Sigma-Aldrich

Aldrich-160512; Fmoc chloride 0.97; CAS No.: 28920-43-6; Synonyms: 9-Fluorenylmethoxycarbonyl chloride; 9-Fluorenylmethyl chloroformate; Fmoc-Cl; Linear Formula: C15H11ClO2; Empirical Formula: C15H11ClO2; find related products, papers, technical documents, MSDS & more at Sigma-Aldrich.

Fmoc chloride 97 28920-43-6

Fmoc chloride

Determining the pKa of key functional groups is critical to understanding the pH-dependent behavior of biological proteins and peptide-based biomaterials. Traditionally, ¹H NMR spectroscopy has been used to determine the pKa of amino acids; however, for larger molecules and aggregating systems, this method can be practically impossible. Previous studies concluded that the C-D stretches in Raman are a useful alternative for determining the pKa of histidine residues. In this study, we report on the Raman application of the C2-D probe on histidine's imidazole side chain to determining the pKa of histidine in a short peptide sequence. The pKa of the tripeptide was found via difference Raman spectroscopy to be 6.82, and this value was independently confirmed via ¹H NMR spectroscopy on the same peptide. The C2-D probe was also compared to other Raman reporters of the protonation state of histidine and was determined to be more sensitive and reliable than other protonation-dependent signals. The C2-D Raman probe expands the tool box available to chemists interested in directly interrogating the pKa's of histidine-containing peptide and protein systems.

pKa Determination of a Histidine Residue in a Short Peptide Using Raman Spectroscopy.

Here, we report a post-synthesis functionalization of the shell of Au nanoclusters (NCs) synthesized using glutathione as a thiolate ligand. The as-synthesized Au NCs are subjected to the post-synthesis functionalization via amidic coupling of dopamine on the cluster shell to tailor photoluminescence (PL) and electrochemiluminescence (ECL) features of the Au NCs. Because the NCs' PL at ca. 610 nm is primarily ascribed to the Au(I)-thiolate (SG) motifs on the cluster shell of the NCs, the post-synthesis functionalization of the cluster shell enhanced the PL intensity of the Au NCs via rigidification of the cluster shell. In contrast to the PL enhancement, the post-synthesis modification of the cluster shell does not enhance the near-infrared (NIR) ECL of the NCs because the NIR ECL at ca. 800 nm is ascribed to the Au(0)-SG motifs in the metallic core of the NCs.

Post-Synthesis Modification of Photoluminescent and Electrochemiluminescent Au Nanoclusters with Dopamine.

Pre-column derivatizations of amino acids often present two major challenges: 1) automation, due to the multi-step manipulations for pH control, reagent addition, mixing and extraction, and 2) effect of matrices in the sample such as salts, buffers and surfactants. Both issues have been addressed in a previous publication on derivatization methods using 9-fluorenylmethyl chloroformate (FMOC) and phenylisothiocyanate (PITC). In this paper, a third method of derivatization, which has recently been developed and published, was studied to address the same issues. The derivatization reagent, naphthalene-2,3-dicarboxaldehyde (NDA), is a modification from o-phthalaldehyde (OPA) and yields more stable derivatives with high fluorescence efficiencies. An autosampler was programmed to mix amino acid samples with cyanide and NDA reagent, allow a programmed reaction time and finally inject onto the HPLC. To study sample matrix effects, amino acid samples were spiked with various concentrations of Tris-HCl, phenol, citrate, sulfosalicylic acid, sodium chloride and sodium dodecyl sulfate. The recoveries of amino acids in varied sample matrices were compared to pure amino acid standards. The matrix effects using the NDA method were similar to those using the FMOC method. Comparisons of all three methods (NDA, FMOC and PITC) are discussed and tabulated.

Matrix effects in the derivatization of amino acids with naphthalene dicarboxaldehyde, 9-fluorenylmethyl chloroformate and phenylisothiocyanate.

