We report the site-specific fluorescent labeling of DNA using Staudinger ligation with high efficiency and high selectivity. An oligonucleotide modified at its 5' end by an azido group was selectively reacted with 5-[(N-(3'-diphenylphosphinyl-4'-methoxycarbonyl)phenylcarbonyl)aminoacetamido]fluorescein (Fam) under aqueous conditions to produce a Fam-labeled oligonucleotide with a high yield (approximately 90%). The fluorescent oligonucleotide was characterized by matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS). Because of the relatively high yield of the Staudinger ligation, simple purification of the product by size-exclusion chromatography and desalting is sufficient for the resulting fluorescent oligonucleotide to be used as a primer in a Sanger dideoxy sequencing reaction to produce fluorescent DNA extension fragments, which are analyzed by a fluorescent electrophoresis DNA sequencer. The results indicate that the Staudinger ligation can be used successfully and site-specifically to prepare fluorescent oligonucleotides to produce DNA sequencing products, which are detected with single base resolution in a capillary electrophoresis DNA sequencer using laser-induced fluorescence detection.

Nitric oxide synthase (NOS) is a heme-containing monoxygenase that catalyzes the oxidation of l-arginine to l-citrulline and NO in two steps. In the second step of the NOS reaction, citrulline and NO are generated from the heme-catalyzed 3-electron oxidation of l-N-hydroxyarginine. To model this unusual reaction, iron porphyrin-catalyzed oxygenations of oximes with O2 were investigated. The oxidation of fluorenone oxime and a stoichiometric amount of hydroxoiron(III) porphyrin (Fe(OH)P, P = TMP and TPFPP) with O2 in benzene generated Fe(NO)P, fluorenone, and O-(9-nitro-9-fluorenyl)fluorenone oxime. The X-ray crystal structure of the oxime ether product suggests that it originated from the dimerization of the fluorenyl iminoxy radicals. Detailed analysis of this reaction showed that the oxime reacted first with Fe(OH)P to generate a 5-coordinate, high-spin oximatoiron(III) porphyrin species [Fe(oximate)P]. The X-ray crystal structure of oximatoiron(III) tetrakis(2,6-dichlorophenyl)porphyrin [Fe(oximate)TDCPP] showed that the oximate ligand was monodentate, O-bound to Fe(III)P. The aerobic oxidation of Fe(oximate)P followed the characteristic kinetics of a metalloporphyrin-catalyzed radical-type autoxidation. O2 surrogates, the π-acids NO and CO, induced the homolysis of Fe(oximate)P to generate Fe(NO)P or Fe(CO)P and the iminoxy radical, implicating a similar reaction mode for O2 with Fe(oximate)P. Fe(oximate)TMP reacted with 18O2 to generate predominantly 18O-labeled fluorenone (75% yield), while the reaction conducted under 16O2 and H2 18O generated only 16O-labeled fluorenone. This reaction is proposed to proceed via an Fe−O bond homolysis of Fe(oximate)TMP followed by O2 insertion to generate 9-nitroso-9-fluorenylperoxyFe(III)TMP, which decomposes via an O−O bond homolysis to generate NO, fluorenone, and oxoFe(IV)P. The implications of this system for the NOS reaction mechanism are discussed.

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