Linker-mediated modulation of the chemiluminescent signal from N(10)-(3-Sulfopropyl)-N-sulfonylacridinium-9-carboxamide tracers.

Four chemiluminescent N-sulfonylacridinium-9-carboxamide active esters (17-20) were prepared from the corresponding acids and coupled to both of the aminated phenobarbital (13) and N-(6-aminohexyl)phenytoin (16) haptens. The level of signal produced by chemiluminescent N-sulfonylacridinium-9-carboxamide phenobarbital and phenytoin tracers in a solid-phase immunoassay format was found to be modulated by at least 20-fold by the judicious choice of the reactive acridinium-hapten linking group.

Resin-supported labeling reagents.

Resin-supported fluorescein, coumarin, acridinium, and biotin active esters were prepared from a new N-hydroxysuccinimidyl resin in high yield. The active esters were used to prepare representative conjugates with estriol, thyroxine, phenytoin, and desipramine haptens without need for purification beyond removal of the spent resin.

Tracermer signal generators: an arborescent approach to the incorporation of multiple chemiluminescent labels.

The synthesis, conjugation, and chemiluminescent evaluation of zero, first, and second order acridinium-based Tracermer signal generators are described. Members of this family of labels have potential use as tracers in diagnostic assays and are structurally similar to arborol dendrimers. Tracermer-BSA conjugates showed up to a sixfold increase in light emission compared to the normal acridinium label.

Preparation of succinimidyl and pentafluorophenyl active esters of 5- and 6-carboxyfluorescein.

A mixture of 5- and 6-carboxyfluorescein was activated with 1-[3-(dimethylamino)propyl]-3-ethylcarbodiimide hydrochloride in the presence of either N-hydroxysuccinimide or pentafluorophenol to give the corresponding succinimidyl and pentafluorophenyl esters. The regioisomeric mixtures were separated to give the 5- and 6-succinimidyl and pentafluorophenyl active esters in > 98% purity.

Quantitative determination of E- and Z-doxepin and E- and Z-desmethyldoxepin by high-performance liquid chromatography.

A high-performance liquid chromatographic (HPLC) method was developed for the quantitative, simultaneous determination of the following four compounds in serum: E-doxepin, Z-doxepin, E-desmethyldoxepin, and Z-desmethyldoxepin. A 3-microns analytical silica column (6 x 100 mm) was employed with the mobile phase 0.025 M phosphate:acetonitrile:n-nonylamine (80:20:1). This HPLC method allows for the accurate measurement of all four isomeric compounds.

Immunoassay reagents for psychoactive drugs. Part 5. Quantitative determination of imipramine and desipramine by fluorescence polarization immunoassay.

Two methods for the quantitative determination of imipramine (IMI) and desipramine (DMI) by fluorescence polarization immunoassay (FPIA) are described. One immunoassay allows for the accurate quantification of imipramine in the presence of desipramine, while the other allows for the accurate quantification of desipramine in the presence of imipramine.

Characterization of protein-hapten conjugates. 1. Matrix-assisted laser desorption ionization mass spectrometry of immuno BSA-hapten conjugates and comparison with other characterization methods.

Several different low molecular weight haptens were conjugated to BSA to produce immunogens useful for antibody development. The extent of BSA modification due to covalent attachment of hapten was estimated by matrix-assisted laser desorption ionization mass spectrometry. The average number of hapten incorporated to immunogen was determined from the difference in the measured molecular weights of the conjugate from nonmodified BSA. The results from mass spectrometry were compared with results obtained from other more traditional methods of immunogen characterization (UV analysis, trinitrobenzenesulfonic acid titrations, and gel electrophoresis). In each case we were able to calculate the average number of hapten covalently bound to BSA for each synthetically prepared immunogen using matrix-assisted laser desorption ionization mass spectrometry. The other methods presented limitations in certain cases.

Immunoassay reagents for thyroid testing. 1. Synthesis of thyroxine conjugates.

Immunoreagents were designed to improve the performance of a commercial fluorescent polarization immunoassay for thyroxine. The thyroxine immunogen was prepared by selective coupling of N-acetyl-L-thyroxine to BSA via an aminocaproic acid spacer arm. The fluorescent tracer was prepared by a multistep reaction sequence which relied on extensive use of orthogonal protecting groups.

Immunoassay reagents for psychoactive drugs. Part 4. Quantitative determination of amitriptyline and nortriptyline by fluorescence polarization immunoassay.

Methods for the quantitative determination of amitriptyline and nortriptyline by fluorescence polarization immunoassay (FPIA) is described. One immunoassay allows for the accurate quantification of amitriptyline in the presence of nortriptyline while the second immunoassay allows for the accurate quantification of nortriptyline in the presence of amitriptyline.

Immunoassay reagents for psychoactive drugs. Part 3. Removal of phenothiazine interferences in the quantification of tricyclic antidepressants.

Phenothiazines and their metabolites are known to interfere in the quantification of tricyclic antidepressants (TCAs). A method for selective chemical modification of phenothiazines by chloramine-T in the presence of TCAs is described. This method allows for accurate quantification of the TCA analyte in a serum sample without interference from the modified phenothiazine.

Immunoassay reagents for psychoactive drugs. II. The method for the development of antibodies specific to imipramine and desipramine.

