from a unique precursor while the alpha-amine and carboxylic groups remain available for modification also makes this method a powerful tool for combinatorial solid-phase synthesis of NO synthase inhibitors.     

Design and synthesis of highly sensitive fluorogenic substrates for glutathione S-transferase and application for activity imaging in living cells

Here we report the development of fluorogenic substrates for glutathione S-transferase (GST), a multigene-family enzyme mainly involved in detoxification of endogenous and exogenous compounds, including drug metabolism. GST is often overexpressed in a variety of malignancies and is involved in the development of resistance to various anticancer drugs. Despite the medical significance of this enzyme, no practical fluorogenic substrates for fluorescence imaging of GST activity or for high-throughput screening of GST inhibitors are yet available. So, we set out to develop new fluorogenic substrates for GST. In preliminary studies, we found that 3,4-dinitrobenzanilide (NNBA) is a specific substrate for GST and established the mechanisms of its glutathionylation and denitration. Using these results as a basis for off/on control of fluorescence, we designed and synthesized new fluorogenic substrates, DNAFs, and a cell membrane-permeable variant, DNAT-Me. These fluorogenic substrates provide a dramatic fluorescence increase upon GST-catalyzed glutathionylation and have excellent kinetic parameters for the present purpose. We were able to detect nuclear localization of GSH/GST activity in HuCCT1 cell lines with the use of DNAT-Me. These results indicate that the newly developed fluorogenic substrates should be useful not only for high-throughput GST-inhibitor screening but also for studies on the mechanisms of drug resistance in cancer cells.     

Bifunctional coumarin derivatives in solution and solid phase synthesis of fluorogenic enzyme substrates

The use of the fluorescent bifunctional compounds 7‐amino‐4‐coumarinyl‐acetic acid 1 , 7‐hydroxy‐4‐coumarinyl‐acetic acid 2 and ethyl 7‐amino‐4‐coumarinyl‐acetate 3 in solution and solid phase synthesis of fluorogenic enzyme substrates was examined. The intramolecularly quenched fluorogenic substrate N‐(7‐amino‐4‐coumarinyl‐acetyl)‐L‐phenylalanyl‐p‐nitroanilide 5 , and the fluorogenic one ethyl 7‐(glutaryl‐L‐phenylalanilamido)‐4‐coumarinyl‐acetate 8 , both suitable for chymotrypsin and/or chymotrypsin like enzymes determination, were prepared in solution. The substrates 7‐oleyloxy‐4‐coumarinyl‐acetic acid 13 and 7‐palmitoyloxy‐4‐coumarinyl‐acetic acid 14 , suitable for the enzymatic study of lipases, were prepared by solid phase technique using 2‐chloro‐chlorotrityl‐resin. The study of the fluorescence properties of the fluorophores 1, 2, 3 , and substrates 5, 8,13,14 showed that the examined bifunctional coumarin derivatives are suitable markers for solution and solid phase synthesis of fluorogenic enzyme substrates.     

Syntheses and spectral properties of longwave absorbing and fluorescing substrates for the direct and continuous kinetic assay of carboxylesterases,phosphatases, and sulfatases

Syntheses, absorption and fluorescence properties for a series of new enzyme substrates are described, which are derived from 7-hydroxycoumarins possessing electron-withdrawing substituents in position 3. The new substrates are advantageous over existing ones in that they exhibit longwave absorption and fluorescence maxima as well as largeStokes' shifts. In addition, theirpK _a values, which are usually between 6.0 and 7.0, allow the direct and continuous kinetic assay of hydrolases such as esterases, phosphatases, and sulfatases.

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Synthesen und spektrale Eigenschaften langwellig absorbierender und fluoreszierender Substrate für die direkte und kontinuierliche kinetische Bestimmung von Carboxylesterasen, Phosphatasen und Sulfatasen

Zusammenfassung Die Synthesen und Absorptions- sowie Fluoreszenzspektren einer Reihe neuer Enzymsubstrate werden beschrieben, welche von 7-Hydroxycumarinen mit elektronenziehenden Substituenten in 3-Stellung abgeleitet sind. Die neuen Substrate bieten gegenüber bisherigen den Vorteil langwelliger Absorptions- und Fluoreszenzmaxima sowie großerStokes-Verschiebungen. Die niedrigenpK _s -Werte, welche zwischen 6 und 7 liegen, erlauben weiters eine direkte und kontinuierliche kinetische Bestimmung von Hydrolasen vom Typ der Esterasen, Phosphatasen und Sulfatasen.

