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At least 19 recordsLinked to original sources
PubMed2001

The effects of the NMR shift-reagents Dy(PPP)2, Dy(TTHA) and Tm(DOTP) on developed pressure in isolated perfused rat hearts. The role of shift-reagent calcium complexes.

The 23Na NMR shift-reagent complexes (Dy(PPP)2, Dy(TTHA), and Tm(DOTP)) bind stoichiometric amounts of Ca2+. Thus, in perfused rat heart systems, a supplementation of Ca2+ is required to maintain the requisite extracellular free calcium concentration ([Ca(o)]f) and to approximate a physiological level of contractile function. The amount of reagent-bound Ca2+ in a heart perfusate that contains a shift-reagent depends on: (1) Ca2+ binding by excess ligand used during the preparation of the shift-reagent; and (2) the Ca2+ binding affinity of the shift-reagent. To address point 1), we introduced a 1H and 31P NMR spectroscopic titration method to quantify directly the concentration of the excess ligand. We also used this method to minimize the amount of excess ligand (L) and thus the amount of Ca*L complex. To address point (2), we determined the stepwise Kd (microm) values of the Ca complexes of the three shift-reagents.: Dy(PPP)2, Kd=0.09, Kd2=7.9; Dy(TTHA), Kd1=10.66, Kd2=10.12; and Tm(DOTP), K(d1)=0.502, Kd2=4.98. The Kd values of the Ca complexes of the phosphonate and triphosphate based shift-reagents, Tm(DOTP) and Dy(PPP)2, respectively, are lower than those of the polyaminocarboxylate-based Dy(TTHA), indicating stronger Ca binding affinities for the former two types of complexes. We have also shown a positive correlation between [Ca(o)]f and left ventricular developed pressure (LVDP) in perfused rat hearts. Dy(TTHA) has shown no effect on LVDP v[Ca(o)]f. The LVDP values in the presence of the phosphonate and triphosphate based shift-reagents, however, were significantly higher than expected from the [Ca(o)]f levels alone. Thus a positive inotropic effect, independent of [Ca(o)]f, is evident in the presence of Tm(DOTP) or Dy(PPP)2.

Animals↗
PubMedDate not supplied

Trifunctional conjugation reagents. Reagents that contain a biotin and a radiometal chelation moiety for application to extracorporeal affinity adsorption of radiolabeled antibodies.

A method of removing radiolabeled monoclonal antibodies (mAbs) from blood using a device external to the body, termed extracorporeal affinity-adsorption (EAA), is being evaluated as a means of decreasing irradiation of noncancerous tissues in therapy protocols. The EAA device uses an avidin column to capture biotinylated-radiolabeled mAbs from circulated blood. In this investigation, three trifunctional reagents have been developed to minimize the potential deleterious effect on antigen binding brought about by the combination of radiolabeling and biotinylation of mAbs required in the EAA approach. The studies focused on radiolabeling with (111)In and (90)Y, so the chelates CHX-A' '-DTPA and DOTA, which form stable attachments to these radionuclides, were incorporated in the trifunctional reagents. The first trifunctional reagent prepared did not incorporate a group to block the biotin cleaving enzyme biotinidase, but the two subsequent reagents coupled aspartic acid to the biotin carboxylate for that purpose. All three reagents used 4,7,10-trioxa-1,13-tridecanediamine as water-soluble spacers between an aminoisophthalate core and the biotin or chelation group. The mAb conjugates were radioiodinated to evaluate cell binding as a function of substitution. Radioiodination was used so that a direct comparison with unmodified mAb could be made. Evaluation of the number of conjugates per antibody versus cell binding immunoreactivities indicated that minimizing the number of conjugates was best. Interestingly, a decrease of radioiodination yield as a function of the number of isothiocyanate containing conjugates per mAb was noted. The decreased yields were presumably due to the presence of thiourea functionality formed in the conjugation reaction. Radiolabeling with (111)In and (90)Y was facile at room temperature for conjugates containing the CHX-A' ', but elevated temperature (e.g., 45 degrees C) was required to obtain good yields with the DOTA chelate. Stability of (90)Y labeled mAb in serum, and when challenged with 10 mM EDTA, was high. However, challenging the (90)Y labeled mAb with 10 mM DTPA demonstrated high stability for the DOTA containing conjugate, but low stability for the CHX-A' ' containing conjugate. Thus, the choice between these two chelating moieties might be made on requirements for facile and gentle labeling versus very high in vivo stability. Application of the trifunctional biotinylation reagents to the blood clearance of labeled antibodies in EAA is under investigation. The new reagents may also be useful for other applications.

