Free Primary Amines Research Articles

S-Nitroso-N-acetylpenicillamine (SNAP) Derivatization of Peptide Primary Amines to Create Inducible Nitric Oxide Donor Biomaterials

An S-nitroso-N-acetylpenicillamine (SNAP) derivatization approach was used to modify existing free primary amines found in fibrin (a natural protein-based biomaterial) to generate a controlled nitric oxide (NO) releasing scaffold material. The duration of the derivatization reaction affects the NO release kinetics, the induction of controlled NO-release, hydrophobicity, swelling behavior, elastic moduli, rheometric character, and degradation behavior. These properties were quantified to determine changes in fibrin hydrogels following covalent attachment of SNAP. NO-releasing materials exhibited minimal cytotoxicity when cultured with fibroblasts or osteoblasts. Cells maintained viability and proliferative character on derivatized materials as demonstrated by Live/Dead cell staining and counting. In addition, SNAP-derivatized hydrogels exhibited an antimicrobial character indicative of NO-releasing materials. SNAP derivatization of natural polymeric biomaterials containing free primary amines offers a means to generate inducible NO-releasing biomaterials for use as an antimicrobial and regenerative support for tissue engineering.

Fluorescent microplate analysis of amino acids and other primary amines in soils

In studies of soil nitrogen (N) cycling, there is growing demand for accurate high-throughput analyses of amino acids and other small organic N compounds. We adapted an existing fluorometric amino acid method based on o-phthaldialdehyde and β-mercaptoethanol (OPAME) for use in 96-well microplates, and tested it using standards and field samples. While we started with an existing protocol, we made one critical change: instead of using a 1-min incubation period, we used a 1-h incubation period to deal with differences in reaction timing among microplate wells and to reduce interference from ammonium. Our microplate method is similar in sensitivity to existing protocols and able to determine leucine standard concentrations as low as ∼0.5 μM. Finally, we demonstrate that the OPAME reagent fluoresces in the presence of primary amines other than amino acids, such as amino sugars and tyramine. Because of this broad sensitivity to primary amines, descriptions of the measured pool should be revised from total free amino acids (TFAA) to total free primary amines (TFPA).

Detection of free primary amines in TFC membranes by microscopic fluorescence imaging

A microscopic imaging method using the fluorescent binding agent, fluorescamine, has been applied to determine the presence and location of primary amines in the selective layer of TFC polyamide RO membranes. The presence of such amines is attributed to incomplete condensation reactions in the preparation of the membrane's active layer for fully aromatic TFC membranes. The concentration of free amines is greatest in the section of the active layer most distant from the external membrane surface. The free amines are effectively removed by combining rinsing with application of oxidation agents. As delivered in spiral wound modules they have already been removed to a large extent. In flat sheet membrane they still contain various extents of free amine depending on the membrane type and the supplier.

Dynamic Surface Properties of Amino-Terminated Self-Assembled Monolayers Incorporating Reversible CO2 Chemistry

An innovative approach to creating a dynamically controllable smart surface is proposed, incorporating the reversible reaction of CO2 with free primary and secondary amines of amino-terminated self-assembled monolayers (SAMs). Solutions of N-(2-aminoethyl)(3-aminopropyl)methyldimethoxysilane (AEAPMDS), N-(6-aminohexyl)-3-aminopropyltrimethoxysilane (AHAPTS), and trimethoxysilylpropyl(polyethleneimine) (PEI-silane) were exposed to CO2, producing the respective carbamate species. The reacted CO2 was readily released by application of moderate heating and reduced pressure. Thermogravametric analysis (TGA) showed a weight loss percentage corresponding to 1 mol of CO2 per 1 mol of diamine at roughly 50 °C, and attenuated total reflectance Fourier transform infrared (ATR FTIR) spectroscopy confirmed the formation of the carbamate species. Well-ordered amino-terminal SAMs of AEAPMDS, AHAPTS, and PEI-silane were prepared on silicon substrates from toluene solutions and were then exposed to CO2. The subsequent cha...

