A new approach to specific labelling of organic compounds with tritium: Catalysed exchange in solution with tritium gas

Evans, E. A.; Sheppard, Hilary C.; Turner, John; Warrell, D. C.

doi:10.1002/jlcr.2590100407

Cited by 109 publications

(28 citation statements)

References 18 publications

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“…1), was synthesized by the catalytic exchange method of Evans et al [9]. The method calls for the stirring of a nucleotide or saccharide derivative in an aqueous medium (basic pH) in the presence of a large mass excess of the hydrogenation catalyst, 5% palladium oxide supported on barium sulfate.…”

Section: Resultsmentioning

confidence: 99%

“…Based on the work of Evans et al [9], five protons of ADAC should be most susceptible to tritium exchange-the C-8 proton of adenine and the four benzylic protons. However, the observed specific activity of 27.5Ci/mmol suggests substitution mainly at the most readily exchanged C-8 proton.…”

Section: Resultsmentioning

confidence: 99%

“…Unlabeled ADAC [2], 7 mg, was dissolved in 0.1 M sodium phosphate, pH 10, and subjected to catalytic exchange [9] using 100 mg of 5% PdO/BaSO 4 under 10 Ci of tritium gas (carried out by the Amersham Corp., U.K., procedure code TR.7). The catalyst was removed by filtration, and labile protons were exchanged, leaving 33 mCi of radioactivity, 25% of which co-migrated with ADAC by thin-layer chromatography (Merck silica gel 60, CHCl 3 -MeOH-HOAc, 10:10:1, silica, R f = 0.14).…”

Section: Preparation Of [ 3 H]adac (2)mentioning

confidence: 99%

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Molecular probes for extracellular adenosine receptors

Jacobson

Ukena

Padgett

et al. 1987

Biochemical Pharmacology

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Derivatives of adenosine receptor agonists (N 6 -phenyladenosines) and antagonists (1,3-dialkyl-8-phenylxanthines) bearing functionalized chains suitable for attachment to other molecules have been reported [Jacobson et al., J. med. Chem. 28, 1334 and 1341]. The "functionalized congener" approach has been extended to the synthesis of spectroscopic and other probes for adenosine receptors that retain high affinity (K i ~ 10 −9 −10 −8 M) in A 1 -receptor binding. The probes have been synthesized from an antagonist xanthine amine congener (XAC) and an adenosine amine congener (ADAC). [ 3 H]ADAC has been synthesized and found to bind highly specifically to A 1 -adenosine receptors of rat and calf cerebral cortical membranes with K D values of 1.4 and 0.34 nM respectively. The higher affinity in the bovine brain, seen also with many of the probes derived from ADAC and XAC, is associated with phenyl substituents. The spectroscopic probes contain a reporter group attached at a distal site of the functionalized chain. These bifunctional ligands may contain a spin label (e.g. the nitroxyl radical TEMPO) for electron spin resonance spectroscopy, or a fluorescent dye, including fluorescein and 4-nitrobenz-2-oxa-1,3-diazole (NBD), or labels for 19 F nuclear magnetic resonance spectroscopy. Potential applications of the spectroscopic probes in characterization of adenosine receptors are discussed."Functionalized congener" refers to a drug derivative that contains a chemical functional group, such as an amine or carboxylic acid, attached through a spacer chain to a position on the pharmacophore, which position is relatively insensitive to steric bulk. The congeners are designed as intermediates for the synthesis of covalent conjugates that retain binding properties at the receptor site for the drug. The aims of this approach are 2-fold: (1) to develop potential new pharmaceutical agents in which the activity of the primary pharmacophore (portion of drug molecule essential for biological activity) may be modulated through distal changes, and (2) to synthesize radioactive and non-radioactive molecular probes for the drug binding site. Since the strategy of this drug design allows groups to be placed at a distance from the binding site of the primary pharmacophore, it is possible to overcome constraints on the size of the attached group [1][2][3][4][5].Sites for functionalization of adenosine receptor ligands have been identified for both agonists (derivatives of N 6 -phenyladenosine) and antagonists (derivatives of 1,3-dialkyl-8-phenylxanthine . A common structure-activity relationship (SAR) feature in both agonist and antagonist series is that the presence of a distal amino group located on the attached chain in many cases results in increased affinity to adenosine receptors.In this study we have examined the parameters of binding of tritiated ADAC and have demonstrated the utility of both ADAC and XAC as synthetic precursors for receptor probes. Biotin-containing probes for adenosine receptors have been reported [6]. ...

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Section: Resultsmentioning

confidence: 99%

Section: Resultsmentioning

confidence: 99%

Section: Preparation Of [ 3 H]adac (2)mentioning

confidence: 99%

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Molecular probes for extracellular adenosine receptors

Jacobson

Ukena

Padgett

et al. 1987

Biochemical Pharmacology

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“…A FORTRAN-77 program was written to simulate the plateau counts per minute values of an experiment based on a given dissociation constant and concentration of binding sites. The 100% free rate, dissociation constant, and concentration of binding sites were then adjusted by a descending simplex algorithm (23) (Sigma) were labeled at their reducing end via an exchange reaction using tritium gas and palladium (5%) on BaSO4 (Aldrich) (9). Specifically, 0.5 ml of 0.24 M sugar solution in 0.1 M Na .…”