Imipramine and desipramine were structurally modified by the attachment of spacer arms to the aromatic ring which were subsequently attached to bovine serum albumin. This approach utilized novel spacer arms and conjugation methods. This method yielded antisera with excellent selectivity and good titers. Rabbits were used to raise the antisera and the antibodies produced were characterized with respect to their cross-reactivity with imipramine, desipramine and hydroxy metabolites as well as selected structurally related compounds.

Immunoassay reagents for psychoactive drugs. I. The method for the development of antibodies specific to amitriptyline and nortriptyline.

Amitriptyline and nortriptyline were structurally modified by the attachment of spacer arms to the aromatic ring which were subsequently attached to bovine serum albumin (BSA). Rabbits inoculated with these conjugates yielded polyclonal antisera with high selectivity and good titers. This approach required novel spacer arms and new conjugation methods. The antisera produced were characterized with respect to their cross-reactivity with amitriptyline, nortriptyline and their hydroxy metabolites as well as selected structurally related compounds.

Structure of (+/-)-(1R*,6S*)-1-benzyloxy-8,11,11-trimethyl-6-phenylthiobicyclo [5.3.1 ]undec-7-en-3-one.

C27H32O2S, Mr = 420.61, monoclinic, P2(1)/c, a = 8.4756(13), b = 14.710(2), c = 18.425(3) A, beta = 99.575(13) degrees, V = 2265.2(6) A3, Z = 4, Dx = 1.23 g cm-3, mu = 1.5567 cm-1, Mo K alpha, lambda = 0.7107 A, F(000) = 904, T = 298 K, R = 0.0630 for 2942 reflections [Fo greater than or equal to 6 sigma (Fo)]. The cyclooctane portion of the [5.3.1] ring system assumes the boat-chair conformation while the cyclohexene portion has the boat conformation. As has been observed in other [5.3.1]undecene systems, ring strain appears to cause a distortion of the geometry of the bridgehead alkene. The bond length C7--C8 [1.343(5) A] is long for an isolated C--C double bond. The Csp2--Csp3 bond lengths at C7 are asymmetric with C7--C6 being 1.498(5) A while C7--C11 is 1.535(5) A. The maximum deviation from ideality for the torsion angles around the double bond is 16.2(4) degrees (absolute value). This twist in the alkene group is reflected in the non-planarity of the group [max. deviation 0.193(6) A for C9] and in the dihedral angle between the nearly planar portions (C6, C7, C8, C11 and C7, C8, C9, C15), which is 9.4(2) degrees.

Structure of (+/-)-(1S*,3S*)-3-hydroxy-8,11,11-trimethyl-9-oxobicyclo[5.3.1]undec-7- en-1-yl benzoate.

C21H26O4, Mr = 342.43, monoclinic, P2(1)/n, a = 7.211 (2), b = 19.521 (6), c = 12.674 (3) A, beta = 95.04 (2) degrees, V = 1777.2 (8) A3, Z = 4, Dx = 1.28 g cm-3, Mo K alpha radiation, lambda = 0.7107 A, mu = 0.8152 cm-1, F(000) = 736, T = 163 K, R = 0.0632 for 3754 reflections [Fo greater than or equal to 4 sigma(Fo)]. The molecules exist as hydrogen-bonded dimers [O14-H14...O16 (related by -x, 1-y, 1-z), O14...O16 2.775 (2), H14...O16 1.93 (2) A, O14-H14...O16 175 (2) degrees] which stack in columns along b. Distortion at the bridgehead double bond is observed. Deviations from ideal values for the torsion angles around the double bond, C7-C8, are as large as 17.8 (2) degrees. The dihedral angle between planes through C6-C7-C8-C11 and C7-C8-C9-C15, the planar portions of the alkene moiety, is 13.4 (9) degrees. The Csp2--Csp3 bond lengths involving C7 are asymmetric with C7-C6 1.501 (2) and C7-C11 1.538 (2) A. The bond length C7-C8 is elongated [1.352 (3) A] for a C-C double bond. The enone system is also non-planar [max. deviation -0.404 (2) A for C10].

Structure of (+/-)-(1R*,4S*,6S*)-1-benzyloxy-4,8,11,11-tetramethyl-6- phenylthio-bicyclo[5.3.1]undec-7-en-3-one.

C28H34O2S, Mr = 434.64, triclinic, P1, a = 8.9868 (7), b = 11.2933 (11), c = 12.3497 (6) A, alpha = 80.937 (6), beta = 73.108 (5), gamma = 87.405 (7) degrees, V = 1184.32 (15) A3, Z = 2, Dx = 1.22 g cm-3, mu = 1.509 cm-1, Mo K alpha radiation, lambda = 0.7107 A, F(000) = 468, T = 298 K, R = 0.0583 for 6924 reflections [Fo greater than or equal to 4 sigma(Fo)]. The cyclooctane portion of the [5.3.1] ring system is in the boat-chair conformation while the cyclohexene portion assumes the boat conformation. The carbonyl group and the alkene group are nearly parallel in the molecule with a dihedral angle of 14.9 (1) degree between planes through the two groups. Ring strain appears to cause distortion in the alkene functionality. The bond length [C7-C8 1.343 (2) A] is long for an isolated C-C double bond. The torsion angles deviate between 3.7 (2) and 18.39 (14) degrees (absolute values) from ideality. This twist in the alkene group is reflected in the non-planarity of the group [max. deviation -0.177 (2) A for C9] and in the dihedral angle between the nearly planar portions (C6,C7,C8,C11 and C7,C8,C9,C16) which is 12.1 (1) degrees.