title: A tetramethyl rhodamine (Tamra) phosphoramidite facilitates solid-phase-supported synthesis of 5'-Tamra DNA.
abstract: 5-Carboxy Tamra 1 was conjugated to 4-hydroxypiperidine with BOP and N-methylmorpholine, and the resulting 5-(N-pipyridyl-4-hydroxy)-Tamra carboxamide 2 was treated with 2-cyanoethyl tetraisopropylphosphorodiamidite to give 5-[N-pipyridyl-4-O-(2-cyanoethyl diisopropylphosphoramidite)]-Tamra carboxamide 3. Solutions of 3 were coupled onto the 5'-hydroxyl of solid-phase-supported DNA fragments with standard amidite coupling techniques. Cleavage and deprotection with aqueous tert-butylamine cocktail gave 5-Tamra-functionalized DNA as well as an additional compound without the Tamra chromophore. A mass spectrum of this product showed the incorporation of tert-butylamine. The extra product was completely suppressed by including a 5 min acetylation step after coupling. A model study of 3 coupled onto thymidine-functionalized CPG showed similar results. NMR and mass spectra of cleaved products confirmed the addition of tert-butylamine to the minor product. Coupling a Tamra active ester onto T CPG which was previously coupled with N-(4-methoxytrityl)piperidyl-4-O-(2-cyanoethyl diisopropylphosphoramidite) 4 produced the same major Tamra-bearing product, which coeluted on reverse phase HPLC with the major product generated with 3.
authors.affiliation: Biosearch Technologies, Inc., 81 Digital Drive, Novato, California 94949, USA. matt@biosearchtech.com
authors.forename: M H
authors.initials: MH
authors.lastname: Lyttle
authors.forename: T G
authors.initials: TG
authors.lastname: Carter
authors.forename: D J
authors.initials: DJ
authors.lastname: Dick
authors.forename: R M
authors.initials: RM
authors.lastname: Cook
mesh_terms: D002851:Chromatography, High Pressure Liquid; D004247:DNA / Q000737:chemistry*; D005452:Fluoresceins / Q000737:chemistry; D005456:Fluorescent Dyes / Q000737:chemistry*; D007202:Indicators and Reagents; D009682:Magnetic Resonance Spectroscopy; D013058:Mass Spectrometry; D009841:Oligonucleotides / Q000138:chemical synthesis*; D010880:Piperidines / Q000138:chemical synthesis*; D012235:Rhodamines / Q000138:chemical synthesis* / Q000737:chemistry*
chemical_list: C420543:5-(N-pipyridyl-4-O-(2-cyanoethyl diisopropylphosphoramidite))-tetramethyl rhodamine carboxamide; D005452:Fluoresceins; D005456:Fluorescent Dyes; D007202:Indicators and Reagents; D009841:Oligonucleotides; D010880:Piperidines; D012235:Rhodamines; C005358:tetramethylrhodamine; D004247:DNA