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PubMed1993

Inhibition of the red blood cell calcium pump by eosin and other fluorescein analogues.

This paper addresses the mechanism of inhibition of the plasma membrane Ca pump by fluorescein analogues and their isothiocyanate derivatives. Eosin (i.e., tetrabromofluorescein) was found to be one of the most potent reversible inhibitors of the erythrocyte Ca pump [half-maximal inhibitory concentration (IC50) < 0.2 microM]; fluorescein itself was about four orders of magnitude less potent (IC50 approximately 1,000 microM). Eosin decreased the maximum influx and thus did not compete with ATP for the Ca pump. Irreversible inhibition produced by the isothiocyanate analogues of eosin and fluorescein [eosin 5-isothiocyanate (EITC) and fluorescein 5-isothiocyanate (FITC), respectively] was also studied. While EITC bound reversibly at the eosin site, two results suggest that EITC does not react covalently at this site: 1) eosin did not alter the time course of the EITC irreversible reaction, and 2) the concentration dependence for reversible EITC inhibition was different from the concentration dependence for irreversible EITC inhibition. ATP did slow the rate of inactivation of both EITC and FITC consistent with the idea that EITC and FITC bind to the ATP site. Our results are consistent with eosin and ATP binding to separate sites and EITC reacting covalently at the ATP site, but not the eosin site.

Adenosine Triphosphate↗
PubMed1991

Comparison of epidermal growth factor (EGF) receptor-receptor interactions in intact A431 cells and isolated plasma membranes. Large scale receptor micro-aggregation is not detected during EGF-stimulated early events.

We have used resonance energy transfer to monitor epidermal growth factor (EGF) receptor micro-aggregation at the surface of intact human epidermoid carcinoma (A431) cells. EGF molecules labeled with fluorescein isothiocyanate and eosin isothiocyanate were demonstrated to bind tightly to cellsurface receptors, to elicit immediate changes in cytosolic free [Ca2+], and to undergo endocytosis. Under conditions which maintain the integrity of the cell, we observed no energy transfer between the donor fluorescein isothiocyanate-labeled EGF molecules and the acceptor eosin isothiocyanate-labeled growth factors bound to receptors. However, after disruption of cells by Dounce homogenization, a significant degree of energy transfer was observed (approximately 10-20%) with membranes, indicative of receptor aggregation. These results suggest that EGF does not cause micro-aggregation of the majority of its receptors on the surface of intact A431 cells within the time period of the early events associated with growth factor action. Moreover, it appears that the A431 cells contain some component which imparts a constraint on the ability of EGF receptors to aggregate, and that some of this component is lost upon the disruption of cells.

Calcium↗
PubMed1995

Welwitindolinone analogues that reverse P-glycoprotein-mediated multiple drug resistance.

Welwitindolinones are a family of novel alkaloids recently isolated from the blue-green alga Hapalosiphon welwitschii as a part of our effort to identify new compounds that overcome multiple drug resistance. The abilities of three structurally similar members of this family to interact with P-glycoprotein have been compared. Similarly to the effects of verapamil, N-methylwelwitindolinone C isothiocyanate (compound 1) attenuated the resistance of MCF-7/ADR cells to natural product anticancer drugs, including vinblastine, taxol, actinomycin D, daunomycin, and colchicine, without affecting the cytotoxicity of cisplatin. These effects of compound 1 were apparent at doses as low as 0.1 microM, indicating that it is considerably more potent than verapamil for reversal of resistance. Welwitindolinone C isothiocyanate (compound 3) demonstrated weaker reversing activity, whereas an analogue of compound 1 in which the isothiocyanate group is replaced by an isonitrile group (compound 2) was inactive. The accumulation of [3H]vinblastine in SK-VLB-1 cells was increased by compound 1 > compound 3 > verapamil >> compound 2. Interestingly, only compound 1 and verapamil enhanced [3H]taxol accumulation by these cells. Photoaffinity labeling of P-glycoprotein with [3H]azidopine in membranes from SK-VLB-1 cells was inhibited by compounds 1 and 3, but not by compound 2. Therefore, the differences in the size and/or the electronegativity of the isothiocyanate and isonitrile moieties appear to dramatically affect the abilities of the compounds to interact with P-glycoprotein.

