Synthesis of 5- and 6-hydroxymethylfluorescein phosphoramidites.
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Base Sequence↗
Biomedical subjects
P G Mattingly
Publications and source records attributed to P G Mattingly.
Evidence of nucleophilic addition to chemiluminescent N-sulfonylacridinium-9-carboxamides from electrospray ionization mass spectrometry.
Addition of nucleophiles to the chemiluminescent acridinium-9-carboxamide 1 resulted in the formation of the acridan adduct 7. The relative ratio of 1/7 present in solution could be determined by electrospray ionization mass spectrometry (ESI-MS) for a number of nucleophiles that are commonly employed during the use of 1 as a label in medical diagnostic assays.
Acridines↗
Evaluation of chemiluminescent estradiol conjugates by using a surface plasmon resonance detector.
A series of chemiluminescent 17beta-estradiol probes were synthesized. Relative equilibrium dissociation constants (K(D)) for the interaction of an anti-E(2) Fab fragment for the probes in solution were evaluated using a single E(2)-analog biosensor surface on a BIAcore surface plasmon resonance instrument. The results show the antibody fragment binds all chemiluminescent conjugates tested with high affinity showing only minor preferences for site of substitution (C6 versus C7), stereochemistry (alpha versus beta), or linker moiety.
Antibodies, Monoclonal↗
O-(Acridinium)hydroxylamine (AHA): a reagent for the preparation of chemiluminescent acridinium oxime (AO)-steroid conjugates.
O-(Acridinium)hydroxylamine (AHA) reacted with a representative sample of oxo-steroids (6-oxoestradiol, estrone, norethindrone, cortisol, progesterone, digoxin dialdehyde, and digitoxin dialdehyde) to produce chemiluminescent acridinium oxime (AO) conjugates in a single step in 37-68% yield after preparative HPLC.
Acridines↗
Synthesis of a chemiluminescent acridinium hydroxylamine (AHA) for the direct detection of abasic sites in DNA.
[structure: see text] The synthesis of a chemiluminescent acridinium hydroxylamine (AHA) for the direct detection of abasic sites in damaged nucleic acids is described. The reagent reacts readily with abasic sites of damaged calf thymus DNA generated in a time-dependent manner under acid/heat depurination conditions. Preliminary results indicate the sensitivity of the direct chemiluminescent detection format is approximately 0.1 abasic sites detected per 10(6) nucleotides using as little as 200 ng of DNA.
Acridines↗
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.
Acridines↗
O-(fluoresceinylmethyl)hydroxylamine (OFMHA): a reagent for the preparation of fluorescent O-(fluoresceinylmethyl)oxime (FMO)-steroid conjugates.
The 5 and 6-isomers of O-(fluoresceinylmethyl)hydroxylamine reacted with a representative sample of oxo-steroids (6-oxoestradiol, estrone, norethindrone, cortisol, progesterone, and digitoxin-dialdehyde) to produce O-(fluoresceinylmethyl)oxime conjugates in a single step in 24-84% yield after preparative high performance liquid chromotography.
Chromatography, High Pressure Liquid↗
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.
Acridines↗
O-(fluoresceinylmethyl)hydroxylamine (OFMHA): a fluorescent reagent for detection of damaged nucleic acids.
5- and 6-O-(Fluoresceinylmethyl)hydroxylamine (OFMHA, 5a, b) were prepared from the corresponding bis-pivaloyl-protected hydroxymethylfluoresceins (1a, b) in 50-70% yield. The hydroxylamine derivatives reacted smoothly with the abasic sites present in acid/heat stressed calf thymus DNA.
Animals↗
Estradiol-mimetic probes. Preparation of 17 alpha-(6-aminohexynyl)estradiol biotin, fluorescein and acridinium conjugates.
3-O-tert-Butyldimethylsilyl-17 alpha-(6-mesyloxyhexynyl)estradiol was converted to the azide in 60-70% yield with NaN3/DMPU, then reduced to the corresponding amine (> 95% yield). Acylation with the N-hydroxysuccinimide esters of biotin, 5-carboxyfluorescein and 10-(3-sulfopropyl)-N-tosyl-N-(3- carboxypropyl)acridinium-9-carboxamide gave the title conjugates. The KDs of the tracers with an estradiol antibody ranged from 97-197 nM.
Acridines↗
Synthesis of 6 beta-aminoestradiol and its biotin, acridinium, and fluorescein conjugates.