Application of a Combined Weak Cation-Exchange/Crown Ether Column: First Demonstrations of a Versatile Tool for Proteome Subselection

The present paper introduces the use of a weak cation-exchange/crown ether column in the proteomics field. The 18-crown-6 ether functionality is well-known to selectively complex ammonium and monoalkylammonium ions, which should make this column highly suitable to trap peptides with free alpha-NH(2) or free epsilon-NH(2) groups from lysine side chains. This unique selection mechanism was put to the test in an N-teromics setup which aims for the enrichment of deliberately acetylated protein N-terminal peptides from a serum digest. It was demonstrated that peptides with free alpha-NH(2) groups and peptides with alpha-amino-acetylated groups can be separated from each other using this weak cation-exchange/crown ether column. The peptides of interest, bearing no free primary amines, were found to be significantly enriched in the column's flow through. At the same time a favorable coenrichment of N-glycosylated peptides was observed. To obtain more insight in the contributions of the two distinct column functionalities, i.e., the weak cation exchanger and the crown ether, the experimental data were checked against a theoretical prediction of the outcome.

Detection of cometary amines in samples returned by Stardust

Abstract— The abundances of amino acids and amines, as well as their enantiomeric compositions, were measured in samples of Stardust comet‐exposed aerogel and foil using liquid chromatography with UV fluorescence detection and time of flight mass spectrometry (LC‐FD/ToF‐MS). A suite of amino acids and amines including glycine, L‐alanine, β‐alanine (BALA), γ‐amino‐n‐butyric acid (GABA), ɛ‐amino‐n‐caproic acid (EACA), ethanolamine (MEA), methylamine (MA), and ethylamine (EA) were identified in acid‐hydrolyzed, hot‐water extracts of these Stardust materials above background levels. With the exception of MA and EA, all other primary amines detected in comet‐exposed aerogel fragments C2054,4 and C2086,1 were also present in the flight aerogel witness tile that was not exposed to the comet, indicating that most amines are terrestrial in origin. The enhanced relative abundances of MA and EA in comet‐exposed aerogel compared to controls, coupled with MA to EA ratios (C2054,4: 1.0 ± 0.2; C2086,1: 1.8 ± 0.2) that are distinct from preflight aerogels (E243–13C and E243–13F: 7 ± 3), suggest that these volatile amines were captured from comet Wild 2. MA and EA were present predominantly in an acid‐hydrolyzable bound form in the aerogel, rather than as free primary amines, which is consistent with laboratory analyses of cometary ice analog materials. It is possible that Wild 2 MA and EA were formed on energetically processed icy grains containing ammonia and approximately equal abundances of methane and ethane. The presence of cometary amines in Stardust material supports the hypothesis that comets were an important source of prebiotic organic carbon and nitrogen on the early Earth.

Synthesis of New Trisubstituted 4‐Aminopiperidines as PAF‐Receptor Antagonists

AbstractTwo novel classes of 4‐aminopiperidines substituted in the 3‐position by groups bearing either a carbamate or a ureido function have been synthesized from ethyl 4‐oxo‐3‐piperidinecarboxylate and 3,3′‐iminobis(propanenitrile), respectively. The key step in this synthesis, the reduction of the piperidinic β‐enamino ester or nitrile, occurred readily. In contrast to published works, the free primary amines could be isolated from the corresponding β‐amino ester or nitrile. Regioselective amidification of the amino group offered two pairs of diastereoisomers which were successfully separated and identified. Measurement of PAF‐receptor antagonist activity gave interesting results with an IC50 close to the micromolar.(© Wiley‐VCH Verlag GmbH & Co. KGaA, 69451 Weinheim, Germany, 2008)

Enantioselective diethylzinc addition to the exocyclic C [dbnd]N double bond of some 4-arylideneamino-3-mercapto-6-methyl-4 H-1,2,4-triazin-5-one derivatives

