Until a few years ago, much more progress had been made in the chemical synthesis of oligo- and polydeoxyribonucleotides (DNA sequences) (see DNA Synthesis) than in oligo- and polyribonucleotides (RNA sequences). There are two main reasons for this. First, until fairly recently, there had been a very much greater demand for DNA than for RNA sequences in biological research. Secondly, due to the presence of the 2 ‘ -hydroxy functions, the chemical synthesis of RNA requires the use of an additional protecting group and is therefore inherently more complicated than the chemical synthesis of DNA. Nevertheless, in the past decade or so, significant progress has been made in synthesizing RNA sequences, in both solution and on a solid support. As in DNA synthesis, the three main factors to be taken into account in synthesizing RNA sequences are (1) the choice of suitable protecting groups, (2) the development of phosphorylation procedures that are suitable for introducing the internucleotide linkages, and (3) the RNA sequences themselves.
1. Protecting the 2 ‘ -Hydroxy Functions
Almost certainly the most critical decision that has to be taken in the chemical synthesis of RNA sequences is the choice (1) of the protecting group (R, as in 1) for the 2 ‘ -hydroxy functions. This protecting group must remain intact until the final unblocking step (Fig. 1) at the end of the synthesis. It must then be possible to remove it under very mild conditions that do not promote the attack of the released 2 ‘ -hydroxy functions (as in 2) on the vicinal (3′ ^ 5′ )-internucleotide phosphodiester linkages, thereby leading to their cleavage or migration (Fig. 2).
Figure 1. Final unblocking step.
Figure 2. (a) Cleavage of the internucleotide linkage under conditions of basic hydrolysis (b) Cleavage and migration of the internucleotide linkage under conditions of acidic hydrolysis.
A number of the groups that have been used to protect the 2 ‘ -hydroxy functions are removed hydrolytically either under basic or acidic conditions. If the 2 ‘ -protecting groups are removed under basic conditions, cleavage but not migration of the internucleotide linkages can occur (2). This is illustrated in Figure 2a. Under basic conditions, the initially formed 2 ‘,3′ -cyclic phosphate 3 undergoes further hydrolysis to give a mixture of the corresponding 2 ‘ – and 3′ -phosphates (5 and 6, respectively). If the 2 ‘ -protecting groups are removed under acidic conditions, both cleavage and migration of the internucleotide linkages can occur (2). This is illustrated in Figure 2b. In the context of RNA synthesis, cleavage of the internucleotide linkages is clearly undesirable, in that it inevitably leads to diminished yields. However, phosphoryl migration, which gives rise to material with one or more (2 ‘ ^ 5′)-internucleotide linkages (as in 8), is a much more serious matter. Even in the case of relatively low molecular weight oligoribonucleotides, it is virtually impossible to remove isomeric contaminants containing unnatural (2 ‘ ^ 5′ )-internucleotide linkages, and thereby obtain pure RNA sequences. Therefore, if acid-labile groups are used to protect the 2 ‘ -hydroxy functions in the synthesis of RNA sequences, such protecting groups must be removed under conditions of acidic hydrolysis that are mild enough to avoid the occurrence of phosphoryl migration.
