Glen Report 36-15: Application Note — Thermal-Aqueous Deprotection of 5′-Trityl Amino Modifiers

Trityl-protected amino modifiers offer an effective strategy for synthesizing amino-labeled oligonucleotides, owing to the versatility of the protecting group.¹ This trityl moiety can either be cleaved by the synthesizer to enable on-column conjugation or be retained as a purification aid for subsequent reverse-phase high-performance liquid chromatography (RP-HPLC) and Glen-Pak™ cartridges. Given these advantages, our assortment of trityl-protected amino modifiers enjoy widespread popularity among our customers.

Over the years, we have published several articles focusing on the utilization of trityl-protected amino modifiers,^1-5 and one of these articles addressed proper retention and removal of the trityl group as needed.⁴ Specifically, MonoMethoxyTrityl (MMT) amino modifiers traditionally necessitated treatment with 20% aqueous acetic acid for an hour at room temperature post-purification to eliminate the MMT moiety. However, recent advancements have introduced an acid-free method, detailed in a recent journal publication, which enables quantitative removal of MMT under neutral aqueous conditions at 60 °C for 60 minutes.⁶ This process involves heating the oligonucleotide to hydrolytically cleave the MMT-amine bond, resulting in the formation of MMT-OH. Although MMT removal can be reversible, the precipitation of MMT-OH, which is insoluble in aqueous conditions, drives the MMT removal to completion (refer to Figure 1). In contrast, the analogous MMT removal conducted in a 1:1 mixture of acetonitrile and water achieved only 55% completion. Furthermore, the authors demonstrated that phosphate buffer saline (PBS) (pH 7.4) and Tris-EDTA (TE) buffer (pH 8.0) did not hinder MMT removal, whereas concentrated ammonium hydroxide did. Notably, this methodology extends to DiMethoxyTrityl (DMT) groups attached to hydroxyl groups, albeit requiring a more rigorous condition of 95 °C for 90 minutes due to the increased difficulty of DMT removal.

Figure 1. Thermal aqueous deprotection 

We successfully validated this new protocol through the following experimental procedures:

• Synthesized 5′-Amino-Modifier C6-T₆.
• Deprotected the synthesized compound using ammonium hydroxide.
• Introduced 1x PBS.
• Evaporated the resulting solution to dryness.
• Resuspended the solid residue in water.
• Subjected the solution to heating at 60 °C for 60 minutes.

The oligonucleotide was analyzed by RP-HPLC post-deprotection, post-resuspension, and post-heating (Table 1). As anticipated, the trityl groups remained intact during the deprotection process. However, during evaporation and subsequent resuspension, approximately 14% of the MMT groups were removed. This result is consistent with the use of our evaporation apparatus, which uses heat to aid in the drying of the deprotected oligonucleotide. When the gaseous ammonia is depleted while water is still present, the trityl removal process gets a head start. Consequently, we recommend the addition of 45 mg of Tris base per mL of deprotection solution during the drying phase for such oligonucleotides to retain the trityl-protecting group.⁴ This quantity corresponds to an initial concentration of 0.37 M, a significant amount of Tris base. At this concentration, Tris base not only ensures that ammonium counterions do not trigger premature detritylation but also serves as a sufficiently basic agent to prevent thermal aqueous cleavage of the MMT groups.

Table 1. 5′-Trityl removal during deprotection and aqueous heating. Oligonucleotide quantities were determined by RP-HPLC. 

We extended the same experimental procedure to DMS(O)MT-NH-T₆ and DMT-T₆, yielding results consistent with those reported in the literature (refer to Table 1). Notably, DMS(O)MT exhibits even greater lability than MMT, suggesting the potential for lower temperatures or shorter incubation times during deprotection. For DMT-ON oligonucleotides, prolonged heating at 95 °C may not always be optimal depending on the oligonucleotide composition; however, it presents another viable option for purification and workup. While we didn't explore the application of our other MMT-protected amino modifiers, such as C12 and C5, we anticipate their behavior to mirror that of the C6 version.

As our experienced customers are aware, oligonucleotides are prone to depurination when exposed to acids, particularly in the absence of protective groups. Hence, the opportunity to circumvent acid treatment entirely in the final step is noteworthy, and we encourage users to explore this alternative approach.

Product Information

Amino-Modifier C6 (10-1906)
5′-DMS(O)MT-Amino-Modifier C6 (10-1907)