The purpose of this study was to develop a specific, sensitive, accurate and reproducible high-performance liquid chromatographic (HPLC) method to measure polymyxin B in human plasma. Derivatization of polymyxin B with fluorescent 9-fluorenylmethyl chloroformate (FMOC-Cl) was performed in the same solid-phase extraction C18 cartridge used for the sample pre-treatment. Reversed-phase HPLC was employed with fluorometric detection. The summed peak areas of polymyxin B1 and B2 derivatives were used for quantification. Stability of polymyxin B FMOC derivatives was examined at room temperature for 6 days. Specificity was investigated against seven potentially co-administered antibiotics. Accuracy and reproducibility of the HPLC assay were determined by inter- and intra-day validation. The derivatives of polymyxin B2 and B1 were well resolved and had retention times of 4.75 and 5.55 min, respectively. Good linearity (r(2) > 0.99) was obtained between 0.125 and 4.00 mg/L polymyxin B in human plasma with good accuracy and reproducibility at the limit of quantification (0.125 mg/L). Intra- and inter-day validation demonstrated good accuracy and reproducibility for quality control samples with nominal concentrations of 0.30 and 3.00 mg/L. FMOC derivatives of polymyxin B were stable for at least 3 days at room temperature. None of the possibly co-administered antibiotics tested interfered with the chromatographic analysis of the polymyxin B FMOC derivatives. A rapid, specific, sensitive, accurate and reproducible HPLC method has been developed and validated to measure polymyxin B in human plasma. The method is suitable for clinical pharmacokinetic studies.

Development and validation of a reversed-phase high-performance liquid chromatography assay for polymyxin B in human plasma.

A pre-column derivatization high-performance liquid chromatographic method for glyphosate analysis has been developed. Derivatization of glyphosate was performed with 4-chloro-3,5-dinitrobenzotrifluoride (CNBF). In pH 9.5 H(3)BO(3)-Na(2)B(4)O(7) media, the reaction of glyphosate with CNBF completed at 60 degrees C for 30min. The labeled glyphosate was separated on a Kromasil C18 column (250mmx4.6mm, 5microm) at room temperature and UV detection was applied at 360nm. The separation of labeled glyphosate was achieved within 15min by gradient elution mode. Compared to other pre-column derivatization, this derivatization was performed more mildly, the derivative was more stable, and the detection limits of a few reagents were higher than CNBF, except 9-fluorenylmethyl chloroformate (FMOC-Cl) using fluorescence and mass spectrometry, however, this reagent avoid to be removed after derivatization like FMOC-Cl. The detection limit of glyphosate was 0.009mgL(-1) (S/N=3) without preconcentration and reach MRL, which is set at the level of 0.1mgL(-1) in China. The method linearity correlation coefficient was 0.9999, in concentrations ranging from 0.3 to 48.5mgL(-1). The proposed method has been applied to the quantitative determination of glyphosate in environmental water with recoveries of 91.80-100.20% and R.S.D. of 2.27-6.80, depending on the sample investigated.

Residue determination of glyphosate in environmental water samples with high-performance liquid chromatography and UV detection after derivatization with 4-chloro-3,5-dinitrobenzotrifluoride.

It is well known that most protein therapeutics such as monoclonal antibody pharmaceuticals and other biopharmaceuticals including cancer biomarkers are glycoproteins, and thus the development of high-throughput and sensitive analytical methods for glycans is essential in terms of their determination and quality control. We previously reported a novel alternative labeling method for glycans involving 9-fluorenylmethyl chloroformate (Fmoc-Cl) instead of the conventional reductive amination procedure. The derivatives were analyzed by high-performance liquid chromatography (HPLC) (Kamoda S, Nakano M, Ishikawa R, Suzuki S, Kakehi K. 2005. Rapid and sensitive screening of N-glycans as 9-fluorenylmethyl derivatives by high-performance liquid chromatography: A method which can recover free oligosaccharides after analysis. J Proteome Res. 4:146-152). This method was rapid and simple; however, it was time-consuming in terms of analysis by HPLC and did not provide so much information such as the detailed structures and mass numbers of glycans. Here we have developed a high-throughput and highly sensitive method. It comprises three steps, i.e., release of glycans, derivatization with Fmoc, and capillary electrophoresis-electrospray ionization mass spectrometry (CE-ESI MS) analysis. We analyzed several glycoproteins such as fetuin, alpha1 acid glycoprotein, IgG, and transferrin in order to validate this method. We were able to analyze the above glycoproteins with the three-step procedure within only 5 h, which provided detailed N-glycan patterns. Moreover, the MS/MS analysis allowed identification of the N-glycan structures. As novel applications, the method was employed for the analysis of N-glycans derived from monoclonal antibody pharmaceuticals and also from alpha-fetoprotein; the latter is known as one of the tumor markers of hepatocellular carcinomas. We were able to easily and rapidly determine the detailed structures of the N-glycans. The present method is very useful for the analysis of large numbers of samples such as a routine analysis.

Capillary electrophoresis-electrospray ionization mass spectrometry for rapid and sensitive N-glycan analysis of glycoproteins as 9-fluorenylmethyl derivatives.