Some of the groups that have been used most widely and indeed most successfully to protect the 2 ‘ -hydroxy functions in the chemical synthesis of RNA sequences are illustrated in Figure 3. Several of these protecting groups [ie, the tetrahydropyran-2-yl (Thp, 9), 4-methoxytetrahydropyran-4-yl (Mthp, 10), and 1-(2-fluorophenyl)-4-methoxypiperidin-4-yl (Fpmp, 13) groups] are acetal systems that undergo hydrolysis under mild conditions (especially in the case of the Fpmp 13 group) of acidic hydrolysis (13). These protecting groups have the considerable advantage that they are completely stable under the ammonolytic conditions that are needed to remove virtually all of the other protecting groups from fully assembled RNA sequences. The 2-nitrobenzyl protecting group 11 (4) is removed photolytically (at wavelengths > 280 nm) and is, of course, also stable to ammonolysis. The fert-butyldimethylsilyl (TBDMS 12) protecting group (5), which has been used very widely in the automated solid-phase synthesis of RNA sequences, has the advantage that it can be removed in the final unblocking step under nonacidic conditions. However, there are at least two distinct disadvantages associated with the use of the TBDMS protecting group. First, it is partially removed under the standard ammonolytic unblocking conditions (concentrated aqueous ammonia, 55°C). Second, as the TBDMS group readily undergoes base-catalyzed migration (6) from the 2 ‘ – to the 3′ -hydroxy function of a ribonucleoside (and vice versa), particular care has to be taken in the preparation of the appropriate monomeric building blocks. Otherwise the synthetic RNA sequences will inevitably be contaminated with material containing (2′ ^5′ )-internucleotide linkages.
Figure 3. Some protecting groups for the 2 -hydroxy functions in RNA synthesis.
Apart from the particular problem associated with 2′ protection, the principles governing the chemic; same as those governing the synthesis of DNA sequences. Similar protecting groups are used for the internucleotide linkages, and the same three principal phosphorylation methods (ie, the phosphotries phosphonate approaches) that are used in the DNA synthesis are also used in the synthesis of RNA s case of DNA synthesis, of these three phosphorylation methods only the phosphotriester approach (1 synthesis of RNA sequences, and the phosphoramidite approach (8-11) again appears to be the meth phase synthesis.
2. Solution-Phase RNA Synthesis
Before a really satisfactory procedure for automated solid-phase synthesis had been developed, relati sequences were prepared by the phosphotriester approach in solution. As an example of this approac used successfully (7) in the preparation of the 3′ -terminal decamer, nonadecamer, and heptatriaconta tRNAAla is illustrated in outline in Figure 4. The achiral Mthp group 10 was used to protect the 2 ‘ -hy and the methoxymethylene group was used to protect the 2 ‘ ,3′ -terminal vicinal diol system (as in 15 functions (as in 14 and 16) were protected with acyl groups [such as the 2-(dibromomethyl)benzoyl ( removable under what were effectively very mild basic conditions. Internucleotide linkages were pro (Ar, 19), and the adenine, cytosine, guanine, and uracil residues were protected as in 20, 21, 22, and uracil residues were protected on O-6 and O-4, respectively, with aryl groups to prevent the occurren coupling steps. Coupling (Fig. 4, step i) was effected with 1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-(1 pyridine solution. After the fully protected RNA sequences had been assembled, unblocking was effe unblocking step (step ii), the 2-chlorophenyl protecting groups were removed from the internucleotid O-aryl groups were removed from the guanine and uracil residues by treatment with (E)-2-nitrobenza tetramethyl-guanidine 26. All of the acyl protecting groups were then removed by treatment with con iii). Finally, the Mthp 10 and the 2 ' ,3'-terminal methoxymethylene protecting groups were removed i acidic hydrolysis. Solution-phase synthesis is labor-intensive and is likely to compete with automate' below) only if relatively large quantities of RNA sequences are required, say, for chemotherapeutic p robust than the Mthp 10 group, and is also removable under milder conditions of acidic hydrolysis. T solution-phase oligonucleotide synthesis it would be advisable to replace the Mthp by the Fpmp or an protecting group.
Figure 4. See text for description of steps i-iv.