4-Arylideneamino-3-mercapto-6-methyl-4 H-1,2,4-triazin-5-ones 2a– f have been evaluated as substrates in the enantioselective diethylzinc addition reaction in the presence of (1 S,2 R)- N-alkyl- N-benzylnorephedrines 3a– d as chiral ligands. The utility of using a dual catalytic system (amino alcohol/halosilane) for the diethylzinc addition reaction has been also examined. The addition products 4-(1-arylpropyl)amino-3-mercapto-6-methyl-4 H-1,2,4-triazin-5-ones 4a– f were obtained in high yields and with enantiomeric excesses of up to 92%. The treatment of arylimines 2a– f with a diethylzinc reagent did not affect the hetero-ring opening although the C N double bond of the lateral chain did undergo an addition reaction to yield the C-ethylated products 4a– f. The reductive cleavage of the 1,2,4-triazinyl heterocyclic ring from addition products 4a– f led smoothly to the corresponding free primary amines 5a– f without a significant loss of enantiomeric purity.

Dendrimer Precursors for Nanomolar and Picomolar Real-Time Surface Plasmon Resonance/Potentiometric Chemical Nerve Agent Sensing Using Electrochemically Crosslinked Ultrathin Films

Nanomolar detection and specific recognition of pinacolyl methylphosphonate (PMP), a hydrolysis product and an analog of a relatively persistent class of toxic nerve agents, has been achieved. In addition, picomolar sensitivity is observed with methylphosphonic acid (MPA), an end-hydrolysis product for all organophosphate-based nerve agents. This is achieved using a combined surface plasmon resonance (SPR) and potentiometry setup. A modified polyamidoamine (PAMAM) carbazole/Cu2+ dendrimer, which is electrochemically crosslinked on a self-assembled monolayer (SAM) modified Au substrate, is used as an active sensing element for trapping the nerve-agent analogs. The ultrathin films have been used to study the anchoring of nerve agents via non-covalent interactions. The carbazole to amine ratio is optimized to ensure free primary amines are available to interact with the analyte and the Cu2+ ions present in the system, which further enhances the selectivity. The carbazole group on the periphery serves a dual purpose: crosslinking the dendrimers to form a conjugated network film, and generating the potentiometric response. The adsorption kinetics are monitored by using an in situ SPR/potentiometric setup. This technique not only offers a real-time dual detection of highly toxic nerve-agent analogs, but also shows viability for future sensor-device applications.

The determination of total dissolved free primary amines in seawater: Critical factors, optimized procedure and artefact correction

The paper reviews the methods described in the literature for the determination of total dissolved free primary amines (TDFPA) by fluorescence. A wide set of reaction conditions can be found, but they rely on few experiments for their validation. Among fluorogenic compounds, o-phthaldialdehyde (OPA) is more sensitive than fluorescamine and was thus examined here. However, the use of mercaptoethanol (ME) in the reaction (as an additional derivatization compound) is able to generate unreliable results, in particular when standardization relies on glycine. We suggest replacing ME with 3-mercaptopropionic acid (MPA) which induces more stable and comparable fluorescence among amine compounds. A systematic study was therefore undertaken to define reagent concentrations and pH effects on the reaction rates for a variety of primary amines with particular focus on amino acids. The reaction rate is increased by increasing OPA concentration and pH, but slowed by excess MPA. Ammonium interference is influenced by several factors, but spectral investigation showed that the choice of conditions can drastically reduce it. The magnitudes of natural and OPA-induced background fluorescence signals have been assessed in various mediums and it is shown that their contribution to the signal amounts to a large fraction, when not most, of the measured fluorescence. A segmented flow method is proposed with a protocol for adequate correction of biases.

