9.6 Reactions of Terminal Phosphate Groups

Differences in the reactivity of terminal and internucleotide phosphate groups in nucleic acids have not been determined in direct experiments because of the extremely low relative amount of the former. However, there is every reason to believe that the differences here will be similar to those observed in the case of mono- and diphosphates; the former are more active as nucleophiles because they carry practically two negative charges at neutral pH.

Nonetheless, it is virtually impossible to conduct such a reaction as alkylation of a nucleic acid selectively at the terminal phosphate group. For the reaction to take place, the terminal phosphate group is activated with the nucleic acid being treated with carbodiimide; in this case, internucleotide phosphate groups are practically not affected. The resulting derivative is not separated. If the reaction is carried out in an alcohol, the terminal phosphate is immediately alkylated. This is how, for example, 5'-terminal phosphate groups in oligoribo- and oligodeoxyribonucleotides were selectively methylated:

The reaction proceeds quantitatively in methanol at room temperature (24-48 h); however, heterocyclic bases also undergo partial methylation. The same method was used to introduce substituents into 5'-phosphate groups in tRNA. For instance. treatment of a trialkylammonium salt of tRNA with diisopropylcarbodiimide in the presence of aniline in a mixture of water, dimethylformamide and term-butyl alcohol at pH 8 yielded an anilide of tRNA. At room temperature, the reaction was 60 -80 % complete within 24-48 h.

Activation of the terminal phosphate in oligo(poly)nucleotides is a widely used procedure for their immobilization on cellulose or Sepharose. Particularly good results are obtained by activation with water-soluble carbodiimides. In this case, the immobilization on hexamethylene diamino-Sepharose, for example, can be performed in aqueous solutions:

This is how oligo(dT), poly(U), poly(A), and other polynucleotides bound to Sepharose (or cellulose) are prepared.

Another method for selective activation of the terminal phosphate in oligo- and polydeoxyribonucleotides was developed recently. A careful study of the reaction between oligodeoxyribonucleotides and mesitylenecarboxyl chloride has shown that only the terminal phosphate is acylated under certain conditions. The reaction is conducted in absolute pyridine for 10 min (20⁰ C). Under such conditions, heterocyclic bases and pentose are not acylated. Internucleotide phosphate groups are acylated, but subsequent treatment with water (when the reaction products are formed) leads to almost instantaneous hydrolysis of the resulting acyl phosphates with departure of the carboxyl anion.

Analysis of the properties of anhydrides of oligo(poly)nucleotides with the sterically hindered carboxylic acid has revealed a number of specific features. First of all, such anhydrides are good phosphorylating agents for sterically unhindered nucleophiles; the reaction proceeds only at the phosphate group. No hydroxamic acids are formed even with hydroxylamine which reacts with acyl phosphates at the phosphate group only. Hence, the steric hindrances for the carbonyl group in mesitylenecarboxylic acid remain and become even more pronounced in the mixed anhydride whose formation involves the terminal (3'or 5') nucleotide phosphate group.

Reactions with weaker nucleophiles (alkyl mercaptans, alcohols, phosphates) proceed more easily in organic solvents:

In this way oligo(poly)deoxyribonucleotides are easily immobilized on hexamethylene diamino-Sepharose:

As opposed to the above-described method for activating the terminal phosphate in polynucleotides by carbodiimides, that based on mixed anhydrides with mesitylenecarboxylic acid is rather simple and much more effective. There is every reason to consider it as one of the most promising ways of controlled modification of nucleic acids at terminal phosphate groups, including introduction of various "active" non-nucleotide groups to create affine reagents ²⁾.

An alternative method for selective modification of the terminal phosphate group in oligo(poly)nucleotides is the reaction with morpholides of alkyl phosphates.

Condensation of methylphosphomorpholide with tri-n-hexylammonium salt of tRNA in pyridine (20⁰ C, 6-7 days) makes it possible to convert the 5'-terminal phosphate in tRNA into methylpyrophosphate:

Similarly, an alkylphosphomorpholide containing a dansyl (5-dimethylaminonaphthalene-l-sulfamido-methyl) group was used to obtain E. coli tRNA^fMet with a fluorescent marker at the 5'-terminal phosphate group (the dansyl group is broadly employed as a fluorescent marker in studying the three-dimensional structure of biopolymers and for other purposes).

Other non-nucleotide groups are selectively introduced at the terminal phosphate in nucleic acids by this method as well.

In recent years, a universal method for modifying the terminal phosphate of oligo(poly)nucleotides in aqueous solutions has been developed.

Two alternative approaches have been devised for selective activation of the phosphomonoester group: the direct derivatization under the effect of water-soluble 1-ethyl-3(3'-dimethylaminopropyl)-carbodiimide (EDC) and the substitution reaction of N-hydroxybenzotriazole phosphodiesters (both types of activation are obtained in an aqueous solution on unprotected oligonucleotides).

EDC-induced derivatization is very simple in experimental terms and proceeds according to the pathways:

It was demonstrated that EDC is the only reagent capable of synthesizing phosphoramidate as well as phosphodiester derivatives in an aqueous solution.

Reactions performed in the optimal pH range and in the presence of a high (2-3 M) concentration of a nucleophile give high yields of products. This method can be recommended as an optimal one for inserting such spacer groups as residues of aliphatic diamines etc. Non-substituted amino groups of the latter may be used for subsequent derivatization.

The oligonucleotide N-hydroxybenzotriazole phosphodiesters are also very promising intermediate compounds for designing a broad series of oligo(poly)nucleotides substituted at the terminal phosphate. These compounds are hydrolytically stable at pH £ 8 and at the same time are readily subjected to aminolysis and alcoholysis in an aqueous solution:

In contrast to the EDC-induced condensation, when all the groups that can be activated by a condensation agent should be protected, in this particular case, one may use amino acids and peptides unprotected at the carboxyl. The nucleophile concentration can be brought down to 0.2-0.3 M.

Both methods of derivatization are simple, effective and mutually complementary.

Thus, the difference in reactivity between the terminal phosphate in polynucleotides and other nucleophilic groups (amino groups, hydroxy groups in carbohydrate moieties, phosphodiester groups), established for some reactions conducted under rigorously defined conditions (solvent, temperature, reaction time), allows one not only to selectively modify the terminal phosphate group but also to activate, also selectively, a given polynucleotide and convert it into a compound capable of forming covalent bonds. This may form the basis of using appropriately modified nucleic acids to solve many problems in molecular biology, such as defining the pathways of nucleic acid functioning, elucidating the topography of cell components (ribosomes, etc.), and so on.