Affinity Labels↗
PubMedDate not supplied

Reagent Selector: using Synthon Analysis to visualize reagent properties and assist in combinatorial library design.

Reagent Selector is an intranet-based tool that aids in the selection of reagents for use in combinatorial library construction. The user selects an appropriate reagent group as a query, for example, primary amines, and further refines it on the basis of various physicochemical properties, resulting in a list of potential reagents. The results of this selection process are, in turn, converted into synthons: the fragments or R-groups that are to be incorporated into the combinatorial library. The Synthon Analysis interface graphically depicts the chemical properties for each synthon as a function of the topological bond distance from the scaffold attachment point. Displayed in this fashion, the user is able to visualize the property space for the universe of synthons as well as that of the synthons selected. Ultimately, the reagent list that embodies the selected synthons is made available to the user for reagent procurement. Application of the approach to a sample reagent list for a G-protein coupled receptor targeted library is described.

Combinatorial Chemistry Techniques↗
PubMed1981

Demonstration of DNA with an extra-sensitive Schiff's reagent and a Schiff-type dye-reagent, toluidine blue O-SO2.

This paper describes a method for the preparation of Schiff's reagent as well as a Schiff-type dye-reagent, toluidine blue O-SO2 for use in Feulgen procedure. The method involves replacement of the usual N HCl by N H2SO4 and the usual amount of potassium metabisulphite. Following this method of preparation, an extra-sensitive Schiff's reagent is obtained which requires only 4-5 min for optimum nuclear colouration even when staining is performed at 5 degrees C. This Schiff's reagent produces perfect Feulgen staining up to 6 months after preparation. Toluidine blue O-SO2, prepared with N H2SO4 and potassium metabisulphite, also produces perfect Feulgen type staining of the DNA-aldehyde molecules of acid-hydrolysed mammalian tissue sections. Toluidine blue O-SO2 when shaken with activated charcoal and filtered produces very satisfactory result. The shell-life of this dye-reagent is just a week. The suitability of the use of N H2SO4 for the preparation of Schiff's reagent as well as a Schiff-type dye-reagent, toluidine blue O-SO2, has been discussed.

Animals↗
PubMedDate not supplied

Pitfalls in characterization of protein interactions using radioiodinated crosslinking reagents. Preparation and testing of a novel photochemical 125I-label transfer reagent.

Much attention has been focused on the study of protein interactions with radioiodinated photo-crosslinking reagents, and pitfalls in using this methodology are discussed. A new photochemical and cleavable heterobifunctional crosslinking reagent, succinimidyl N-14-(2-hydroxybenzoyl)-N-11-(4-azidobenzoyl)-9-oxo-8,11,14-triaza -4,5- dithiatetradecanoate (SHAD) was prepared, and its potential as a label transfer reagent was tested in model systems. SHAD was radioiodinated, and the labeled reagent (125I-SHAD) was converted to an amide (125I-HADM, as a mimicry of conjugation to protein 1) and photolyzed. When compared to the widely used SASD reagent (sulfosuccinimidyl 2-[[(4-azidosalicyl)-amino]ethyl]-1,3- dithiopropionate, Pierce), SHAD has a number of decisive advantages. The amide of 125I-SASD (125I-ASDM) was generated and photolyzed, and it was found that at least 50% of the radioactivity is released from 125I-ASDM after 3 min of irradiation, whereas only approximately 10% is liberated from 125I-HADM under similar conditions. Furthermore, 125I-HADM was photolyzed in the presence of excess amine (mimicry of crosslinking to protein 2), and the product was cleaved by reduction (mimicry of label transfer). The transformations in the course of photolysis were monitored by UV spectroscopy and TLC analysis, and a high degree of reagent cleavage upon reduction was demonstrated. 125I-SHAD was used to crosslink Lys78-plasminogen and fibrin. 125I-SHAD was conjugated to Lys78-plasminogen in the dark. Fibrinogen and thrombin were added, and Lys78-plasminogen was crosslinked to the fibrin clot by exposure to light.(ABSTRACT TRUNCATED AT 250 WORDS)

Azides↗
PubMed1976

An alcohol-soluble Schiff's reagent: a histochemical application of the complex between Schiff's reagent and phosphotungstic acid.