Polymer–filler interactions in kaolin/nylon 6,6 composites containing a silane coupling agent

The properties of calcined and hydrous kaolin filled nylon 6,6 composites have been investigated with respect to particle size and surface treatment with an amino-silane coupling agent (A1100). The mechanical properties of untreated kaolin filled nylon 6,6 composites are kaolin type (hydrous and calcined) independent. However, A1100 surface treatment leads to a distinction between calcined and hydrous kaolin composites, promoting adhesion only with calcined kaolin. Treated calcined kaolin filled nylon 6,6 exhibits improved strength and elongation properties, which are influenced by the silane loading level relative to the filler surface area. At high silane loading levels a plasticing effect was observed with impact strength decreasing. The wet strength of A1100 treated calcined kaolin composites is almost double that of the untreated condition. As A1100 does not promote adhesion with hydrous kaolin wet and dry strength properties are relatively unaffected, with the mode of failure being interfacial. Evidence of interfacial chemical bonding was found only in A1100 treated calcined kaolin. It is thought that the interaction is controlled by an acid base interaction between surface free primary amines and carboxylic acid polymer end groups with amide formation at the interface. The distinction between treated calcined and hydrous kaolin composites is due to the availability of surface free amines. Calcined kaolin has a relatively neutral surface on which the amines are not inhibited in their interaction with nylon functional groups. Hydrous kaolin has an acidic surface with bronsted activity that protonates surface amines inhibiting their interaction with nylon 6,6.

Reductive Amination of Aliphatic Carbonyls Using Low‐Valent Titanium Reagent: A Convenient Route to Free Primary Amines.

Reductive Amination of Aliphatic Carbonyls Using Low‐Valent Titanium Reagent: A Convenient Route to Free Primary Amines.

Reductive Amination of Aliphatic Carbonyls using Low–valent Titanium Reagent: A Convenient Route to free Primary Amines

A simple procedure for reductive amination of aliphatic carbonyls, giving direct access to free primary amines using low–valent titanium (LVT) reagent has been reported.

Child cerebrospinal fluid analysis by capillary electrophoresis and laser-induced fluorescence detection

Capillary electrophoresis with laser-induced fluorescence detection (CE-LIF) was used to analyze a 50-μl sample of cerebrospinal fluid from leukaemic children treated with high doses of methotrexate. Free amino acids and primary amines are labelled with fluorescein isothiocyanate prior to analysis. Electropherograms containing more than 50 peaks were obtained in less than 22 min. Twenty-one peaks were identified, and 19 were quantitated. Observed differences in individual amino acid levels are compared with healthy reference values. The results indicate that CE-LIF is useful as a selective, rapid and sensitive tool for the determination of free amino acids and amines in clinical biology studies.

New photolabile amino protecting groups: photogeneration of amines from [(3′,5′-dimethoxybenzoinyl)oxy]carbonyl carbamates

A novel class of photoiabile amino protecting groups, based on [(3′,5′-dimethoxybenzoinyl)oxy]carbonyl groups, is presented and their scope and versatility demonstrated by the photogeneration of free primary and secondary amines.

Potential role of lipoxygenases in defense against insect herbivory

The potential role of the plant enzyme lipoxygenase in host resistance against the corn earwormHelicoverpa zea was examined. Lipoxygenase is present in most of the common host plants ofH. zea, with highest activity in the leguminous hosts such as soybean and redbean. Treatment of dietary proteins with linoleic acid and lipoxygenase significantly reduced the nutritive quality of soybean protein and soy foliar protein. Larval growth was reduced from 24 to 63% depending upon treatment. Feeding byH. zea on soybean plants caused damage-induced increases in foliar lipoxygenase and lipid peroxidation products. Larvae feeding on previously wounded plant tissue demonstrated decreased growth rates compared to larvae feeding on unwounded tissue. Midgut epithelium from larvae feeding on wounded tissues showed evidence of oxidative damage as indicated by significant increases in lipid peroxidation products and losses in free primary amines. The potential role of oxidative and nutritional stress as a plant defensive response to herbivory is discussed.

A method for detection of hydroxyl radicals in the vicinity of biomolecules using radiation-induced fluorescence of coumarin.