Experimental and theoretical influence of multivalent cations on the analysis of glyphosate and aminomethyl phosphonic acid (AMPA) was studied in pure water and in one surface water. The procedure chosen, based on derivatization with FMOC-Cl, HPLC separation, and fluorescence detection, appears highly affected at cations concentrations current in natural waters. A detailed speciation study performed with the VMINTEQ software strongly suggests that the complexes formed between analytes and cations do not dissociate during the reaction and do not react with the derivatization agent, so that only the free forms are derivatized. These results point out the necessity of a pre-treatment to prevent these interferences, even in low salinity waters. The different ways conceivable are discussed in terms of kinetic and thermodynamic considerations.

Influence of complexation phenomena with multivalent cations on the analysis of glyphosate and aminomethyl phosphonic acid in water.

Functionalization of the surfaces of silica particles is often the first step in their various applications. An improved heterogeneous Fmoc-Cl fluorescent assay using an aqueous solution was developed to detect the number of amino groups on solid-phase supports. The fluorescent Fmoc-Cl method is 50-fold more sensitive than the current UV assay using an organic solvent. This method, together with the homogeneous fluorescamine and OPA assays, is used to detect amino groups on the silica particle surface. The accuracy and effect factors of these methods were examined and the assays were optimized. The results showed that the amine groups on silica particles can produce stronger fluorescence than small amine molecules in solution, because the porous structure of the particle surface is a more hydrophobic environment. The number of active amino groups that can be conjugated with biomolecules is much less than the total number of amino groups on the silica particle. Compared with physical methods, chemical assays involving direct reaction with amino groups would furnish the closest result to the number of active amino groups on the particle surface.

Fluorescent quantification of amino groups on silica nanoparticle surfaces.

The efficiencies of three derivatisation reagents that react with either the amine (9-fluorenylmethyl chloroformate (FMOC)) or the carboxylic acid group (butanol) of amino acid or with both types of functional groups (propyl chloroformate) were compared in the analysis of amino acids by liquid chromatography-electrospray-tandem mass spectrometry (LC-ESI-MS/MS). Separation of 20 amino acids derivatised with these three reagents was studied on reversed-phase chromatography. Linearity, repeatability and limits of detection of the LC-ESI-MS/MS method were determined by analysing FMOC-, butanol- and propyl chloroformate-derivatised lysine, beta-aminobutyric acid, threonine and glutamic acid. The limits of detection for the derivatised amino acids (7.5-75fmol) were as much as 2-60 times lower than those of the corresponding underivatised molecules. The best linearity was observed for amino acids derivatised with propyl chloroformate or butanol (r(2)=0.996-0.999, range=100-8500nmolL(-1)). Propyl chloroformate was the best suited of the reagents tested for the analysis of amino acids with LC-MS/MS and was used for the analysis of amino acids in rat brain microdialysis samples.

Comparison of different amino acid derivatives and analysis of rat brain microdialysates by liquid chromatography tandem mass spectrometry.

A literature overview is given of HPLC methods currently in use to determine amino acids as their 9-fluorenylmethyloxycarbonyl (FMOC) derivatives. On the basis of the detailed literature overview an exhaustive derivatization study was performed with 22 amino acids, applying photodiode array (DAD) and fluorescence (FL) detection simultaneously, in order to clear up the controversial points of FMOC derivatization. Model investigations have been carried out as a function of the reaction time and reaction conditions, such as the molar concentration of the reagent, the molar ratios of the reactants, the pH and the solvent composition of the reaction medium. Special emphasis was put (i) on the evaluation of the blank values of the reagents, as a function of the composition and that of the pH of the reaction medium, (ii) on the unambiguous quantitation of all amino acids, including the less reactive aspartic and glutamic acids, as well as on the formation and transformation of histidine and tyrosine, existing partly, as single (N-FMOC-histidine, N-FMOC-tyrosine), partly as double labeled species (N,NH-FMOC-histidine, N,O-FMOC-tyrosine). Reproducibilities of 22 amino acids, including both histidine and tyrosine derivatives, obtained under optimum derivatization conditions are presented (at 0.5mM FMOC concentration corresponding to the molar ratios of [FMOC]/[amino acids](T)=5.5/1 (note: the superscript 'T' means the total of amino acids), with acetonitrile containing reagents, at pH 9, derivatization time=20 min), and characterized with the relative standard deviation percentages of their responses (<or=3.98%, RSD). Quantitation limit (LOQ) of amino acid FMOC derivatives proved to be 1 pmol, except for serine, glycine, valine (2.5 pmol), for cystine, N,NH-FMOC-histidine and for the total of N-FMOC-tyrosine and N,O-FMOC-tyrosine (5 pmol) and for tryptophan (10 pmol).

