Glen Report 31.25: Technical Brief – Universal Support III PS Cleavage and Dephosphorylation

Since 2001, Glen Research has been offering a version of Universal Support III PS (USIII) as a matrix option for oligonucleotide synthesis. Due to the mild and anhydrous conditions that may be used for oligonucleotide cleavage and phosphate elimination, this product has proven to be a truly universal support and an excellent complement to UnySupport products. In particular, USIII is well-suited for the synthesis of RNA. 

Figure 1

Figure 1. Universal Support III


Oligos synthesized on USIII are typically cleaved and deprotected in two separate steps. First, oligos are released from the support in 2M anhydrous ammonia in methanol at room temperature for 20-60 mins. Subsequently, deprotection is completed based on the protecting groups of the nucleobases and the sensitivity of any modifications. A detailed protocol can be found at: https://www.glenresearch.com/media/productattach/import/tbn/TB_Universal_Support_III.pdf.

Over the years, a number of customers have inquired whether the first step could be skipped or whether alternative procedures were possible. The reason a separate cleavage condition is necessary is because the cleavage reaction for the universal linker requires 1) an amide to form a weak intramolecular hydrogen bond and 2) the presence of the cyanoethyl group of the first phosphoramidite addition. Any environment that interferes with the former or accelerates the removal of the latter will affect the yield. As such, preliminary elimination of cyanoethyl deprotecting groups using hindered bases such as diethylamine or DBU will prevent cleavage and result in no yield.

 

To confirm how critical our recommended two-step procedure is, we evaluated four alternative methods of cleavage/dephosphorylation. The results are summarized in Table 1. In short, anhydrous conditions will give better yields than aqueous conditions. The choice is yours: a faster, single-step cleavage and deprotection reaction, or higher final yields with a two-step cleavage and deprotection reaction. For those performing UltraMild syntheses, a third option would be to cleave and deprotect using 50 mM potassium carbonate in methanol at room temperature for 4 hr (UltraMild Cap Mix A) or overnight (regular capping).

Reagent Time (min) Temp (⁰C) Relative yield (%)

AMA*

15

RT

51

AMA

10

65

57

EMAM

10

65

58

8 M MeNH2/EtOH*

15

RT

85

2M NH3/MeOH*

60

RT

100

Table 1. Cleavage and dephosphorylation of USIII. Five 20 nt oligos were synthesized, and yield was quantified by OD measurement at A260 following deprotection. *Suspensions were filtered to remove support matrix, and deprotection was continued by adding AMA and heat (65 ⁰C, 10 min). AMA, ammonium hydroxide/aqueous methylamine 1:1; EMAM, ethanolic methylamine/aqueous methylamine 1:1; RT, room temperature.


Product Information

Universal Support III PS (26-5010)