Optimized method for the synthesis and purification of adenosine--folic acid conjugates for use as transcription initiators in the preparation of modified RNA

Abstract

We present an optimized synthetic strategy for the attachment of molecules to 5'-adenosine monophosphate (AMP), which can then be used to label the 5'-end of RNA by T7 RNA polymerase mediated in vitro transcription. Through the use of a boronate affinity gel, we have developed an efficient route to the preparation of folate conjugated AMP with high yields and purity. Affi-Gel boronate is an affinity resin that selectively binds nucleoside and nucleoside derivatives at pH>7.5 and releases them at pH<6.5. This resin is used to efficiently bind and purify ribonucleotides such as AMP. This allows for the addition of a large excess of reactants and reagents in order to drive the reaction to completion and then allow easy purification without HPLC. The synthesis can be successfully scaled up to produce large quantities of AMP conjugates.

Figure 1

General two step method for the preparation of 5′ adenosine linker conjugates for use as initiators in the synthesis of RNA conjugates by in vitro transcription using T7 RNA polymerase and the φ 2.5 promoter.

Figure 2

Schematic description of the Affi-Gel boronate purification method. AMP conjugates are loaded onto the column at high pH (> 7.5) and binds to the boronate by the 5-membered ring structure shown. The purified products are released at low pH (< 6.5)

Figure 3

HPLC profile of reaction 2 (Table 1) indicating the identity of the major peaks as determined by LCMS (HPLC method B).

Figure 4

Time course for formation and hydrolysis of the key imidazolide intermediate. (A) HPLC profile after 3 h reaction shows ~ 60 % conversion to the imidazolide intermediate. (B) HPLC profile after 12 h reaction shows near complete hydrolysis to starting material. (HPLC method B)

Figure 5

HPLC profile (260 nm) for the optimized reactions of AMP with (A) TOTDDA linker (B) HDA linker. Conversion is almost quantitative and results in high yields after Affi-Gel boronate purification. Peak identities were determined by LCMS. (HPLC method A)

Figure 6

HPLC profile (260 nm) for the optimized reactions of folic acid with: (A) TOTDDA-AMP (B) HDA -AMP. Conversion is almost quantitative and results in high yields after Affi-Gel boronate purification. Peak identities were determined by LCMS. (HPLC method A)

Scheme 1

Preparation of Folate -5′ AMP conjugates via TOTDDA (3) and HDA (4) linkers.