S a bonus, the removal of the DMT group can also be performed on the cartridge, allowing full length, DMT-OFF product to be isolated without any additional downstream steps. While the DMT group is an excellent hydrophobic handle for purification on cartridges, in theory, any modification of equivalent or greater hydrophobicity should be able to serve the same purpose. In this article, we aim to assess what other 5’hydrophobic modifications may be compatible with the Glen-Pak purification process. The
retention times (RT) observed during RP-HPLC analysis offer good estimates of hydrophobicity for SPE cartridge purification purposes. Figures 1 and 2 show the retention times of many 5′-labeled T6 sequences, relative to a 5′-DMT-T6 standard. This represents a starting point to determine which modifications are hydrophobic enough to consider purifying with cartridges. RP-HPLC was performed with a gradient of acetonitrile (ACN) in 0.1 M triethylammonium acetate (TEAA). Figure 1 data was generated with a gradient of 3-40 % ACN over 20 minutes while Figure 2 data was generated with a gradient of 3-60 % ACN also over 20 minutes.
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Looking at the data, the observed retention times support what we already know. Our User Guide to Glen-PakTM Purification provides detailed purification methods that cover, among others, the 5′-modifications of DMS(O)MT-Amino-Modifier C6, 5′-Amino-Modifier C6, Cyanine 5, and Dabcyl.1 The RT’s for these 5′- modifications are either later than or comparable to that of the DMT-T6.520-18-3 Formula For instance, Cyanine 5 (Figure 3) has an RT that is slightly later than that of the DMT-T6, and the 5′-Cyanine oligonucleotide is indeed compatible with the Glen-Pak purification method.59-67-6 IUPAC Name On the other end of the spectrum, the 5′-Fluorescein, TET, and HEX labels, which we know do not work with this purification process,1 have much earlier RTs compared to that of the DMT-T6.PMID:29262012 These fluorescein type dyes are simply not very hydrophobic, and our recommendation has always been to use Poly-PakTM II Cartridges instead, where a somewhat different reverse phase purification is carried out.2 We can now apply this knowledge to other modifications. Based on the RT data and the relevant hydrophobicity to the DMT-T6, we anticipate that the rhodamine dyes AquaPhluor593 and 639 (AP593/ AP639) should work fine with Glen-Pak cartridges (Figure 1). Quenchers (BHQ1, BHQ-2, Eclipse, and BBQ-650) also appear to be compatible (Figure 2). With a relatively late RT, Palmitate should work as well. Furthermore, DBCO- and Yakima Yellow-labeled oligonucleotides are less hydrophobic and are probably more challenging for Glen-Pak purification. RPHPLC purification may be a safer/better alternative for these two modifications. Finally, the JOE modification (Figure 3) yielded a RT consistent with the rest of the fluorescein family and will not be compatible with Glen-Pak cartridges. Although this is not a comprehensive list of all the labels we offer, we believe the major groups are covered, and the compatibility of other groups can be deduced accordingly. For example, if the Palmitate (Figure 3) is compatible with Glen-Pak purification, other fatty groups like Stearyl and Cholesterol should also work. Likewise, if AP593 (Figure
3) is compatible, then fellow rhodamine TAMRA is likely to work as well. As noted earlier, cartridge purification is a crude but convenient purification method. Even if the purification handles are sufficiently hydrophobic, performance can still vary based o.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