ATP Binding Cassette Transporter, Subfamily B, Mem↗
PubMed1990

19F NMR study of the myosin and tropomyosin binding sites on actin.

Actin was labeled with pentafluorophenyl isothiocyanate at Lys-61. The label was sufficiently small not to affect the rate or extent of actin polymerization unlike the much larger fluorescein 5-isothiocyanate which completely inhibits actin polymerization [Burtnick, L. D. (1984) Biochim. Biophys. Acta 791, 57-62]. Furthermore, the label resonances in the 376.3-MHz 19F NMR spectrum were unaffected by actin polymerization. However, the binding of the relaxing protein tropomyosin resulted in the fluorinated Lys-61 resonances broadening out beyond detection due to a substantial increase in the effective correlation time of the label. Similarly, the binding of myosin subfragment 1 to F-actin resulted in the dramatic broadening of the labeled Lys-61 resonances. Thus, Lys-61 on actin appears to be closely associated with the binding sites for both tropomyosin and myosin, suggesting that both these proteins can compete for the same site on actin. The other region of actin known to be involved in myosin binding, Cys-10, was found to be more remote from the actin-actin interfaces than Lys-61. Labels on Cys-10 exhibited substantially greater mobility than fluorescein 5-isothiocyanate attached to Lys-61 which appeared to be held down on the surface of the actin monomer. This may sterically hinder the actin-actin interaction about 1 nm from the tropomyosin/myosin binding site.

Actins↗
PubMed1982

Rapid, one step staining procedures for analysis of cellular DNA and protein by single and dual laser flow cytometry.

Detailed, simplified techniques are described for simultaneous staining and analysis of DNA and protein in a number of mammalian cell types. Cell staining involves the addition of appropriate dye mixtures to unfixed or ethanol-fixed cells and subsequent analysis of cell populations in the staining reagents generally within 10 to 20 minutes. The approach is novel in that no centrifugation steps are involved during the staining procedure, thus, eliminating cell clumping and cell loss and making the procedures appropriate for samples containing limited numbers of cells. For single wavelength analysis, staining of DNA and protein in ethanol-fixed cells was accomplished with a dye solution containing propidium iodide, fluorescein isothiocyanate and RNase. After 20 minutes at room temperature cells were analyzed using the 488 nanometer (nm) laser excitation line. For dual laser analysis the following dye combinations were employed without RNase: mithramycin-rhodamine 640, mithramycin-substituted rhodamine isothiocyanate, Hoechst 33342-rhodamine 640 and Hoechst 33342-rhodamine isothiocyanate. Unfixed cells were also stained with the Hoechst 33342-rhodamine 640 dye combination. Mithramycin was excited at 457.9 nm, Hoechst 33342 at 333-363 nm, and the rhodamine dyes at 568 nm. Cell types analyzed included Chinese hamster ovary cells, cultured mouse colon 26 cells, mouse embryo forelimb bud cells, and rat cell samples obtained by lung lavage.

Animals↗
PubMed1995

Acute and long-term effects of nine chemicals on the Japanese medaka (Oryzias latipes).

