A novel and versatile phosphoramidite, N-Fmoc-O1-DMT-O2-cyanoethoxydiisopropylamino-phosphinyl-3-am ino-1,2-propanediol (1, Fig. 1), has been synthesized and used to incorporate primary aliphatic amines into synthetic oligonucleotides. Its convenient preparation and use in solid phase oligonucleotide synthesis is described. Using phosphoramidite 1, an amino-modified oligonucleotide probe complementary to M13mp18 DNA was constructed with five primary amines attached to the 5'-terminus. The amino-modified oligonucleotide was subsequently labeled with biotin and employed in a dot-blot hybridization assay. As little as 0.5 ng of M13mp18 target DNA was colorimetrically detected.

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Diethylpyrocarbonate (DEP), an amino acid modifying reagent, causes complete inactivation of particulate and deoxycholate-solubilized benzodiazepine-receptors from calf cerebral cortex. No heterogeneity was observed in DEP-sensitivity of the receptors. Protection from DEP-induced inactivation was provided by the centrally active benzodiazepines, diazepam and nitrazepam, but not by the peripherally active Ro5-4864, suggesting that DEP modifies a residue which is essential for the central actions of benzodiazepines. GABA did not protect against inactivation or influence the protection afforded by diazepam, indicating that the DEP-modifiable residue is independent of GABA binding sites, or that GABA binding sites are also sensitive to DEP. DEP-induced inactivation of benzodiazepine-receptors proceeds much faster at pH 10.1 than at pH 8.1 or 6.0, indicating the modification of a high pKa side group, possibly the phenol of a tyrosyl residue. This postulation is in accord with our previous findings with the modifying reagents tetranitromethane and N-acetylimidazole.

Antibodies specific for benzodiazepines were raised in rabbits by immunization with a conjugate of a benzodiazepine derivative, Ro 7-1986/1, with bovine serum albumin. The presence of anti-Ro 7-1986/1 antibodies in the sera was demonstrated by a radioimmunoassay using the radioligand [3H]flunitrazepam ([3h]FNZ). The antibodies displayed a high-affinity for [3H]FNZ (KD = 0.073 +/- 0.003 nM) and and cross-reacted with a broad spectrum of benzodiazepine derivatives. Benzodiazepine levels in samples of sera and urine of benzodiazepine-treated humans were determined. Due to the high sensitivity of the assay only minute volumes (microliter quantities) of body fluids are employed and, therefore, no extraction of the drugs is required. Nitrazepam and diazepam levels as low as 20 picograms can be easily observed. Intoxicating levels of benzodiazepines can be detected by a single measurement in less than 10 min. This radioimmunoassay is advantageous for pharmacokinetic studies, toxicological examinations and forensic medicine due to its high sensitivity, wide-range specificity and technical simplicity.