Amino acid analysis by high-performance liquid chromatography after derivatization with 9-fluorenylmethyloxycarbonyl chloride Literature overview and further study.

This study proposes a sensitive method for the simultaneous separation and concentration of 9 pairs of amino acid enantiomers by combining poly(ethylene oxide) (PEO)-based stacking, β-cyclodextrin (β-CD)-mediated micellar electrokinetic chromatography (MEKC), and 9-fluoroenylmethyl chloroformate (FMOC) derivatization. The 9 pairs of FMOC-derivatized amino acid enantiomers were baseline separated using a discontinuous system, and the buffer vials contained a solution of 150 mM Tris-borate (TB), 12.5% (v/v) isopropanol (IPA), 0.5% (w/v) PEO, 35 mM sodium taurodeoxycholate (STDC), and 35 mM β-CD, and the capillary was filled with a solution of 1.5 M TB, 12.5% (v/v) IPA, 35 mM STDC, and 35 mM β-CD. Based on the difference in viscosity between the sample zone and PEO solution and because of the STDC sweeping, the discontinuous system effectively stacked 670 nL of the 9 pairs of FMOC-derivatized amino acid enantiomers without losing chiral resolution. Consequently, the limits of detection for the 9 pairs of FMOC-derivatized amino acid enantiomers were reduced to 40-60 nM. This method was successfully used to determine d-Tryptophan (Trp), l-Trp, d-Phenylalanine (Phe), l-Phe, d-Glutamic acid (Glu), and l-Glu in various types of beers.

On-line sample preconcentration by sweeping and poly(ethylene oxide)-mediated stacking for simultaneous analysis of nine pairs of amino acid enantiomers in capillary electrophoresis.

Optogenetic tools provide users the ability to photocontrol the activity of cells. Commonly, activation is achieved by expression of proteins from photosynthetic organisms, for example, microbial opsins (e.g., ChR2). Alternatively, a sister approach, synthetic optogenetics, enables photocontrol over proteins of mammalian origin by use of photoswitches, visible light (typically), and genetic modification. Thus, synthetic optogenetics facilitates interrogation of native neuronal signaling mechanisms. However, the poor tissue penetration of visible wavelengths impedes the use of the technique in tissue, as two-photon excitation (2PE) is typically required to access the near-infrared window. Here, we describe an alternative technique that uses 2PE-compatible photoswitches (section 1) for photoactivation of genetically modified glutamate receptors (section 2). Furthermore, for fast, multi-region photoactivation, we describe the use of 2P-digital holography (2P-DH) (section 3). We detail how to combine 2P-DH and synthetic optogenetics with electrophysiology, or with red fluorescence Ca2+ recordings, for all-optical neural interrogation. The time required to complete the methods, aside from obtaining the necessary reagents and illumination equipment, is ~3 weeks.

Holographic two-photon activation for synthetic optogenetics.

A high-performance liquid chromatographic method (HPLC) with combined diode array and fluorescence detection of amino acids and amines in various cheese samples is described. The proposal is based on acidic deproteinization, derivatization and gradient optimization studies, resulting in the identification and quantification of 21 amino acids and 9 amines from a single solution, by one injection. The optimized, simple protocol consists of deproteinization (1M perchloric acid), centrifugation, filtration and the subsequent derivatization with the o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate (OPA-ET-FMOC) reagent. The method can be characterized with a linearity of wide concentration range (6.25-1000 pM/injection), a good chromatographic reproducibility (average: 2.69% RSD) and an excellent recovery (average: 100.2%; average 3.84% RSD). The developed method was successfully applied in the determination of the amino acid and amine contents of port salut cheese, blue cheese and smoked cheese samples.

Simultaneous analysis of amino acids and amines as their o-phthalaldehyde-ethanethiol-9-fluorenylmethyl chloroformate derivatives in cheese by high-performance liquid chromatography.

For the successful implementation of Distributed Drug Discovery (D(3)) (outlined in the accompanying Perspective), students, in the course of their educational laboratories, must be able to reproducibly make new, high quality, molecules with potential for biological activity. This article reports the successful achievement of this goal. Using previously rehearsed alkylating agents, students in a second semester organic chemistry laboratory performed a solid-phase combinatorial chemistry experiment in which they made 38 new analogs of the most potent member of a class of antimelanoma compounds. All compounds were made in duplicate, purified by silica gel chromatography, and characterized by NMR and LC/MS. As a continuing part of the Distributed Drug Discovery program, a virtual D(3) catalog based on this work was then enumerated and is made freely available to the global scientific community.