A schedule for staining partially hydrated PAS-positive structures using non-aqueous solutions has been devised. Tissues are dewaxed, taken down to 70% alcohol, oxidised for 10 min in a 1% w/v alcoholic solution of periodic acid, treated with an alcoholic solution of phosphotungstic acid-Schiff reagent complex (PTA-Schiff reagent) for 25 min, washed in alcohol, cleared in xylene and mounted in a synthetic medium. The PTA-Schiff reagent complex prepared from de Tomasi Schiff reagent by precipitation with PTA may be stored in the deep freeze for many months and dissolved freshly in alcohol for use. The PTA-Schiff reagent used as above allows staining of highly water soluble materials such as dextran. From blocking and digestion studies the mode of action seems similar to de Tomasi Schiff reagent. The partial hydration of the tissues prior to reaction was found to be essential for effective staining.

Alcohols↗
PubMed1996

Blood glucose reagent strip tests in the operating room: influence of hematocrit, partial pressure of oxygen, and blood glucose level--a comparison of the BM-test 1-44, BM-Accutest, and Satellite G reagent strip systems.

OBJECTIVE: The objective of our study was to assess the influence of hematocrit (HCT), partial pressure of oxygen (PO2), and blood glucose level upon results obtained with three different blood glucose reagent strip tests used in conjunction with the appropriate meter: BM-Test 1-44, BM-Accutest, and Satellite G. METHODS: Our study was designed as a consecutive sample study of patients undergoing coronary artery surgery. The setting was the hospital theater and intensive care unit. We conducted blood analysis for HCT, PO2 and blood glucose on 20 consecutive patients undergoing coronary artery surgery using three blood glucose reagent strip testing systems and a laboratory analysis of plasma glucose. RESULTS: All three blood glucose reagent strip tests showed a significant bias when compared with plasma glucose: BM-Test 1-44, 0.89 mmol/L; BM-Accutest, -1.27 mmol/L; Satellite G, 0.75 mmol/L (p < 0.05). The error found when using the Satellite G system was worse than that of either of the other two systems. Results obtained with the BM-Accutest strips were unaffected by PO2 (p = 0.745). Blood glucose value and HCT both had an influence on the results of all three blood glucose strip systems. CONCLUSIONS: Caution must be taken when using reagent strip systems in the operating room or intensive care setting because, of the three systems tested, all showed a significant bias, all were influenced by blood glucose level and HCT, and only the BM-Accutest reagent strips used with the Accutrend meter was unaffected by PO2.

Bias↗
PubMed1996

Development of a novel derivatization reagent for the sampling and analysis of total isocyanate group in air and comparison of its performance with that of several established reagents.

Analytical reference standards generally are not available for non-monomeric isocyanate species, making accurate identification and quantitation by high-performance liquid chromatography (HPLC) difficult. A successful derivatizing reagent must react rapidly with all isocyanate groups, the derivatized isocyanate must be detectable selectively and at very low levels, and the detector used for quantitation must give a response proportional to the number of derivatized isocyanate groups present. A novel derivatizing reagent, 1-(9-anthracenylmethyl)piperazine (MAP), was prepared in an attempt to achieve these goals. Derivatives were prepared by reacting five mono- and difunctional isocyanates with MAP and three other established isocyanate derivatizing reagents. These reagents included 1-(2-methoxyphenyl)piperazine (MOPP),9-(methylaminomethyl)anthracene (MAMA), and tryptamine (TRYP). The relative reactivities of MAP, MOPP, TRYP, and MAMA with phenyl isocyanate were found to be 100, 88, 30, and 25, respectively. Average molar absorptivities at the absorbance maxima +/- compound-to-compound variabilities were, for MAP: 1.47 x 10(5) +/- 3.50%; MAMA: 1.38 x 10(5) +/- 7.07%: and TRYP: 3.98 x 10(4) +/- 13.1%. Average fluorescence responses were, for MAP: 100 +/- 32.6%; MAMA: 41.0 +/- 58.8%; and TRYP: 2.27 +/- 15.6%. A comparison of MAP and MOPP ureas by HPLC/ultraviolet (UV)/electrochemical (EC) gave average responses for UV, EC, and EC/UV for MAP: 117 +/- 7.3%, 52.1 +/- 6.6%, and 0.447 +/- 10.7%, respectively; for MOPP: 24.3 +/- 62.5%, 76.7 +/- 28.5%, and 4.28 +/- 59.1%, respectively. The favorable performance of MAP warrants its further study as a reagent for the determination of total isocyanate group in air.

Air Pollutants, Occupational↗
PubMed1994

Reagents for bioorganic synthesis. 6. The design and synthesis of two novel polyfunctional organic reagents for biomacromolecular modification.

