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11.4 Methods for Chemical Synthesis of Oligodeoxyribonucleotides

As already mentioned in the introduction, each internucleotide (intermonomer) linkage event during chemical synthesis boils down to acylation of the hydroxyl group of one monomer (nucleoside component) with the phosphate of another (nucleotide component):

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Here, at least two things must be accomplished: (1) protection of all functional groups of the nucleoside and nucleotide components, not subject to phosphorylation; and (2) activation of the phosphate in the nucleotide component.

We shall now consider the most widely used methods for introducing protective groups and preparing nucleoside and nucleotide components. Covered as a separate topic will be methods for activating the phosphate group and the synthesis procedures involved in the diester, triester and phosphite-triester methods for producing oligodeoxyribonucleotides.

11.4.1 Nucleoside and Nucleotide Components for Synthesis by the Phosphodiester and Phosphotriester Methods

As can be seen from the above schemes illustrating the formation of natural internucleotide linkages, the nucleotide chain grows in length as a result of the interaction between the groups in the carbohydrate moiety: namely, 3'- or 5'-hydroxyl group of the nucleoside component and, respectively, 5'- or 3'- phosphate group of the nucleotide component.

To make sure that the reaction proceeds in a predicted fashion, the other reactive sites in these components (hydroxyl and phosphate groups in the carbohydrate moieties, which must remain intact, as well as amino groups of the heterocyclic bases) must be protected or, in other words, blocked. This can be accomplished in two ways: (1) by blocking all reactive groups in the nucleoside and nucleotide components with subsequent selective deblocking of the hydroxyl and phosphate groups, respectively, to create an internucleotide linkage during the following step of the synthesis; and (2) by selective protection of those groups in both components which should not participate in the reaction. Examples illustrating these two approaches will be given in what follows.

The protective groups introduced into the nucleotide and nucleoside components during the steps preceding the internucleotide linkage formation must meet the following basic requirements: (1) the introduction and removal conditions must be mild to avoid undesired side processes; (2) no elimination must take place during formation of internucleotide linkages and subsequent isolation of the reaction products; and (3) ways must be devised for selective elimination and, sometimes, introduction of a given protective group.

Distinction between protective groups is usually made according to the ways of their elimination. The two most common types include those eliminated by acid and alkaline hydrolysis. The third type includes protective groups eliminated by other means (hydrogenolysis, with the aid of anions, in the presence of a zinc-copper couple in dimethylformamide, etc.).

11.4.1.1 Blocking of the Hydroxyl Groups of Pentose and Amino Groups in Heterocyclic Bases

It has already been pointed out that the two types of groups to be blocked in the nucleoside and nucleotide components prior to formation of an internucleotide linkage are, firstly, the hydroxyls that must remain intact and, secondly, the amino groups of heterocyclic bases, which may interact with the activated phosphate group of the nucleotide component.

Notably, acylation of the amino groups in heterocyclic bases enhances the solubility of components in absolute pyridine or another organic solvent in which the oligonucleotide synthesis is carried out. The acylation of nucleotides has already been covered at length with examples of complete selective blocking of the above groups and principles of selection of blocking groups for different heterocyclic bases. Reactions with acid halides or anhydrides proceed quantitatively for all practical purposes, both at the amino groups of heterocycles and hydroxy groups of pentose. Selective removal of protective groups from carbohydrate moieties is achieved by virtue of different rates of cleavage of ester and amide bonds. Protective groups meeting all requirements have been identified with due attention paid to the stability of the amide bonds in the nucleoside and nucleotide components acylated at their heterocyclic bases under conditions of oligonucleotide synthesis as well as deblocking (usually involving treatment with concentrated ammonia) for each constituent heterocyclic base of the nucleic acid. These are isobutyric, 2-methylbutyric and, less commonly, benzoyl group in the case of guanine, benzoyl group in case of adenine, and anisoyl or benzoyl group in the case of cytosine.

Table 11-1 gives some information (introduction and removal conditions) about the acyls most frequently used as protective groups in oligonucleotide synthesis.

Acetylation is a common procedure for blocking the secondary hydroxyl of pentose in the nucleotide and nucleoside components. The primary 5'-hydroxyl group can be blocked selectively in a forward reaction with trityl chloride and its substituents. Methoxytrityl groups are used most often because they are easier to remove from oligonucleotides than an unsubstituted trityl group.

Table 11-1. Major Protective Groups Used in oligonucleotide Synthesis.

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11.4.1.2 Protection of Phosphate Groups

The incorporation of substituents into the phosphate groups of nucleosides is usually achieved through reactions with alcohols or amines in the presence of condensation agents. The most widely used protective group is the b-cyanoethyl one which is removable by mild alkaline treatment (see Table 11-1). Another method for obtaining nucleotides protected at the phosphate group is phosphorylation of nucleosides with active phosphate derivatives, such as halides of p-chlorophenylphosphate. Selective removal of the p-chlorophenyl group can be effected during alkaline hydrolysis without any cleavage of the phosphate-nucleoside linkage (see Table 11-1). The table also shows some other groups used for protection of phosphate groups in nucleotides.

11.4.1.3 Preparation of the Nucleoside and Nucleotide Components

As has already been mentioned, the nucleotide chain usually can have its length increased during oligonucleotide synthesis as a result of interaction between the 3'- or 5'-hydroxyl of the nucleoside component and, respectively, the 5'- or 3'-phosphate group of the nucleotide component.

The function of both components can be performed by monomeric nucleosides and nucleotides as well as oligomers, or oligonucleotides, insofar as any one of these can be entered into phosphorylation reactions of the above-mentioned type after appropriate preparation.