Ninety-six-hour acute and 28-day larval survival and growth tests were conducted with nine organic chemicals, using the Japanese medaka (Oryzias latipes) as the test organism. The nine tested chemicals were allyl isothiocyanate, aniline, benzyl acetate, 4-chloroaniline, 2-chloroethanol, 2,4-diaminotoluene, 1,2-dibromoethane, 2,4-dichlorophenoxyacetic acid (2,4-D), and phenol. The derived 96-h LC50 values for medaka for all chemicals ranged from 0.077 mg/L for allyl isothiocyanate to 2,780 mg/L for 2,4-D. The chronic values for six of the nine chemicals tested ranged from 0.013 mg/L for allyl isothiocyanate to 42.5 mg/L for 2,4-D. Acute-to-chronic ratios for these six chemicals ranged from 1.4 for 2-chloroethanol to 70.9 for 2,4-D. Growth of medaka was significantly reduced in the lowest exposure concentration during 28-dy larval tests with aniline, 4-chloroaniline, and 2,4-diaminotoluene. The estimated maximum acceptable toxicant concentration was reported as less than the lowest exposure concentration of 4.6, 2.2 and 40.3 mg/L for tests with aniline, 4-chloroaniline and 2,4-diaminotoluene, respectively. Chronic values for 2-chloroethanol and medaka were 12.6 mg/L during an embryo-larval test and 22.1 mg/L during the 28-day larval test.

2,4-Dichlorophenoxyacetic Acid↗
PubMedDate not supplied

Is cysteine residue important in FITC-sensitive ATP-binding site of P-type ATPases? A commentary to the state of the art.

Treatment of P-type ATPases (from mammalian sources) by fluorescein isothiocyanate (ITC) revealed the ITC label on a lysine residue that was than considered as essential for binding of ATP in the ATP-binding site of these enzymes. On the other hand, experiments with site directed mutagenesis excluded the presence of an essential Iysine residue that would be localized in the ATP binding sites of ATPases. Other previous studies, including those of ourselves, indicated that the primary site of isothiocyanate interaction may be the sulfhydryl group of a cysteine residue and this may be essential for binding of ATP. In addition considerable knowledge accumulated since yet also about the differences in stability of reaction product of isothiocyanates with SH- or NH2- groups. Based upon evaluation of the data available up to now, in present paper the following tentative roles for lysine and cysteine residues located in the ATP-binding site of P-type ATPases are proposed: The positively charged micro-domain of the lysine residue may probably attract the negatively charged phosphate moiety of the ATP molecule whereas the cysteine residue may probably be responsible for recognition and binding of ATP by creation of a proton bridge with the amino group in position 6 on the adenosine ring of ATP.

Adenosine Triphosphatases↗
PubMedDate not supplied

Inhibition of (Na/K)-ATPase by electrophilic substances: functional implications.

The effect of electrophilic substances: p-bromophenylisothiocyanate (PBITC); fluoresceinisothiocyanate (FITC); [4-isothiocyanatophenyl-(6-thioureidohexyl)-carbamoylmethyl] -ATP (ATPITC); 2,4,6-trinitrobezenesulfonic acid (TNBS); 1-(5-nitro-2-furyl)-2-phenylsulfonyl-2-furylcarbonyl ethylene (FE1); 1-(5-phenylsulfonyl-2-furyl)-2-phenylsulfonyl-2-furylcarb onyl ethylene (FE2) and 1-(5-phenylsulfonyl-2-furyl)-2-phenylsulfonyl-2-tienocarb onyl ethylene (FE3) on the sarcolemmal (Na/K)-ATPase isolated from guinea-pig hearts was studied. FITC and PBITC were found to inhibit competitively the activation of (Na/K)-ATPase by ATP. Being for the enzyme inhibitor and substrate at the same time ATPITC does not offered clear kinetic behavior. However, the activation of (Na/K)-ATPase by sodium and potassium ions was inhibited non-competitively by all three isothiocyanates. These data indicated that isothiocyanates may interact predominantly in the ATP-binding site of the enzyme molecule. In contrary to isothiocyanates TNBS and FE1 (FE2 and FE3 were ineffective) inhibited the activation of (Na/K)-ATPase by ATP non-competitively i.e., their interaction in the ATP-binding site seemed to be improbable. Nevertheless, TNBS and FE1 both manifested affinities to that moiety of (Na/K)-ATPase molecule which is binding potassium. More specific was the effect of FE1 that showed clearly competitive inhibition of potassium-stimulation of the enzyme activity. FE1 exerted also an ouabain-like effect on the mechanical activity of isolated perfused guinea-pig heart. This result indicates that FE1 seems to exert a selective inhibition of the (Na/K)-ATPase not only in vitro but also in integrated cardiac tissue.