Amination of 3,17 beta-Bis[(2-trimethylsilylethoxy) methoxy]-1,3,5(10)-estratriene-6-one (2) using NaCNBH4 and NH4OAc afforded 3,17-bis(SEM)-6-aminoestradiol (3) as a mixture of alpha and beta-isomers in 60:40 ratio. Hydrolysis of the mixture of 3 using HF and separation by preparative high-performance liquid chromatography afforded pure 6 beta-aminoestradiol (4) in good yield. The relative stereochemistry of the amino group in 4 was established by NMR. The biotinylated estradiol probe (7), chemiluminescent probe (9), and fluorescent probe (11), were prepared from 6 beta-aminoestradiol (4) and the corresponding biotin, 10-(3-sulfopropyl)-N-tosyl-N-(3-carboxypropyl)acridinium-9-carboxamide, and 5-carboxyfluorescein N-succinimidyl esters in 43-63% yields and > 99% purity.
Acridines↗
An efficient stereoselective synthesis of 6-alpha-aminoestradiol: preparation of estradiol fluorescent probes.
6-Oxoestradiol (2) was protected as its bis[(2-trimethylsilylethoxy)methyl] ether (4) and converted to the corresponding oxime (4). The oxime (4) on reduction with zinc in ethanol afforded the bis-SEM ether of 6-alpha-aminoestradiol (5) in 96% epimeric excess. Subsequently, 5 was hydrolyzed with HF to 6-alpha-aminoestradiol (6) in good yield. The absolute stereochemistry of the amino group in 6 was established by NMR and confirmed by X-ray crystallography on the corresponding 4-bromobenzamide derivative (9). Treatment of amine (6) with 6-(t-butoxycarbonylamino)hexanoic acid succinimidyl ester (10) followed by hydrolysis produced the amine (12) with a C-6 linker. The fluorescent probes (7 and 13) were prepared from 6 and 12 respectively, in 54-60% yield and > 99% purity.
Crystallography, X-Ray↗
Digoxin dialdehyde reductive aminations. Structure proof of the perhydro-1,4-oxazepine product.
Digoxin dialdehyde reportedly undergoes reductive amination with primary amines to form a perhydro-1,4-oxazepine; however, no structural proof has been published to substantiate this belief. A digoxin perhydro-1,4-oxazepine derivative has been isolated from the reductive amination of digoxin dialdehyde and its structure determined by mass spectroscopy and NMR measurements. Comparison of the NMR, mass spectroscopy, and HPLC of two compounds obtained from the degradation of the digoxin reductive amination product with synthesized perhydro-1,4-oxazepine diastereomers showed them to be identical. We conclude that, under appropriate conditions, reductive amination products can be obtained, but caution that other products may be produced as well, especially under the conditions of bioconjugation to proteins.
Amines↗
Chemiluminescent 10-methyl-acridinium-9-(N-sulphonylcarboxamide) salts. Synthesis and kinetics of light emission.
10-Methyl-acridinium-9-(N-sulphonylcarboxamide) salts are prepared via acylation of sulphonamides with acridine-9-carboxylic acid chloride and subsequent N-alkylation with methyl triflate. Substituents on the sulphonamide component were varied to show the effect of steric and electronic factors on the kinetics of light output. The lifetime of the chemiluminescence light output ranged from 1 to 50 seconds for the 15 compounds reported. The long-term stability of the new compounds was superior to the phenyl ester counterparts.
Acridines↗
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.
Acridines↗
Design of acridinium-9-carboxamides and anti-acridinium antibodies for chemiluminescent signal enhancement.
A novel system of signal enhancement is presented in which every labeled antibody is capable of generating a signal. Three chemiluminescent acridinium-9-carboxamide haptens (1, 2, and 3) which incorporated differences in charge and location of the linker were designed and synthesized. Anti-acridinium polyclonal antibodies for each hapten were screened using surface plasmon resonance instrumentation to determine specificity for each hapten. Anti-acridinium 2 antibodies were found to be non-cross-reactive to acridinium 1. This property was exploited to design secondary antibody conjugates which would bind to primary antibodies labeled with 2 yet could still be labeled with the structurally similar acridinium 1. Consequently, both layers contributed to the overall chemiluminescent signal. This format is an advance over other signal amplification formats which employ non-signal-generating, labeled antibodies to construct multilayered systems.
Acridines↗
Preparation of 5- and 6-(aminomethyl)fluorescein.
5(6)-Carboxyfluorescein is protected as the diacetate then reduced to 5(6)-(hydroxymethyl)fluorescein diacetate. The separated isomers are subjected to a Mitsunobu reaction with dibenzyl imidodicarbonate, yielding diprotected 5- and 6-(aminomethyl)fluorescein diacetate. Methanolysis of the acetates followed by deprotection with HBr/acetic acid gives 5- and 6-(aminomethyl)fluorescein hydrobromide.
Acetic Anhydrides↗
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.
Chromatography, High Pressure Liquid↗