Ed at the 5′ terminus. So, it was natural for us to apply the 6-FAM approach to our branched product line and we introduced 6-fluorescein phosphoramidite (5) and the equivalent 3′-(6-fluorescein) CPG (6). These two products offer the benefits of 6-FAM (simplicity, isomeric purity and minimal side reactions) along with the ability to purify derived oligos by Glen-PakTM DMT-ON purification. Many years ago, we also introduced 3′-(6-FAM) CPG (4), which is based on the
well used 1,2-diol backbone. We still offer this product and its popularity has never waned. However, as we have demonstrated in past newsletters, this type of backbone is susceptible to an elimination reaction during deprotection that leads to loss of label. M o r e r e c e n t l y, w e i n t r o d u c e d 6-fluorescein serinol phosphoramidite (7) and the equivalent 3′-6-fluorescein serinol CPG (8) based on our increasingly popular serinol-based 1,3-diol linkage. 3 These products offer equivalent performance levels to phosphoramidite (4) and CPG (5). EXPERIMENTAL DESIGN In order to take a close look at the performance of these eight fluorescein products, we have carried out a simple set of experiments designed to illustrate the strength and weakness of each product. In the case of the phosphoramidites, each was added to the 5′ terminus of a simple T6 oligo. Where apropriate, the final DMT group was removed and the crude oligo
FIGURE 2: HYDROLYSIS OF C=S
O O O O HN S NH ODMT O P O CNEt N(iPr)2 O O O
was analyzed by reverse phase HPLC.738606-46-7 manufacturer In the case of each support, a T6 oligonucleotide was synthesized followed by deprotection and DMT-ON purification using Glen-Pak cartridges. The DMT-ON purification was designed to remove any early failures so that we could focus entirely on the 3′ linkage to fluorescein. In all cases, the oligonucleotides were deprotected using ammonium hydroxide at 55 for 17 hours to closely mimic the deprotection of regular oligonucleotides.
RESULTS 5′ Terminus As expected, the 5′-fluorescein phosphoramidite (3) performed impeccably, yielding a product oligonucleotide, which was essentially 100% pure.95094-00-1 web The results for 6-Fluorescein, as shown in Figure 3a, and 6-Fluorescein Serinol Phosphoramidites were the same at close to 100% purity.PMID:31424785 However, the results for Fluorescein Phosphoramidite looked even worse than we had expected. The chromatogram showed several impurity
peaks but the UV/Visible spectrum of all components confirmed the presence of fluorescein, as shown in Figure 3b. 3′-Terminus Oligonucleotide synthesis with the label at the 3′ terminus is much more difficult than at the 5′ terminus. The label itself is challenged by all of the reagents of oligonucleotide synthesis in multiple cycles. Similarly, the linker is susceptible to side reactions. In our experiments, three of the supports, 3′-(6-Fluorescein), 3′-(6-FAM) and 3′-6-Fluorescein Serinol performed very well with only minor side reactions being observed at the linker. One surprise for us was that 3′-(6-FAM), attached by a 1,2-diol based linker and perfectly set up for an elimination reaction, did not lead to any appreciable loss of label in this experiment, as shown in Figure 3c. On the other hand, 3′-Fluorescein exhibited even more side reactions at the thiourea linker than expected, as shown in Figure 3d, but, again, all peaks contained fluorescein. In addition to the expected hydrolysis and ammonolysis side products illustrated in Figure 2, mass spectroscopic analysis showed there were also comp.MedChemExpress (MCE) offers a wide range of high-quality research chemicals and biochemicals (novel life-science reagents, reference compounds and natural compounds) for scientific use. We have professionally experienced and friendly staff to meet your needs. We are a competent and trustworthy partner for your research and scientific projects.Related websites: https://www.medchemexpress.com