Distributed Drug Discovery, Part 3: using D(3) methodology to synthesize analogs of an anti-melanoma compound.

The purpose of this study was to explore the transfer of drug solutions from stomach to small intestine and its impact on intraluminal drug concentrations in humans. The collected intraluminal data were used as reference to evaluate simulations of gastrointestinal transfer currently implemented in different in vitro and in silico absorption models. Gastric and duodenal concentrations of the highly soluble and non-absorbable compound paromomycin were determined following oral administration to 5 healthy volunteers under the following conditions: fasted state, fed state and fed state in the presence of a transit-stimulating (domperidone) or transit-inhibiting (loperamide) agent. Based on the obtained intraluminal concentration-time profiles, gastrointestinal transfer (expressed as the half-life of gastric emptying) was analyzed using physiologically-based parameter estimation in Simcyp®. Subsequently, the observed transfer profiles were used to judge the implementation of gastrointestinal transfer in 2 in vitro simulation tools (the TNO Intestinal Model TIM-1 and a three-compartmental in vitro model) and the Simcyp® population-based PBPK modeling platform. The observed duodenal concentration-time profile of paromomycin under fasting conditions, with a high average Cmax obtained after 15 min, clearly indicated a fast transfer of drug solutions from stomach to duodenum (estimated gastric half-life between 4 and 13 min). The three-compartmental in vitro model adequately reflected the in vivo fasted state gastrointestinal transfer of paromomycin. For both TIM-1 and Simcyp®, modifications in gastric emptying and dilutions were required to improve the simulation of the transfer of drug solutions. As expected, transfer from stomach to duodenum was delayed in the fed state, resulting in lower duodenal paromomycin concentrations and an estimated gastric half-life between 21 and 40 min. Administration of domperidone or loperamide as transit-stimulating and transit-inhibiting agent, respectively, did not affect the fed state gastric half-life of emptying. For the first time, the impact of gastrointestinal transfer of solutions on intraluminal drug concentrations was directly assessed in humans. In vitro and in silico simulation tools have been validated and optimized using the in vivo data as reference.

Gastrointestinal transfer: in vivo evaluation and implementation in in vitro and in silico predictive tools.

The aim of this study was to synthesize a peptide prodrug of glucosamine (GlcN) with increased gut permeability through the gut peptide transporter 1 (PepT1). Glycine-Valine ester derivative of GlcN (GVG) was synthesised using solid phase synthesis followed by characterization and evaluation of its physicochemical and intestinal stability. In addition, GVG was evaluated for its ability to be biotransformed to GlcN in the liver homogenate. In vitro absorption of the new prodrug through everted rat gut was also assessed. GVG demonstrated significant and meaningful increased gut permeability as compared with GlcN. It showed favorable stability in the gut and a quick cleavage to GlcN after exposure to the liver homogenate. In conclusion, a novel prodrug of glucosamine with superior gut permeability compared to GlcN was developed and successfully tested in vitro.

Synthesis and characterization of a new Peptide prodrug of glucosamine with enhanced gut permeability.

Repeated applications of glyphosate may contaminate the soil and water and threaten their quality both within the environmental system and beyond it through water erosion related processes and leaching. In this study, we focused on the transport of glyphosate and its metabolite aminomethylphosphonic acid (AMPA) related to soil erosion at two slope gradients (10 and 20°), two rates of pesticide with a formulation of glyphosate (Roundup®) application (360 and 720 mg m(-2)), and a rain intensity of 1.0 mm min(-1) for 1 h on bare soil in hydraulic flumes. Runoff and erosion rate were significantly different within slope gradients (p<0.05) while suspended load concentration was relatively constant after 15 min of rainfall. The glyphosate and AMPA concentration in the runoff and suspended load gradually decreased. Significant power and exponent function relationship were observed between rainfall duration and the concentration of glyphosate and AMPA (p<0.01) in runoff and suspended load, respectively. Meanwhile, glyphosate and AMPA content in the eroded material depended more on the initial rate of application than on the slope gradients. The transport rate of glyphosate by runoff and suspended load was approximately 14% of the applied amount, and the chemicals were mainly transported in the suspended load. The glyphosate and AMPA content in the flume soil at the end of the experiment decreased significantly with depth (p<0.05), and approximately 72, 2, and 3% of the applied glyphosate (including AMPA) remained in the 0-2, 2-5, and 5-10 cm soil layers, respectively. The risk of contamination in deep soil and the groundwater was thus low, but 5% of the initial application did reach the 2-10 cm soil layer. The risk of contamination of surface water through runoff and sedimentation, however, can be considerable, especially in regions where rain-induced soil erosion is common.

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