     

Design and synthesis of a novel fluorescent protein probe for easy and rapid electrophoretic gel staining by using a commonly available UV‐based fluorescent imaging system

A new fluorescent molecular probe, methyl 3‐(3,5‐bis((bis(pyridin‐2‐ylmethyl)amino)‐methyl)‐4‐hydroxyphenyl)‐2‐(5‐(dimethylamino)naphthalene‐1‐sulfonamido) propanoate, dizinc(II) chloride salt (Dansyl‐ 1 ‐Zn(II)), which possesses Zn(II) complexes and a dansyl group, was designed and synthesized to enable the detection of proteins in solution and in high‐throughput electrophoresis by using a UV‐based detection system. Dansyl‐ 1 ‐Zn(II) exhibited weak fluorescence in the absence of proteins and strong green fluorescence at approximately 510 nm in the presence of BSA upon irradiation with light at a wavelength of 345 nm. Compared with conventional protocols for in‐gel SDS‐PAGE protein staining (e.g. silver staining, SYPRO Ruby, and Oriole), the operating times of which range from 90 min to overnight, Dansyl‐ 1 ‐Zn(II) allowed 1‐step protein staining (SDS‐PAGE →Staining →Detection) and shortened the operating time (35 min) with high sensitivity (LOD: 1 ng or less) under 312‐nm or 365‐nm light excitation with orange or red emission filters, respectively. Moreover, Dansyl‐ 1 ‐Zn(II) was successfully applied to protein identification by MS via in‐gel tryptic digestion, Western blotting, and Native‐PAGE. Accordingly, Dansyl‐ 1 ‐Zn(II) may facilitate highly sensitive and high‐throughput protein detection, and it may be widely applicable as a convenient tool in various scientific and medical fields.     

Expanding the scope of PNA-encoded libraries: divergent synthesis of libraries targeting cysteine, serine and metallo-proteases as well as tyrosine phosphatases

Seven PNA-encoded combinatorial libraries targeting proteases and phosphatases with covalent reversible and irreversible mechanism-based inhibitors were prepared. The libraries were synthesized using modified PNA monomers, which dramatically increase the water solubility of the libraries in biologically relevant buffers. The libraries were shown to selectively inhibit targeted enzymes.     

Interpretation of mass spectrometry data for high-throughput proteomics

Recent developments in proteomics have revealed a bottleneck in bioinformatics: high-quality interpretation of acquired MS data. The ability to generate thousands of MS spectra per day, and the demand for this, makes manual methods inadequate for analysis and underlines the need to transfer the advanced capabilities of an expert human user into sophisticated MS interpretation algorithms. The identification rate in current high-throughput proteomics studies is not only a matter of instrumentation. We present software for high-throughput PMF identification, which enables robust and confident protein identification at higher rates. This has been achieved by automated calibration, peak rejection, and use of a meta search approach which employs various PMF search engines. The automatic calibration consists of a dynamic, spectral information-dependent algorithm, which combines various known calibration methods and iteratively establishes an optimised calibration. The peak rejection algorithm filters signals that are unrelated to the analysed protein by use of automatically generated and dataset-dependent exclusion lists. In the "meta search" several known PMF search engines are triggered and their results are merged by use of a meta score. The significance of the meta score was assessed by simulation of PMF identification with 10,000 artificial spectra resembling a data situation close to the measured dataset. By means of this simulation the meta score is linked to expectation values as a statistical measure. The presented software is part of the proteome database ProteinScape which links the information derived from MS data to other relevant proteomics data. We demonstrate the performance of the presented system with MS data from 1891 PMF spectra. As a result of automatic calibration and peak rejection the identification rate increased from 6% to 44%.Abbreviations 2-DE Two-dimensional gel electrophoresis - MALDI Matrix-assisted laser desorption ionisation - PMF Peptide mass fingerprinting - MS Mass spectrometry - TOF Time of flight     

Analytical method development for directed enzyme evolution research: A high throughput high-performance liquid chromatography method for analysis of ribose and ribitol and a capillary electrophoresis method for the separation of ribose enantiomers

