The 3'-terminus of a synthetic oligonucleotide has proved to be attractive for chemical modification experiments. In a previous Glen Research Report, we described the use of a protected ribonucleoside support for use as a precursor for the introduction of a 3'-phosphate and for the attachment of carrier molecules, e.g., poly-lysine. To these processes for modification of the 3'-terminus, we now add a new technique for 3'-phosphorylation and a new product, 3'-Amino-Modifier C3-CPG, for use in the attachment of biotin or a fluorescent label.
Glen Research CPR-1 has proved to HPLC. be fast and convenient for chemical phosphorylation of the 5'-terminus of oligonucleotides. In addition, this reagent has proved its utility for simple phosphorylation of the 3'-terminus. It is introduced as the first addition to any nucleoside support, followed by normal synthesis of the target oligonucleotide. After the standard ammonia deprotection, the linkage decomposes and is B-eliminated from the target molecule, leaving a phosphate group at the 3'-terminus.
Glen Research offers a variety of reagents for amino-modification of the 5'-terminus and we now introduce a product for amino-modification of the 3'-terminus. 3'-Amino-Modifier C3- CPG is used in the same way as normal nucleoside CPG for oligonucleotide synthesis. After deprotection, the product oligonucleotide contains a primary amine with a 3 carbon spacer at the 3'-terminus. This product is therefore useful for the introduction of a label at the 3'-terminus; a process which had previously been difficult to achieve. An interesting benefit of this approach is that during the development of, for example, a diagnostic probe, the 5'-terminus can also be labelled with 32P to provide an additional highly sensitive marker.
n alternative to the use of the 5'-Amino-Modifier C6, in which the primary amine is protected with the acid-labile MMT group, is the use of the analogue with the primary amine protected with the base-labile trifluoroacetyl (TFA) group. While it is desirable in most cases to use the MMT group to purify the intermediate amino-modified oligonucleotide by a RP technique, it may be occasionally attractive to use the TFA-analogue in which the protecting group is removed during the ammonia deprotection step. Using the latter strategy, the crude intermediate is reacted with biotin or a fluorescent label to give a product which must be purified by RP HPLC.