The design, synthesis, structure, and properties of two tetrafunctional organic reagents, namely, 2,2'-sulfonylbis[3-(carboxymethylamino)-(E,E)-N-(2-chloroeth yl)propenamide] (SBCCP) (1) and 2,2'-sulfonylbis[3-(carboxymethylamino)-(E,E)-N-(2-oxoeth yl)propenamide] (SBCOP) (2) are reported. Reagents 1 and 2 contain reactive bis(alkyl halide) and bis(aldehyde) functions, respectively, as well as bis(carboxylic acid) moieties. The reagents have potential applications for biomacromolecular cross-linking, in particular for the cross-linking of hemoglobin subunits. Single-crystal X-ray diffraction data of synthetic intermediates (4,5, and 12) provided information about structural features and tether lengths of the target reagents.

Aldehydes↗
PubMed2005

Fluorous reagents and scavengers versus solid-supported reagents and scavengers, a reaction rate and kinetic comparison.

Reactions using fluorous reagents and scavengers are compared side-by-side with their solid-supported counterparts. Fluorous triphenylphosphine is used in the bromination reaction of alcohols, fluorous thiol is used as an electrophile scavenger for alpha-bromoketones, fluorous isatoic anhydride is used as a nucleophile scavenger for primary and secondary amines. Reactions involving fluorous reagents and scavengers occur in homogeneous media with solution-phase reaction kinetics. Reactions with solid-supported reagents and scavengers occur in a heterogeneous media, and the reaction kinetics are greatly affected by the nature of the solid-support and reaction environment. Significantly larger amounts of reagents and more time are usually required to complete the solid-supported reaction.

Amines↗
PubMed1988

Construction of dry reagent chemistries: use of reagent immobilization and compartmentalization techniques.

The development of dry reagent systems has provided convenience to the user as well as devices that are more versatile and suitable for a variety of analyses. Most dry reagent chemistries are usually less than 7 cm2 by 300 micron thick packaged as discrete test devices. This reduces spoilage of unused reagents. Sample volumes needed for analysis are usually in the range of 3-30 microliter, with 10 microliter most commonly used. The use of such small volumes makes these devices suitable for neonatal and geriatric patients where large sample volumes are not often available. Hence, 150 microliter of serum (approximately 300 microliter blood) is sufficient for at least 15 different analyses on a sample. Dry reagent chemistries are easy to store, readily available for use, and disposable. Only application of a sample is needed to start an analysis.

Blood Chemical Analysis↗
PubMed1993

Demonstrating instrument-reagent flexibility: a carbamazepine enzyme immunoassay reagent system.

Versatility of immunoassay reagents is beneficial to laboratories seeking cost-effective combinations of tests and automated instrumentation. In such cases, both immunoassay analytical performance and instrument independence must be assessed. Considering this, we determined the compatibility of a new carbamazepine EMIT 2000 reagent system with two fully automated but different kinetic rate analyzers (Hitachi 704 and Cobas MIRA), comparing results to a reagent-dedicated fluorescent polarization automated device (TDx) as reference. In order to more stringently assess reagent antibody specificity, we tested recovery of purified carbamazepine spiked into sera pooled from different hospital groups (normal, renal failure, hepatic failure, term pregnancy, cord blood). Cross-reactivity was additionally tested using patient sera containing various amounts of tricyclic antidepressants, compounds structurally but not functionally related to carbamazepine. Despite distinct operational differences between analyzers, precision (< 5.5% CV) and accuracy (> 95% recovery) compared well to the TDx method. However, when carbamazepine was spiked into sera from patients with hepatic failure or at term pregnancy, all three methods measured a negative bias in recovery of 16-20%. No significant cross-reactivity was observed at normal therapeutic concentration of certain tricyclic compounds, though measurable cross-reactivity was detected when present at toxic serum concentrations. We conclude that the EMIT carbamazepine immunoassay is adaptable to the different kinetic rate analyzers studied. Analytical specificity should, furthermore, be assessed in the context of interferences likely to be clinically encountered.

Antibody Specificity↗
PubMed1978

A comparative study of five commercial reagents for the Coulter Model S: a proposed method for reagent evaluation.

A comparative study of five competitive commercial reagents for the Coulter Model S was performed jointly by two large, busy hospital laboratories with active quality control programs. Both laboratories, independently using the same reagent systems, studied 20 consecutive blood samples from patients from their own respective hospital for 20 days. Data from both laboratories were analyzed by standard and nonstandard statistical methods, and the results from both laboratories were compared. Although all reagent systems performed reasonably well in a clinical setting, highly significant statistical differences in their precisions were demonstrated by using univariate and multivariate techniques. The statistical method developed in this study can be applied to other systematic investigations that compare reagent systems.