Adenosine Triphosphate↗
PubMed1992

Synthesis and pharmacology of irreversible affinity labels as potential cocaine antagonists: aryl 1,4-dialkylpiperazines related to GBR-12783.

As part of a program aimed at designing irreversible antagonists of the stimulant and reinforcing properties of cocaine, derivatives of GBR-12783 containing electrophilic substituents were synthesized. GBR-12783, a potent and selective inhibitor of both stimulant binding and dopamine transport, was modified to incorporate either isothiocyanate or maleimido groups at the meta- or para-positions in one phenyl ring of the geminal diphenyl portion of the molecule. The effect of these compounds, as well as their respective amino- or nitro-substituted precursors, on stimulant binding to rat striatal tissue was studied using the [3H]methylphenidate radioreceptor assay. Under the assay conditions used, the compounds were found to have IC50s (nM) ranging from 11.9 (m-nitro) to 1677 (p-maleimido); the parent compound, GBR-12783, had an IC50 of 12.0. Using a washout technique (repeated washing with 100 mM KCl) which completely removed the tightly bound, but reversible GBR-12783, both the m- and p-isothiocyanate compounds were found to irreversibly inhibit binding of [3H]methylphenidate to the stimulant recognition site. The m-maleimido derivative also irreversibly inhibited binding, albeit with lower efficacy than was observed with the isothiocyanate compounds. Neither the p-maleimido, nor the amino or nitro intermediates, were capable of irreversible inhibition.

Animals↗
PubMed1984

Group-directed modification of bacteriorhodopsin by arylisothiocyanates. Labeling, identification of the binding site and topology.

Group-directed hydrophobic modification of membrane-integrated protein segments by arylisothiocyanates is applied to bacteriorhodopsin. Labeling of purple membrane with phenylisothiocyanate and 4-N,N'-dimethylamino-azobenzene-4'-isothiocyanate results in covalent modification of a unique lysine epsilon-amino group of bacteriorhodopsin. Lysine residue 41, located in the amino-terminal chymotryptic fragment, has been identified as the arylisothiocyanate binding site by established sequencing techniques. The phenylisothiocyanate binding site is not accessible for the aqueously soluble analog p-sulfophenylisothiocyanate. Furthermore, the acid-induced bathochromic shift of the bound chromophore reagent is not observed following acidification of 4-N,N'-dimethylamino-azobenzene-4'-isothiocyanate-labeled purple membrane. The modification thus occurs in the hydrophobic membrane domain, providing further evidence for intramembraneous disposition of the modified protein segment. Light-induced proton translocation is preserved in reconstituted vesicles containing either phenylisothiocyanate-modified or 4-N,N'-dimethylamino-azobenzene-4'-isothiocyanate-modified bacteriorhodopsin.

Amino Acid Sequence↗
PubMed1992

High affinity acylating antagonists for muscarinic receptors.

The muscarinic antagonists pirenzepine and telenzepine were derivatized as alkylamino derivatives at a site on the molecules corresponding to a region of bulk tolerance in receptor binding. The distal primary amino groups were coupled to the cross-linking reagent meta-phenylene diisothiocyanate, resulting in two isothiocyanate derivatives that were found to inhibit muscarinic receptors irreversibly and in a dose-dependent fashion. Preincubation of rat forebrain membranes with an isothiocyanate derivative followed by radioligand binding using [3H]N-methylscopolamine diminished the Bmax value, but did not affect the Kd value. The receptor binding site was not restored upon repeated washing, indicating that irreversible inhibition had occurred. IC50 values for the irreversible inhibition at rat forebrain muscarinic receptors were 0.15 nM and 0.19 nM, for derivatives of pirenzepine and telenzepine, respectively. The isothiocyanate derivative of pirenzepine was non-selective as an irreversible muscarinic inhibitor, and the corresponding derivative prepared from telenzepine was 5-fold selective for forebrain (mainly m1) vs. heart (m2) muscarinic receptors.

