The use of oligonucleotides modified with aliphatic amino groups continues to grow as immobilization to surfaces becomes even more important than labelling with tags. The selection of commercially available 3'-amino-modifiers has been dominated by two products1,2 containing branched linkers in which the amino group is protected with the fluorenylmethoxycarbonyl (Fmoc) group. These are supplied by Glen Research and are the products 3'-Amino-Modifier C3 CPG (1) and 3'-Amino-Modifier C7 CPG (2), as shown in Figure 1. Since the Fmoc group is base-labile, it offers some advantages but also disadvantages.
An amino support without an Fmoc group has been described by Lyttle and coworkers3 and is now commercially available (Biosearch Technologies, Inc.). Under normal circumstances, an aliphatic amide cannot be simply hydrolyzed under conditions appropriate for oligonucleotide deprotection. The novel support (3) contains an amide group which is hydrolyzed to give the aliphatic amine because of the participation in the hydrolysis of the neighboring carboxylic acid group. This approach does require extended cleavage conditions (ammonium hydroxide at 55°C for 17 hours) but the deprotection of the bases is obviously achieved simultaneously.
Another interesting approach was described in 19924 and has remained commercially dormant since then. In this approach, the nitrogen destined to become the 3'-amino group is included in a phthalimide (PT) group which is attached to the support through an amide group attached to the aromatic ring. The structure of the support (4) is shown in Figure 1. This simple linkage is very stable to all conditions of oligonucleotide synthesis and contains no chiral center. Again, using an extended ammonium hydroxide treatment (55°C for 17 hours), the cleavage of the amine from the phthalimide is accomplished along with the deprotection of the oligonucleotide.
A comparison of the yields of crude oligonucleotides produced with 3'-Amino-Modifier C7 CPG (2) and 3'-PT-Amino-Modifier C6 CPG is shown in Table 1 and the HPLC purity of the oligonucleotides is shown in Figure 2 and Figure 3. The results indicate that the yield of product from the 3'-PT-Amino-Modifier C6 CPG is about 20% lower if deprotected with ammonium hydroxide. However, the purity of amino-modified product is significantly higher due to the absence of the acetyl capped product.
|Support||Synthesis Scale||Oligo Length||Yield (A260 Units)|
|3'-Amino-Modifiπer C7 CPG||1 µmole||20mer||119|
|3'-PT Amino-Modifier C6 CPG||1 µmole||20mer||91|
3'-Amino-Modifier C3 CPG has been discontinued.
3'-Amino-Modifier C7 CPG 500 has been discontinued.