Lyttle M H, Carter T G, Dick D J, Cook R M
Biosearch Technologies, Inc., 81 Digital Drive, Novato, California 94949, USA.
J Org Chem. 2000 Dec 29;65(26):9033-8. doi: 10.1021/jo0011134.
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.
5-羧基四甲基罗丹明1通过BOP和N-甲基吗啉与4-羟基哌啶共轭，所得的5-(N-哌啶基-4-羟基)-四甲基罗丹明甲酰胺2用2-氰基乙基四异丙基磷二酰胺处理，得到5-[N-哌啶基-4-O-(2-氰基乙基二异丙基亚磷酰胺)]-四甲基罗丹明甲酰胺3。用标准亚磷酰胺偶联技术将3的溶液偶联到固相支持的DNA片段的5'-羟基上。用叔丁胺水溶液混合物进行切割和脱保护，得到5-四甲基罗丹明功能化的DNA以及一种不含四甲基罗丹明发色团的额外化合物。该产物的质谱显示了叔丁胺的掺入。通过在偶联后加入一个5分钟的乙酰化步骤，额外的产物被完全抑制。将3偶联到胸腺嘧啶功能化的CPG上的模型研究显示了类似的结果。切割产物的NMR和质谱证实了叔丁胺添加到次要产物中。将四甲基罗丹明活性酯偶联到先前与N-(4-甲氧基三苯甲基)哌啶基-4-O-(2-氰基乙基二异丙基亚磷酰胺)4偶联的T CPG上，产生了相同的主要含四甲基罗丹明产物，该产物在反相HPLC上与用3产生的主要产物共洗脱。