Acylation↗
PubMed1995

Crotavirin, a potent platelet aggregation inhibitor purified from the venom of the snake Crotalus viridis.

A potent platelet aggregation inhibitor in the venom of Crotalus viridis snake was purified to homogeneity by gel filtration chromatography and reverse phase high-performance liquid chromatography. This purified principle, named crotavirin, is a single-chain polypeptide with a mol. wt of 9200 as estimated by SDS-polyacrylamide gel electrophoresis. It inhibited the aggregation of human washed platelets induced by collagen, thrombin and thomboxane analogue (U46619) with a similar IC50 (approximately 1.0 micrograms/ml, 0.11 microM). The binding of fluorescein isothiocyanate-conjugated crotavirin to platelets was abolished in the presence of divalent cation chelator, EDTA, indicating that divalent cation is essential for crotavirin's binding. A monoclonal antibody, 7E3, raised against platelet glycoprotein IIb-IIIa complex blocked the binding of fluorescein isothiocyanate-conjugated crotavirin to platelets, whereas the other monoclonal antibody against glycoprotein IIb-IIIa, 10E5, had no inhibitory effect. In addition, crotavirin inhibited in a concentration-dependent manner the binding of fluorescein isothiocyanate-conjugated rhodostomin, a member of the disintegrin family, to platelets. Its binding to platelets was blocked by disintegrins, e.g. trigramin and rhodostomin. It is concluded that crotavirin is a potent platelet aggregation inhibitor, which acts specifically on an epitope of glycoprotein IIb-IIIa, leading to the blockade of fibrinogen binding to glycoprotein IIb-IIIa and eventually the blockade of platelet aggregation.

Animals↗
PubMed1998

Gene transfer to vein graft wall by HVJ-liposome method: time course and localization of gene expression.

BACKGROUND: A novel gene transfer method using liposomes with a viral envelope of hemagglutinating virus of Japan (HVJ) has been reported to be very effective for gene transfection into somatic cells and might be applicable to improve the patency of vein grafts. The present study examined the time course and localization of gene expression to assess the feasibility of ex vivo gene transfer into the vein graft by the HVJ-liposome method. METHODS: The HVJ-liposome complex containing either beta-galactosidase plasmid DNA (deoxyribonucleic acid) or no genes (controls) (experiment 1) or fluorescein isothiocyanate-labeled oligonucleotides either with or without HVJ-liposomes (experiment 2) was infused into rabbit vein grafts and allowed to incubate before autologous transplantation to carotid arteries. RESULTS: In experiment 1, all grafts incubated with beta-galactosidase plasmid with HVJ-liposomes showed the blue staining of X-gal 7 days after operation, whereas the controls did not. The blue granules were present in the medial and adventitial tissue and were still present after 14 days. In experiment 2, many fluorescein isothiocyanate-labeled nuclei were observed in the graft wall 2 and 4 days after operation and remained present mainly in the media of HVJ-liposome-treated grafts after 7 and 14 days, when no fluorescein isothiocyanate activity was observed without HVJ-liposome treatment. CONCLUSIONS: These results demonstrated the feasibility of ex vivo transfection to the medial and adventitial tissue of the vein graft by the HVJ-liposome method and suggest the possibility of its clinical application to prevent vein graft failure.

Animals↗
PubMed2000

Localization of N-methyl-D-aspartate NR2B subunits on primary sensory neurons that give rise to small-caliber sciatic nerve fibers in rats.