3. Solid-Phase RNA Synthesis
The advantages of automated solid-phase synthesis in the preparation of relatively small (ie, milligra are fully discussed in the early DNA Synthesis. The same advantages apply to the automated solid-p Indeed, apart from the final unblocking procedures, the protocols involved in solid-phase DNA and R are closely similar. Thus, the first step in the synthetic cycle of solid-phase RNA synthesis (8-11) is 1 protecting group (Fig. 5a, step i). It is essential that this step should proceed both rapidly and quantit phenylmethyl (DMTr, 27) and 9-phenylxanthen-9-yl (Px, 28) (11), which are particularly acid-labile to be the 5'-protecting groups of choice. Such "trityl" groups have an additional advantage in that the be estimated spectrophotometrically, and the efficiency of each coupling step can thereby be monitoi of DNA sequences, the adenine, cytosine, and guanine base residues are protected with #-acyl group respectively), and the uracil moieties are left unprotected (as in 32). The base moieties of modified ri protected in an appropriate manner. Again, as in the solid-phase synthesis of DNA sequences, 2-cyai blocks 35 are generally used, and the 3'-terminal ribonucleoside residue (as in 33) is usually attachec linker to the solid support [P, usually controlled-pore glass (CPG) or polystyrene]. The other second; function is conveniently protected with an acyl (e. g., benzoyl) group, and it does not matter which o actually linked to the solid support.
Figure 5. Reagents: i, 3% Cl3C·CO2H, CH2Cl2; ii, 1H-tetrazole, MeCN; iii, Ac2O, 2,6-lutidine, 1-methylimidazole, TH v, NH3, EtOH–H2O; vi, conc. aq. NH3, 55°C; vii, Et3N·3HF; viii, 0.5 M aq. NaOAc buffer (pH 3.25), 30°C.
In solid-phase RNA synthesis, it is essential that the 2 ‘ -protecting groups should be completely stable the "detritylation" steps at the beginning of each synthetic cycle (Fig. 5a, step i); it is also highly des should be completely stable under the ammonolysis conditions that lead to the detachment of the ass solid support, the removal of the #-acyl protecting groups from the base moieties, and the removal o from the internucleotide linkages in the penultimate unblocking step (Fig. 5b, step v or vi). At preser to be the most suitable groups for the protection of the 2 ‘ -hydroxy functions in solid-phase RNA syn phosphoramidite building blocks 35a and 35b are available commercially. In the case of TBDMS-pr adenine, cytosine, and guanine residues are now usually protected (9) with particularly base-sensitive 29a, 30a, and 31a, respectively]; in the case of the Fpmp-protected phosphoramidites 35b, the adenin are protected (11) with more robust acyl groups (as in 29b, 30b, and 31b, respectively). The reasons consideration of the unblocking steps involved (Fig. 5b). The TBDMS-protected RNA sequences 38 normal way, and are then treated with aqueous alcoholic ammonia (9) (step v) under mild conditions the loss of TBDMS-protecting groups. The resulting partially protected intermediates 39a. R’ = H th are treated directly with triethylamine trihydrofluoride (step vii) (8, 9) or with tetra-n-butylammoniu: solution to remove all of the TBDMS-protecting groups and give the fully unblocked products 40. T 38b are not "detritylated," but are treated with concentrated aqueous ammonia under much more dra partially protected material 39b, R’ = DMTr without any loss of the Fpmp-protecting groups. This 2 ‘ completely stable to base-catalyzed hydrolysis and to any contaminant ribonucleases. A considerable is that the latter material may be regarded as stabilized RNA and can readily be purified (ie, freed fro it is fully unblocked under mild conditions of acidic hydrolysis (step viii). If the final unblocking ste carefully controlled conditions (pH 3.25, 30°C), is not unnecessarily prolonged, cleavage and migrat can be avoided. Relatively high molecular weight RNA sequences have been prepared successfully f 35a and 35b. The coupling rates observed are somewhat slower than for the corresponding DNA pho efficiencies appear to be slightly lower. It is not yet clear which of the two 2 ‘ -protecting groups 12 an
Finally, the increasing demand for synthetic RNA sequences will undoubtedly stimulate further resea improvements in the methodology of both solid-phase and solution-phase synthesis. Indeed, it has re phase synthesis based on protected ribonucleoside phosphoramidites of general structure 41 leads to now forms the basis of a custom synthesis service.