A novel method is described to quantitate radiation-induced hydroxyl radicals in the vicinity of biomolecules in aqueous solutions. Coumarin-3-carboxylic acid (CCA) is a non-fluorescent molecule that, upon interaction with radiation in aqueous solution, produces fluorescent products. CCA was derivatized to its succinimidyl ester (SECCA) and coupled to free primary amines of albumin, avidin, histone-H1, polylysine, and an oligonucleotide. When SECCA-biomolecule conjugates were irradiated, the relationship between induced fluorescence and dose was linear in the dose range examined (0.01-10 Gy). The fluorescence excitation spectrum of irradiated SECCA-biomolecule conjugates was very similar to that of 7-hydroxy-SECCA-biomolecule conjugates, indicating the conversion of SECCA to 7-hydroxy-SECCA following irradiation. Control studies in environments that excluded certain radiation-induced water radicals for both the conjugated and unconjugated forms of irradiated SECCA demonstrated that: (1) the induction of fluorescence is mediated by the hydroxyl radical; (2) the presence of oxygen enhances induced fluorescence by a factor of about 1.4, and (3) other primary water radicals and secondary radicals caused by interaction of primary water radicals with biomolecules do not significantly influence the induced fluorescence. The data indicate that the induction of fluorescence on SECCA-biomolecule conjugates records specifically the presence of the hydroxyl radical in the immediate vicinity of the irradiated biomolecule. The method is rapid and sensitive, uses standard instrumentation, and the sample remains available for further studies.

Reversible protection of lysine to facilitate the purification of protected peptide segments

The polarity of temporarily free lysine side chains can be exploited to aid in the purification of protected peptides. The amine groups can easily be reprotected after the purification step. Impurities in commercial TFE 1 can cause trifluoroacetylation of free primary amines.

Real-time determination of dissolved free amino acids and primary amines in seawater by time-resolved fluorescence

Dissolved free amino acids (DFAAs) and ammonium are important nutrients in marine microbial food webs. DFAA concentrations are most often studied by high-pressure liquid chromatography of fluorescent derivatives with ortho-phthaldialdehyde (OPA). This procedure is laborious, requires extensive sample handling, and yields results hours or days after sampling. A fluorescent monitoring system for DFAAs and other primary amines was developed that is automated and continuous, and provides results in real time. A gently pumped stream of seawater is reacted with an OPA-mercaptoethanol-buffer reagent and passed over the tip of a fiber optic probe which supplies pulsed laser light at 337 nm. The fluorescent signal, with a maximum at 455 nm, is transmitted by the probe to an optical multichannel analyzer which uses a linear photodiode array. A plot of fluorescent intensity vs. time is recorded. The signal is linear in the range of 1–500 nM alanine equivalents. The signals with ammonium and urea are weak, not linear with respect to concentration, and not expected to contribute significantly to the total signal of well-oxygenated seawater. Ammonium can be resolved from a mixture of amino acids by time-resolved fluorescence. The fluorescent lifetime of the ammonium derivative is 9 ns; that of all amino acids tested is 21 ns, as is that of the bulk seawater fluorescence. A surface transect from San Francisco to Hawaii showed hundreds of nanomoles per liter of alanine equivalents in the California Current, declining to ∼45 nmol l −1 in the central gyre, and rising sharply at Pearl Harbor. Loose correlation was observed with chlorophyll and bioluminescence. The system has demonstrated utility in monitoring bulk amino acid concentrations while under way at sea.

Nonradioactive labeling of synthetic oligonucleotide probes with terminal deoxynucleotidyl transferase

Synthetic oligonucleotides were tailed at the 3′ end using terminal deoxynucleotidyl transferase. Nucleotide triphosphates with free primary amines at the end of side chains were compared for their tailing efficiency and/or detection sensitivity, using biotin-11-dUTP as a reference. Free primary amines were tagged with activated biotin or fluorescein isothiocyanate. The probes were then detected with either streptavidin-alkaline phosphatase complex or anti-fluorescein antibodies and alkaline phosphatase-conjugated secondary antibodies. Tailing conditions were optimized and the probes were tested for detection of Escherichia coli ST 1a enterotoxin DNA and rotavirus RNA.