Blood Cell Count↗
PubMed1994

[Complementary-addressed photomodification of nucleic acids by arylazide and perfluoroarylazide oligonucleotide derivatives. III. Oligonucleotide reagents with a photoactive group at the end or inside the chain; tandem reagents].

Photomodification of target oligonucleotides with reagents bearing p-azidotetrafluorobenzamide group at various positions of the oligonucleotide address was investigated. The photoactive group was attached to the 5'- or 3'-terminal phosphate or at the C5-position of a deoxyuridine residue at the 5'-end or inside the oligonucleotide chain. The reagents with the internal photoactive group modified the target with 50-55% efficiency (fraction of covalent adducts reagent-target), whereas the derivatives with a terminal reactive group were more effective (70%). The main point of the modification was the guanosine residue of the target which located near to the photoactive group and was not involved into the duplex formation. Tandems of reagents which are complementary to neighbouring sites of the target modify predominantly the same guanosine residue, with up to 80% extent.

Azides↗
PubMed2000

How borane reagents based on alpha-pinene control stereoselectivity in the reductions of carbonyl groups with different stereogenic elements. 3. The effect of the relative size and conformation of the carbonyl substituents on the stereoselectivity of the Ipc(2)BCl, Eap(2)BCl, B-t-Bu-IpcBCl, and B-t-Bu-EapBCl reagents. A semiempirical study

The reductions of a more elaborate prostereogenic nonchiral and chiral ketones with the representative borane reagents derived from (+)-alpha-pinene confirm that the overall stereoselectivity, similarly to the previously studied reductions of benzaldehyde and acetophenone, is also controlled, early along the reaction coordinate, by the structure of the borane reagent. The stereoselectivity reflects the energy differences among syn-1, 3-interactions of the smallest of the three boron substituents in the reacting borane conformation with the two carbonyl substituents in the substrate. For the B-alkyl-IpcBCl reagents and the R(S)-CO-R(L) substrates, these are the interactions of the B-Cl with the C-R(S) and C-R(L) bonds. To minimize developing intermolecular syn-1,3-interactions with the reagent's B-Cl bond during the reaction, in the conformationally mobile R(S)-CO-R(L) systems, the R(L) and R(S) groups seek to adopt a more reactive conformation even at the expense of the unfavorable intramolecular interactions.

Journal Article↗
PubMed2005

Development of highly potent D-glucosamine-based chiral fluorescent labeling reagents and a microwave-assisted beta-selective glycosidation of a methyl glycoside reagent.

We synthesized new chiral fluorescence labeling reagents having a 2,3-anthracenedicarboximide group from D-glucosamine, and it was possible to introduce target alcohols at the anomeric positions of the reagents with beta-selectivity by glycosidations. Especially, it was possible to use methyl glycoside reagent as a glycosyl donor with a Lewis acid and microwave irradiation, and it gave selectively beta-glycoside while the reaction without microwave irradiation gave alpha- and beta-mixed glycosides. Those reagents showed very high chiral discrimination ability, and they made it possible to separate the eight stereoisomers of 4,8,12,16-tetramethylheptadecanol by HPLC after derivatizations.

Carbohydrate Conformation↗
PubMed1989

Protection of hexaprenyl-diphosphate synthase of Micrococcus luteus B-P 26 against inactivation by sulphydryl reagents and arginine-specific reagents.

Hexaprenyl-diphosphate synthase from Micrococcus luteus B-P 26 has been shown to comprise two essential components, designated as components A and B. Treatment of the synthase with sulphydryl reagents (N-ethylmaleimide, iodoacetamide or p-chloromercuribenzoate) or arginine-specific reagents (2,3-butanedione, 1,2-cyclohexanedione or phenylglyoxal) resulted in a rapid loss of the component B activity. In contrast, component A was resistant to treatment with such reagents, retaining the initial activity almost completely. Farnesyl diphosphate, isopentenyl diphosphate, farnesyl monophosphate and inorganic pyrophosphate protected the synthase against the inactivation by N-ethylmaleimide, farnesyl diphosphate being the most effective. The presence of Mg2+ was essential for the protection by isopentenyl diphosphate and inorganic pyrophosphate. For protection of the synthase activity against the inactivation by 2,3-butanedione, the presence of farnesyl diphosphate, isopentenyl diphosphate and Mg2+ was more effective than that of the individual substrates and Mg2+. Inorganic pyrophosphate provided substantial protection. In the absence of component A, the component B activity was not protected by any substrates or its analogue. These results suggest that the catalytic site of the synthase is formed by cooperative interaction between components A and B, and that cysteine and arginine residues on component B play important roles in the synthase activity.

Aldehydes↗