Analytical methods were developed for a directed enzyme evolution research programme, which pursued high performance enzymes to produce high quality l-ribose using large scale biocatalytic reaction. A high throughput HPLC method with evaporative light-scattering detection was developed to test ribose and ribitol in the enzymatic reaction, a β-cyclobond 2000 analytical column separated ribose and ribitol in 2.3 min, a C₁₈ guard column was used as an on-line filter to clean up the enzyme sample matrix and a short gradient was applied to wash the column, the enzymatic reaction solution can be directly injected after quenching. Total run time of each sample was approx. 4 min which provided capability of screening 4 × 96-well plates/day/instrument. Meanwhile, a capillary electrophoresis method was developed for the separation of ribose enantiomers, while 7-aminonaphthalene-1,3-disulfonic acid was used as derivatisation reagent and 25 mM tetraborate with 5 mM β-cyclodextrin was used as electrolyte. 0.35%of d-ribose in l-ribose can be detected which can be translated into 99.3% ee of l-ribose. Derivatisation reagent and sample matrix did not interfere with the measurement.     

Development of a water-soluble 3-formylBODIPY dye for fluorogenic sensing and cell imaging of sulfur dioxide derivatives

A new water-soluble, highly fluorogenic 3-formylBODIPY dye that enables the sensing of SO₂ derivatives in aqueous buffers and cancer cells is reported. The quaternary ammonium group appended through the meso-position of the BODIPY dye ensures water solubility. The probe exhibits high specificity for cytosolic (bi)sulfites and fluoresces brightly in human lung adenocarcinoma cells (A549).     

Design and parallel solid-phase synthesis of ring-fused 2-pyridinones that target pilus biogenesis in pathogenic bacteria

A new method for the solid-phase synthesis of enantiomerically enriched highly substituted ring-fused 2-pyridinones 13 has been developed. The synthesis mediates introduction of substituents at two positions in the 2-pyridinone ring in a diverse manner and is suitable for parallel synthesis. (19)F NMR spectroscopy was used as a tool to monitor each of the five steps in the reaction sequence. The optimized conditions thus obtained were then used to prepare a library of 20 2-pyridinones with high yields. The library members were chosen from a statistical multivariate design to ensure diversity and reliable data for structure-activity relationships. Screening of the library against the bacterial periplasmic chaperone PapD was performed using surface plasmon resonance. Three new 2-pyridinones with a higher affinity for the chaperone PapD than the previous best 13[10,1] were found, and important structural features could be deduced.     

Synthesis and anticholinesterase activity of some new fluorogenic analogues of organophosphorus nerve agents

Eighteen new fluorogenic analogues of organophosphorus nerve agents were synthesised and characterised. They included analogues of tabun, sarin, cyclosarin, soman, VX, and Russian VX, with the 7-oxy-4-methylcoumarin or 7-oxy-4-(trifluoromethyl)coumarin leaving group. These analogues inhibited acetylcholinesterase (AChE) effectively in vitro and therefore have potential as tools for the identification of novel organophosphatases in biological systems. Analogues of VX and Russian VX with the 7-amino-4-methylcoumarin group, although poor AChE inhibitors, may have utility for screening enzyme libraries for phosphoramidases capable of cleaving P-N bonds.     

Synthesis and characterization of a new fluorogenic substrate for alpha-galactosidase

Alpha-galactosidase A hydrolyzes the terminal alpha-galactosyl moieties from glycolipids and glycoproteins in lysosomes. Mutations in α-galactosidase cause lysosomal accumulation of the glycosphingolipid, globotriaosylceramide, which leads to Fabry disease. Small-molecule chaperones that bind to mutant enzyme proteins and correct their misfolding and mistrafficking have emerged as a potential therapy for Fabry disease. We have synthesized a red fluorogenic substrate, resorufinyl α-d-galactopyranoside, for a new α-galactosidase enzyme assay. This assay can be measured continuously at lower pH values, without the addition of a stop solution, due to the relatively low pK _a of resorufin (~6). In addition, the assay emits red fluorescence, which can significantly reduce interferences due to compound fluorescence and dust/lint as compared to blue fluorescence. Therefore, this new red fluorogenic substrate and the resulting enzyme assay can be used in high-throughput screening to identify small-molecule chaperones for Fabry disease. Zhen-Dan Shi and Omid Motabar contributed equally to this work     