In the present study we have used immunohistochemical staining and retrograde tracing techniques to investigate the relationship between the N-methyl-D-aspartate receptor NR2B subunits and small-diameter primary afferent dorsal root ganglion neurons that give rise to the sciatic nerve fibers. Three days after an intra-sciatic nerve injection of tetramethyl rhodamine isothiocyanate-conjugated wheat germ agglutinin which labels small-diameter primary afferents, many NR2B and wheat germ agglutinin-double-labeled cells ( approximately 70% of wheat germ agglutinin-labeled neurons) were observed in the L5 dorsal root ganglia. Three days after an intra-sciatic nerve injection of fluorescein isothiocyanate-conjugated Bandeiraea simplicifolia agglutinin isolectin B4 which labels predominantly non-peptidergic C-fiber primary afferents, NR2B and Bandeiraea simplicifolia agglutinin isolectin B4 double-labeled neurons ( approximately 90% of Bandeiraea simplicifolia agglutinin isolectin B4-labeled neurons) were also observed in the L5 dorsal root ganglion. Three days after an intra-sciatic nerve injection of fluorescein isothiocyanate-conjugated cholera toxin B subunit, only approximately 40% of cholera toxin B subunit-labeled neurons were NR2B positive and those labeled neurons tended to be small-sized. When calcitonin gene-related peptide and NR2B were labeled by a double immunofluorescent staining technique, we found that the majority of calcitonin gene-related peptide-positive neurons was NR2B immunoreactive (>90% of calcitonin gene-related peptide-positive neurons, and approximately 60% of NR2B-positive neurons) as well. Size frequency analysis also demonstrated that NR2B subunits were predominantly localized on the small and medium-sized neurons. These results suggest that NR2B subunits are predominantly expressed on small diameter primary afferents, and these NR2B containing N-methyl-D-aspartate receptors may play a role in the modulation of neurotransmitter release from primary afferent terminals.

Animals↗
PubMed1984

High-performance liquid chromatographic resolution of enantiomers of 1-phenyl-2-aminopropanes (amphetamines) with four chiral reagents.

High-performance liquid chromatography (HPLC) was employed for resolution of enantiomers of chiral ring-substituted 1-phenyl-2- aminopropanes (amphetamines) and 1-phenylethylamine following derivatization with four chiral reagents: (R)-(+)-1-phenylethyl isocyanate ( PEIC ), (-)-alpha-methoxy-alpha-(trifluoromethyl)phenylacetyl chloride ( MTPA X Cl), 2,3,4,6-tetra-O-acetyl-beta-D-glucopyranosyl isothiocyanate ( GITC ), and 2,3,4-tri-O-acetyl-alpha-D- arabinopyranosyl isothiocyanate ( AITC ). Reactions were accomplished under mild conditions (25-70 degrees C) and were complete for all substrates within 60 min. Derivatization with the sugar isothiocyanates ( GITC and AITC ) and the acyl chloride ( MTPA . Cl) was carried out in methylene chloride or acetonitrile in the presence of a base catalyst while derivatization with the isocyanate ( PEIC ) was performed in methylene chloride. The diastereomeric derivatives were separated by reversed-phase HPLC (C18) with a methanol-water mobile phase. In general, HPLC resolution of the diastereomeric reaction products of GITC or AITC , and MTPA .Cl with the amine substrates was more complete (usually greater than 98% baseline separation) than HPLC resolution of the diastereomeric reaction products of PEIC .

Amphetamines↗
PubMed1996

Micellar electrokinetic chromatography separations and analyses of biological samples on a cyclic planar microstructure.

