A P P L I C AT I O N N O T E

2’-ACE RNAsynthesis
chemistry

DharmaconTM 2’-ACE RNA synthesis chemistry is based on anovel protecting group scheme1. A new class of silyl ethers
is used to protect the 5’-hydroxyl (5’-SIL) in combination
with an acid-labile orthoester protecting group on the
2’-hydroxyl (2’-ACE)2. This set of protecting groups is thenused with standard phosphoramidite solid-phase synthesis
technology3. The structures of the protected and functionalized
ribonucleoside phosphoramidites currently in use are as
illustrated in Figure 1.
RNA oligonucleotides are synthesized in a stepwise fashion using
the nucleotide addition reaction cycle illustrated in Figure 2.
Each nucleotide is added sequentially (3’- to 5’-direction) to asolid support-bound oligonucleotide. The first nucleoside atthe 3’-end of the chain is covalently attached to a solid support.
The nucleotide precursor, a ribonucleoside phosphoramidite,and activator are added (step i in Figure 2), coupling the secondbase onto the 5’-end of the first nucleoside. The support iswashed and any unreacted 5’-hydroxyl groups are cappedwith acetic anhydride to yield 5’-acetyl moieties (step ii).
The P(III) linkage is then oxidized to the more stable and ultimately
desired P(V) linkage (step iii). At the end of the nucleotide
addition cycle, the 5’-silyl group is cleaved with fluoride (step iv).
The cycle is repeated for each subsequent nucleotide.

2’-ACE RNA synthesis chemistry

The 2’-orthoester groups are the last protecting groups tobe removed. The structure of the 2’-ACE protected RNA
immediately prior to 2’-deprotection is as represented
in Figure 3.

Figure 1: Protected RNA nucleoside phosphoramidites for
Dharmacon 2’-ACE RNA synthesis chemistry.

Figure 3: Structure of 2’-ACE protected RNAimmediately prior to2’-deprotection

The ethylene glycol monoacetate orthoester group (Figure 1)
has the following innovative properties. It is stable to theconditions of nucleoside phosphoramidite synthesis andoligonucleotide synthesis. However, after oligonucleotide
synthesis the oligonucleotide is treated with methylamine
which not only cleaves the oligo from the solid support
but also removes the acetyl groups from the orthoesters.
The resulting 2-ethyl-hydroxyl substituents on the orthoester
are less electron withdrawing than the acetylated precursor.
As a result, the modified orthoester becomes more labile toacid-catalyzed hydrolysis (refer to mechanism in Figure 4).
Specifically, the rate of cleavage is approximately 10 times
faster after the acetyl groups are removed. Therefore,
this orthoester possesses sufficient stability in order tobe compatible with oligonucleotide synthesis and yet,when subsequently modified, permits deprotection toFigure 2: Outline of the RNA synthesis cycle.

be carried out under relatively mild aqueous conditions
compatible with the final RNA product.

Following synthesis, the methyl protecting groups on

The hydrophilic character of the 2’-protecting groups

the phosphates are cleaved in 30 minutes utilizing 1 M

predicts that 2’-protected RNA of any length or sequence

disodium-2-carbamoyl-2-cyanoethylene-1, 1-dithiolate

will be readily soluble in water. Base composition andtrihydrate (S2Na2) in DMF . The deprotection solution islength have been found to have no effect on the solubility

washed from the solid support bound oligonucleotide usingof the 2’-protected RNA oligos. To date, all lengths andwater. The support is then treated with 40% methylaminesequences synthesized with 2’-ACE chemistry havein water for 10 minutes at 55 °C5. This releases the RNA

been water soluble. This enables the routine handling ofoligonucleotides into solution, deprotects the exocyclic

2’-protected RNA in water. When ready to remove theamines and modifies the 2’-ACE groups. The oligonucleotides

2’-protecting groups, the orthoesters are readily hydrolyzed

can be analyzed by anion exchange HPLC at this stage.

under acid catalysis by the mechanism in Figure 46.

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22’-ACE RNA synthesis chemistry

As the 2’-protecting groups are very hydrophilic and readilysolvated by water, acid-catalyzed hydrolysis proceeds

References

to completion regardless of sequence or length. 5’-SIL-2’

1. S.A. Scaringe, Ph.D. Thesis, University of Colorado, 1996.

ACE oligonucleotide chemistry is a definitive advance in(b) S.A. Scaringe, M.H. Caruthers, “Silyl Ether Protection ofRNA synthesis technology. Nucleoside coupling yields

the 5’-Hydroxyl during Solid Phase Oligonucleotide

are comparable to DNA and are routinely < 60 seconds.

Synthesis,” in preparation. (c) S.A. Scaringe,

The final acid deprotection is mild, fast and amenable

M. H. Caruthers, “5’-O-Silyl Ethers in Conjunction withto subsequent use with minimal handling. Analysis andAcid-labile 2’-O-orthoesters for the Solid Phase Synthesis

purification via PAGE or HPLC are possible with anyof RNA.” in preparation.

sequence while in the stable 2’-protected form. This property
minimizes opportunities to degrade the RNA, while also
making it possible to analyze troublesome sequences withstrong secondary structure. 5’-SIL-2’-ACE chemistry has
enabled the routine synthesis of RNA oligonucleotides ofunprecedented quality.

2. S.A. Scaringe, F.E. Wincott, and M.H. Caruthers, Novel RNA
Synthesis Method Using 5’-Silyl-2’-Orthoester Protecting
Groups. J. Am. Chem. Soc., 120, 11820-11821 (1998).
3. (a) M.D. Matteucci, M.H. Caruthers, J. Am. Chem. Soc.
103, 3185-3191 (1981). (b) S.L. Beaucage, M.H. Caruthers,Tetrahedron Lett. 22, 1859-1862 (1981).
4. B.J. Dahl, K. Bjergarde, et al., Acta Chem. Scand.
44, 639-641 (1990).
5. (a) M.P. Reddy, N.B. Hanna, and F. Farooqui, Tetrahedron
Lett. 25, 4311-4314 (1994). (b) F. Wincott, A. DiRenzo, et al.,Nucleic Acids Res. 23, 2677-2684 (1995).
6. (a) B.E. Griffin, M. Jarman, et al., Tetrahedron Lett. 23,2301-2313 (1967). (b) B.E. Griffin, M. Jarman, et al.,Tetrahedron Lett. 23, 2315-2331 (1967).

Figure 4: Generalized mechanism for acid catalyzed hydrolysisof 2’-orthoesters.

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