Design and synthesis of an isopenicillin N synthase mimic

Towards the aim of creating a functional mimic of isopenicillin N synthase, a small molecule designed to coordinate around iron(II) and model the enzyme active site has been prepared in nine synthetic steps from 2,6-bis(hydroxymethyl)pyridine, (S)-(+)-mandelic acid and pivaldehyde. One aspartate, two histidines and a water ligand in the natural enzyme are replaced by an α-hydroxy acid, pyridine and aniline in the model compound. Additionally, a free thiol designed to simulate the enzyme substrate, δ-(l-α-aminoadipoyl)-l-cysteinyl-d-valine, is linked to the ligand by a three carbon chain. We postulate that in the presence of molecular oxygen, the complex formed between this synthetic ligand and iron(II) will display oxidative chemistry similar to that observed in the active site of isopenicillin N synthase.     

High‐abundance protein depletion: Comparison of methods for human plasma biomarker discovery

Affinity depletion of abundant proteins from human plasma has become a routine sample preparation strategy in proteomics used prior to protein identification and quantitation. To date, there have been limited published studies comparing the performance of commercially available depletion products. We conducted a thorough evaluation of six depletion columns using 2‐DE combined with sophisticated image analysis software, examining the following criteria: (i) efficiency of high‐abundance protein depletion, (ii) non‐specific removal of other than the targeted proteins and (iii) total number of protein spots detected on the gels following depletion. From all the products investigated, the Seppro IgY system provided the best results. It displayed the greatest number of protein spots on the depleted plasma gels, minimal non‐specific binding and high efficiency of abundant protein removal. Nevertheless, the increase in the number of detected spots compared with the second best performing and cheaper multiple affinity removal column (MARC) was not shown to be statistically significant. The ProteoPrep spin column, considered to be the “deepest” depletion technique available at the time of conducting the study, surprisingly displayed significantly fewer spots on the flow‐through fraction gels compared with both the Seppro and the MARC. The spin column format and low plasma capacity were also found to be impractical for 2‐DE. To conclude, we succeeded in providing an overview of the depletion columns performances with regard to the three examined areas. Our study will serve as a reference to other scientists when deciding on the optimal product for their particular projects.     

Multicomponent synthesis of epoxy-tetrahydronaphthyridine and structural diversification by subsequent fragmentation

Three-component reaction of an α-isocyanoacetamide 7, an homoallylamine 8 and an aldehyde 9 in methanol at room temperature provides oxa-bridged tricycle 4 in good to excellent yield as a mixture of two separable diastereoisomers. In this one-pot multicomponent process, one C-N, one C-O and three C-C bonds are formed with concomitant creation of five asymmetric centers and three rings. Fragmentation of epoxy-tetrahydronaphthyridine 4 affords differentially substituted 5,6,7,8-tetrahydro-1,7-naphthyridine (5, 6) depending on the reaction conditions, providing thus additional structural diversity. A one-pot three-component synthesis of 5,6,7,8-tetrahydro-1,7-naphthyridine (6) from 7, 8 and 9 is also documented.     

Microchip electrophoresis-SDS methods with high-resolution and silver stain sensitivity for quality screening and quantitation of protein products

Two microchip electrophoresis (ME)-SDS methods have been developed for high throughput quantitation and quality screening of protein products. Both methods utilize a commercial microchip instrument to separate dodecyl sulfate-coated proteins within 1 min. In the high-resolution ME-SDS method, improved separation selectivity is achieved using a mixture of sieving polymers. Proteins of similar sizes, such as different fragment antigen-binding (Fab) assemblies can be readily resolved and individually quantified. A high-sensitivity ME-SDS method was also developed with sensitivity comparable to that of SDS-PAGE with silver staining. In this method, protein molecules are derivatized with a fluorescence reagent prior to analysis. LIF detection of the covalently attached fluorophore enables accurate quantitation of low-expressing proteins and detection of minor species at 0.04% level (1 ng/mL loading concentration). Both the high-resolution and the high-sensitivity ME-SDS methods can be applied to crude fermentation samples. The utilities of these methods in process development and formulation stability study are presented.