Micellar electrokinetic capillary chromatography (MECC) separations and analyses of biological samples on a planar glass microchip capillary electrophoresis device with laser-induced fluorescence solute detection are discussed. A cyclic channel system which permits dead volume free repeated column switching and thus the use of various channel lengths together with a relatively low applied separation voltage is described. It features an unbiased, dead volume free electrokinetic sample inlet system of approximately 12 pL. Because of the small cross section and favorable heat dissipation in glass microstructures, MECC separations with an electric field strength of up to 2000 V/cm achieving efficiencies of submicrometer plate heights can be performed. After a separation length of 2 cm, six fluorescein isothiocyanate labeled amino acids are shown to be separable within a few seconds and with an imprecision for peak areas (or heights) and detection times of < 2% and < 0.5%, respectively. Without application of electrokinetic solute stacking, the detection limit of fluorescein isothiocyanate labeled arginine is 3.3 nM, corresponding to approximately 40 zmol injected. Furthermore, the feasibility of directly applying human urine and serum samples onto the uncoated channel system is demonstrated and first data of the successful performance of a chip-based MECC immunoassay for serum theophylline are presented. Compared to MECC in conventional fused-silica capillaries, MECC analyses on microchips can be performed 1-2 orders of magnitude faster, with higher efficiency and at no expense of accuracy and precision. Furthermore, versatility is shown to be much increased with the use of a cyclic rather than a single-path channel system. The MECC separation efficiency of fluorescein isothiocyanate labeled amino acids is shown to be comparable to that obtained by gel electrophoresis performed in the same chip layout.

Amino Acids↗
PubMedDate not supplied

Trifunctional agents as a design strategy for tailoring ligand properties: irreversible inhibitors of A1 adenosine receptors.

The 1,3-phenylene diisothiocyanate conjugate of XAC (8-[4-[[[[(2-aminoethyl)amino]carbonyl]methyl]- oxy]phenyl]-1,3-dipropylxanthine, a potent A1 selective adenosine antagonist) has been characterized as an irreversible inhibitor of A1 adenosine receptors. To further extend this work, a series of analogues were prepared containing a third substituent in the phenyl isothiocyanate ring, incorporated to modify the physiochemical or spectroscopic properties of the conjugate. Symmetrical trifunctional cross-linking reagents bearing two isothiocyanate groups were prepared as general intermediates for cross-linking functionalized congeners and receptors. Xanthine isothiocyanate derivatives containing hydrophilic, fluorescent, or reactive substituents, linked via an amide, thiourea, or methylene group in the 5-position, were synthesized and found to be irreversible inhibitors of A1 adenosine receptors. The effects of the 5-substituent on water solubility and on the A1/A2 selectivity ratio derived from binding assays in rat brain membranes were examined. Inhibition of binding of [3H]-N6-(2-phenylisopropyl)-adenosine and [3H] CGS21680 (2-[2-[4-carboxyethyl)phenyl]ethyl]amino] adenosine-5'-N-ethylcarboxamide) at central A1 and A2 adenosine receptors, respectively, was measured. A conjugate of XAC and 1,3,5-triisothiocyanatobenzene was 894-fold selective for A1 receptors. Reporter groups, such as fluorescent dyes and a spin-label, were included as chain substituents in the irreversible binding analogues, which were designed for spectroscopic assays, histochemical characterization, and biochemical characterization of the receptor protein.

Adenosine↗
PubMedDate not supplied

Functionalized tricarbocyanine dyes as near-infrared fluorescent probes for biomolecules.

The syntheses of three novel functionalized tricarbocyanine dyes are described. These dyes containing isothiocyanate and succinimidyl ester functional groups are reactive toward primary amines and can be used as fluorescent probes for biologically pertinent compounds such as amino acids and functionalized dideoxynucleotides. The absorption and fluorescence maxima occur in the near-IR region of the spectrum (770-810 nm). The succinimidyl ester proved to be very sensitive to hydrolysis and was generated in situ to label amino acids. The isothiocyanates were less susceptible to hydrolysis and were conjugated using organic modified [40% (v/v) acetonitrile] buffers to amino acids. A dye with an alkyl isothiocyanate moiety showed conjugation to amino-functionalized dideoxynucleotide triphosphates.

Amino Acids↗