Described below are the most widely used approaches for the preparation of the nucleoside and nucleotide components.

Nucleoside Component. By definition, a nucleoside component is a compound undergoing phosphorylation in the course of internucleotide linkage formation by a nucleotide component. Accordingly, nucleoside components are prepared in such a manner that all of their reactive groups are blocked, except for one of the hydroxyls which must be involved at a later stage in internucleotide linkage formation. Here are some examples (for symbols, see Table 11-1) illustrating the protection of the heterocyclic amino groups.

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Today, the most effective yet simple way to produce N-benzoyl derivatives of nucleosides is a one-step procedure with intermediate silylation (without separation of the reaction products) of deoxyribose hydroxyls. The synthesis of N-benzoyldeoxyadenosine by this method is as follows:

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Nucleosides containing a single deprotected hydroxyl (secondary or primary) group, at which phosphorylation during oligonucleotide synthesis may occur, are produced according to scheme (a) or (b):

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If a nucleotide is used as the starting nucleoside component, as is often the case with oligodeoxyribonucleotide synthesis, its phosphate group must be blocked. To this end, the reaction with b-cyanoethanol in the presence of dicyclohexylcarbodiimide (DCC) is usually conducted.

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Nucleotide Component. Usually, the respective 3'- or 5'-phosphates called, as we already know, nucleotide components, act as phosphorylating agents during synthesis by the phosphodiester and -triester methods. The procedures for preparing these phosphates depend on the exact way in which the nucleotide chain is to be elongated.

The most readily available source of the nucleotide component is 5-deoxythymidylic acid:

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In other deoxyribonucleotides, the amino group of the heterocyclic base residue is blocked first, then the 3'-O-acyl group is replaced by a 3'-O-acetyl one, because the latter can be easily eliminated in a selective manner during alkaline treatment of the synthesized oligonucleotide, which is required for subsequent elongation of the oligonucleotide chain.

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Similarly, sequential acylation, deacylation and acetylation give d[pibG(Ac)] and d[pbzA(Ac)] (for symbols designating protective groups, see Table 11-1).

The above procedures for obtaining nucleotide components by introducing protective groups are by far the most common approaches to ester synthesis. There is a great many of blocking techniques differing by the type of protective groups as well as ways to introduce and remove them. These techniques, however, are not as frequently used in oligonucleotide synthesis and are preferred only by a few groups of specialists.

11.4.1.4 Mechanisms of the Intemucleotide Linkage Formation

We already know that the chemical synthesis of oligonucleotides involves condensation of specially prepared nucleoside and nucleotide components. For the latter to react effectively enough, the phosphate group of the nucleotide component must be activated. The activating agents in the phosphodiester method are arylsulfonyl chlorides and, primarily, 2,4,6-tri-isopropylphenylsulfonyl and mesitylenesulfonyl chlorides. At the early stages of the method's evolution N,N'-dicyclohexylcarbodiimide was used.

The mechanisms of the reactions involved in activation of the phosphate group by arylsulfonyl chlorides have been described elsewhere, but nothing was said about the paths of internucleotide linkage formation and the role of absolute pyridine in which the reaction is conducted. The latter compound stabilizes the intermediately forming "active metaphosphate". It has been speculated that the stable form emerging in this fashion is essentially an internal salt (betaine) in which the pyridine residue is rather rapidly exchanged. This is borne out by the fact that the stable form of metaphosphate can be obtained only if the reaction is conducted in absolute pyridine or if it is present in the reaction mixture in sufficient amounts. This exchange probably involves a transition state having a trigonal bipyramid structure, and two pyridine molecules are most likely to participate in the stabilization.

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Phosphorylation of the nucleoside component occurs at the moment when the trigonal bipyramid structure receives the hydroxyl of the nucleoside component instead of a pyridine molecule (a similar pattern is observed in the interaction between metaphosphate and water (yielding an appropriate nucleotide). In what follows, the pyridine-stabilized metaphosphate will be designated as ROPO2. C5H5N.

Condensation of the nucleotide and nucleoside components in the presence of triisopropylphenylsulfonyl (or mesitylensulfonyl) chloride leads to formation of a dinucleoside phosphate in absolute pyridine.

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However, the phosphodiester group in the latter is a stronger nucleophile than the hydroxyl of the nucleoside and immediately reacts with the next molecule to yield a trisubstituted pyrophosphate. This compound is a less active phosphorylating agent but reacts (albeit slowly and non-quantitatively) with the nucleoside to give a dinucleoside phosphate; that is, the phosphorus of the ionized phosphate group is attacked, and the departing group is the represented by anion of the stronger acid.

As can be inferred from the findings by Knorre and coworkers concerning the mechanism of internucleotide linkage formation, this reaction is accompanied by several side processes, primarily hydrolysis of betaine and formation of a triphosphate.

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The latter is rather unstable and, for example, can be easily hydrolysed with formation of a dinucleoside phosphate having a 3'-3' internucleotide linkage not existing in nature; thus, this side process is not only accompanied by the cleavage of the internucleotide linkage formed during synthesis, but also "contaminates" the end product by giving a compound with a closely similar structure, which is difficult to eliminate.

No satisfactory condensation agents for phosphotriester synthesis existed by the late seventies when Narang proposed to use tetrazolide of triisopropylsulfonic acid (TPSTe). Later, other azolides of arylsulfonic acids were proposed, such as

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The mechanism of phosphodiester activation by arylsulfonyl azolides seems to include formation of a mixed phosphodiester anhydride with subsequent nucleophilic substitution at the phosphorus atom to give a diphosphate azolide. The latter reacts with the hydroxyl of the nucleoside component to form internucleotide linkage.

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