Functional group protection and deprotection in oligonucleotide synthesis

Artificial synthesis of nucleic acids has now become an important technique in biochemistry and a great deal of technical experience has been accumulated today [1]. The key to the efficient synthesis of nucleic acids lies in the proper protection and deprotection of functional groups during the synthesis of nucleic acids. Therefore, it is not too much to say that the history of nucleic acid synthesis research is the history of the research and development of protecting groups. Even with the rapid technological advances, the fact that the choice of protecting groups still greatly influences the synthesis of nucleic acids cannot be denied.

Nucleic acid synthesis strategy

The structures of peptides/sugar chains/nucleic acids are completely different, but the basic strategies of artificial synthesis between them have many similarities, which are summarized as follows:

(1) Prepare monomer for proper protection

(2) Deprotection of reaction point

(3) Next, let the monomer combine

(4) Repeat steps (2) and (3) as many times as necessary

(5) Unprotect all

The side chains of most peptides have multiple functional groups, while the sugar chains have a complex steric structure, for which most of the hydroxyl groups must be protected separately. Although the protection of nucleic acids is much simpler than that of peptides and sugar chains, it still requires a great deal of effort to generalize it into a common method.

In nucleic acid synthesis, the functional groups that need to be protected are the amino group of the nucleic acid base and the hydroxyl group of the sugar group. The detachment conditions of these two are tested orthogonally, i.e., one is in the detached condition and the other is also in the steady state, and vice versa. The following will describe the common methods for protecting base synthesis.

Protective group of nucleic acid base

Protecting the amino groups of adenine (A), guanine (G) and cytosine (C) is necessary to avoid unnecessary side reactions during the nucleic acid chain extension reaction. Thymine (T) and uracil (U) do not need to be protected because they do not contain functional groups for the reaction.

Figure 1. 5 nucleic acid bases, with the amino groups that need to be protected marked

Acyl groups such as benzoyl (Bz), acetyl (Ac), and isobutyryl are commonly used as protecting groups for these amino acids. Among them, benzoyl is most frequently used because it is quite stable under both acid treatment and alkaline hydrolysis conditions, and does not undergo cleavage or side reactions even in chain extension reactions of nucleotides (or deoxyribonucleotides).

In recent years, various nucleosides with protecting groups have been commercially available, thus making them commonly used. If you want to synthesize a nucleic acid with a specific structure not available on the market, then making your own protective nucleoside is the way to go.

In order to link a protecting group, a two-step reaction is often used. Simply put, the amino group of the nucleic acid base is acylated together with the hydroxyl group of the sugar group by first adding an excess of acyl chloride to the nucleoside to be protected, provided that the base is present. Then, the ester group is removed by alkaline solution to obtain the aminoacylated derivative.

Figure 2. Example of amino protection

In addition, nucleic acids with base benzoate esterification can be obtained by a one-step reaction of pentafluorophenyl benzoate with cytidine [2].

Figure 3. Benzoylation with activated ester

In the last stage of the synthesis, these acyl groups need to be deprotected in this step of the reaction. In most cases, the acyl groups can be deprotected by using the condition of 5 h at 50°C with concentrated ammonia. The exception to this is in cases where the amino group of guanine is protected by an isobutylene group. Therefore, it is best to use the dimethylformamide group (DMF), which is more easily removed by concentrated ammonia, as the protecting group at this time[3]. This protecting group can be linked by adding an excess of dimethylformamide diethyl acetal to dimethylformamide (DMF) at room temperature by stirring.

Figure 4. DMF Protector of guanine

If deprotection is difficult, acetyl (Ac), phenoxyacetyl (Pac), 4-isopropylphenoxyacetyl (iPrPac), etc., which is known as a "super mild protecting group", should be chosen. The upper protection method is the same as the mechanism of the acylation reaction mentioned above. All these protecting groups can be deprotected at room temperature with a mild reaction condition of a mixture of 33% ammonia and 40% methylamine[4]. However, when using compounds protected by ultramild protecting groups, care needs to be taken to avoid slow decomposition in the solution state during long-term storage.