Section: Methodsmentioning

confidence: 99%

Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli

et al. 1991

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We have directly measured the stoichiometry of maltodextrin-binding sites in LamB. Scatchard plots and computer fitting of flow dialysis (rate-of-dialysis) experiments clearly establish three independent binding sites per LamB trimer, with a dissociation constant of approximately 60 ,uM for maltoheptaose. The current model for LamB's function as a specific pore is discussed with respect to the symmetry in LamB's kinetic properties and the implications of our results.LamB (also called maltoporin) is a trimeric outer membrane protein that functions as a pore for the passage of maltose and maltodextrins [linear a(1-4)-linked D-glucose polymers] across the outer membrane of Escherichia coli (10,24,25,27). A variety of experiments have shown that LamB acts as a passive pore with a binding site for its substrates (2,3,7,8,13,18,19). Binding of maltodextrins inhibits pore activity and has allowed measurements of LamB's binding affinity for maltose and longer maltodextrins. In whole cells, LamB has been shown to act as a saturable pore for certain chromogenic substrate analogs with a measurable Km and Vmax (14).The number of channels (and maltodextrin-binding sites) per LamB trimer has widely been presumed to be either one or three. This is partially based on images of negatively stained two-dimensional crystals of LamB that show three stain-filled channels merging into one (17). Genetic studies of dominance between mutant LamB proteins have suggested that each LamB monomer forms a single independent pore (11). Here, we confirm the results of this genetic study and show that maltodextrin binding to LamB can be detected in solution and the stoichiometry of binding can be measured. MATERIALS AND METHODSFlow dialysis. Flow dialysis (rate of dialysis) was carried out as described by Colowick and Womack (6), using a homemade dialysis apparatus consisting of two chambers separated by a dialysis membrane. The upper chamber was loaded with 1 ml of concentrated, purified LamB (between 11 and 35 mg/ml) in a solution containing 50 mM Tris * Cl, 100 mM NaCl, 5 mM EDTA, 3 mM NaN3, 10 mM MgCl2, and 0.5% sodium dodecyl sulfate (SDS), pH 7.5. To start the experiment, -50 ,uCi of high-specific-activity [1-3H]maltodextrin (2.5 to 8 Ci/mmol) was added to the upper chamber.The bottom chamber (volume, -100 RI) was constantly flushed with buffer at 1.1 ml/min, and fractions were collected every minute. Both upper and lower chambers were constantly stirred. The radioactivity in the fractions was determined by liquid scintillation counting and used as a measure of the free [1-3H]maltodextrin concentration. Unlabeled maltodextrin was added at 5-min intervals to the * Corresponding author. The results of the flow dialysis experiments were analyzed by two methods. The first, Scatchard plot analysis, required estimating the rate of [1-3H]maltodextrin elution in the absence of LamB binding sites (the "100% free" rate). In general, at the highest plateau (lowest specific activity), most of the [1-3H]maltodextrin is blocked from the LamB binding site...

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“…Donors were tested for the presence of anti-Candida antibody in serum by electrosyneresis and/or by hemagglutination (19), and only seropositive donors were considered as sensitized. Some adult donors were immunised by pneumococcal Sll antigen, subcutaneously at a dose of 50 gg in 1 ml of saline, [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30] (Sigma Chemical Co.) at a final concentration of 1 mg/ml in carbonate buffer was added to each well. Plates were incubated for 1 h at 370C.…”

Section: Methodsmentioning

confidence: 99%

Specific in vitro antimannan-rich antigen of Candida albicans antibody production by sensitized human blood lymphocytes.

Durandy¹,

Fischer²,

Griscelli³

1983

J. Clin. Invest.

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A B S T R A C T We have developed a new antigenic system for the induction of specific in vitro antibody response in man. The antigen used was purified from the cell wall of Candida albicans strain A and contained >96% polysaccharide mannan. Peripheral blood mononuclear cells from Candida-sensitized donors produced specific antimannan antibodies during a 7-d culture in the presence of mannan absorbed with methylated bovine serum albumin. Two methods were used to detect antimannan antibody responses. Antimannan antibody-producing cells were identified by radioautography with tritiated mannan. Antibody concentration in culture supernatants was measured by an enzyme-linked immunosorbent assay. In both methods, specific IgM and IgG (but not IgA) antibodies were detected. The antibody production to mannan was specific, since an antigenically unrelated polysaccharide (pneumococcal antigen S III) did not bind to methylated bovine serum albumin-mannan-induced blast cells and did not induce antimannan antibodycontaining cells. Furthermore, a pulse with an excess of unlabeled mannan abolished [3H]mannan binding, whereas an excess of unlabeled S III did not. Similarly, no antimannan antibody was obtained in influenza virus-stimulated cultures and mannan-stimulated cultures were not inducing antiinfluenza antibodies. The antimannan antibody production was shown to be a T cell-dependent phenomenon. The T helper effect appeared to be radiosensitive. It was under a genetic restriction as it occurred only in autologous or semiidentical but not in allogeneic situations. This system is relatively simple, reproducible, and well suited for

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A new approach to specific labelling of organic compounds with tritium: Catalysed exchange in solution with tritium gas

Cited by 109 publications

References 18 publications

Molecular probes for extracellular adenosine receptors

Molecular probes for extracellular adenosine receptors

Stoichiometry of maltodextrin-binding sites in LamB, an outer membrane protein from Escherichia coli

Specific in vitro antimannan-rich antigen of Candida albicans antibody production by sensitized human blood lymphocytes.

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