Figure 5. Ultra mild protective groups (from left to right: acetyl, phenoxy acetyl, 4-isopropyl phenoxy acetyl）

Protective group of glycosyl group

The hydroxyl groups of DNA are located at the deoxyribose 3' and 5' sites, while the hydroxyl groups of RNA are located at the ribose 2', 3' and 5' sites. However, in the general synthesis method, this is not protected because the 3' site is the reaction site.

In most cases, the hydroxyl group in the 5' site is protected with 4,4'-dimethoxytriphenylmethyl (DMTr). The reason for this is that the triphenylmethyl cation is stabilized by two methoxy groups and they are easily cut off by the action of weak acids. Moreover, since this group is very bulky, it selectively protects the primary hydroxyl group at the 5' position.

Figure 6. 4,4 '- Dimethoxytriphenylmethyl (DMTr)

Nucleic acid synthesis usually links the DMTr group through the reaction of an amino-protected nucleoside with 4,4'-dimethoxytrityl chloride (DMTr-Cl) under basic conditions. The hydroxyl groups at the 2' and 3' positions of the secondary are different, so that only the hydroxyl group at the 5' position can be protected.

Fig. 7. Protection of the 5' position

The deprotection of the DMTr group requires only a dichloromethane solution of 2-3% dichloroacetic acid or trichloroacetic acid, and the reaction is completed in a few minutes. Since the cation of the deprotected dimethoxytrityl group is shown in orange color, the reaction process can be monitored by the naked eye. The primary prerequisite for the artificial synthesis of RNA is the protection of the hydroxyl group at the 2' position. Tert-butyldimethylsilyl (TBDMS) is the most commonly used protecting group for the hydroxyl group at the 2' position[5]. The reaction is usually carried out by adding 1 equivalent of TBDMS-Cl for the amino group of the nucleotides base as well as for nucleosides with a protecting group at the 5' position of the ribose. However, in this case, by-products of methylsilylation at the 3' position are also produced, so the reaction subsequently requires the separation and purification of both by silica gel column chromatography.

Figure 8. Protection of the TBDMS group

Triisopropylmethylsiloxymethyl (TOM) was also used for the protection of the 2'-position hydroxyl group. Both TBDMS and TOM groups can be deprotected by treatment with fluoride ions such as tetrabutylammonium fluoride (TBAF).

reference：

1. Colin B. Reese. Oligo- and poly-nucleotides: 50 years of chemical synthesis[J].Org. Biomol. Chem., 2005,3, 3851-3868https://doi.org/10.1039/B510458K

2. Jean Igolen,Christophe Morin. Rapid syntheses of protected 2'-deoxycytidine derivatives[J]. J. Org. Chem.,2002,45(23). https://doi.org/10.1021/jo01311a061

3. L. J. MCBRIDE,R. KIERZEK,S. L. BEAUCAGE,M. H. CARUTHERS. ChemInform Abstract: Nucleotide Chemistry. Part 16. Amidine Protecting Groups for Oligonucleotide Synthesis.[J]. Chemischer Informationsdienst,1986,17(32).https://doi.org/10.1021/ja00268a052

4. Reddy M.P.,Hanna N.B.,Farooqui Firdous. Fast cleavage and deprotection of oligonucleotides[J]. Tetrahedron Letters,1994,35(25).https://doi.org/10.1016/s0040-4039(00)73341-7

5. Usman N.,Ogilvie K. K.,Jiang M. Y.,Cedergren R. J.. The automated chemical synthesis of long oligoribuncleotides using 2'-O-silylated ribonucleoside 3'-O-phosphoramidites on a controlled-pore glass support: synthesis of a 43-nucleotide sequence similar to the 3'-half molecule of an Escherichia coli formylmethionine tRNA[J]. Journal of the American Chemical Society,1987,109(25).  https://doi.org/10.1021/ja00259a037