MXPA96004355A - Oligonucleotides and used modified intermediaries in nucleic acids therapeuti - Google Patents

Abstract

The present invention provides nuclease resistant 3'-carbon modified oligonucleotides which can be used in the field of therapeutics and diagnostics of nucleic acids. The modified oligonucleotides of the present invention have at least one modified internucleotide linkage, wherein the divalent oxygen moiety at the 3'-position of the internucleotide linkage is substituted by a portion of tetravalent carbon. The carbon 3'-modified internucleotide linkage is preferably a 3'-methylene or 3'-hydroxymethylene linkage. Also provided is a monomeric nucleoside method and nucleoside intermediates for making the modified oligonucleotides in the present invention.

Description

ACIDS nr.?t q THERAPEOTICDS FIELD OF THE PWBKaQN This invention relates to the field of therapeutic materials and in particular to the field of therapeutic nucleic acids.

Traditional approaches in drug development have focused on the use of therapeutic agents capable of interacting directly with proteins involved in disease states and other morbid states. Medicines that arise from this tradition include, for example, synthetic hormones (to simulate the function of hormones based on proteins present in a desirable way in the body), antibiotics (which attack foreign proteins, mainly microorganisms) and vitamins (which provide the building blocks required by certain proteins to perform their usual functions in the body) in addition to many others. More recently, therapeutic agents have been designed in the form of oligonucleotides to regulate, control or otherwise have impacts of another REF: 23228 way, indirectly, on the function of proteins by altering the genetic concentration of the blueprint or machinery that controls the synthesis of all proteins. Because each gene contains the information necessary to produce many copies of a particular protein, each of these therapeutic nucleic acid agents can affect a greater number of protein molecules through their indirect interaction compared to a traditional macromolecular drug that is based on direct interaction with the target protein. Therapeutic nucleic acid compounds can act in many different ways, but the most commonly fall into one of two categories. The first category includes oligonucleotides that simulate or enhance in some way the desired genetic effect. The activity stimulated by this type of nucleic acid therapeutic compound is usually referred to as "gene therapy". The second category of nucleic acid therapeutic compounds includes inhibitory oligonucleotides in which the therapeutic nucleic acid compound inhibits the production of unwanted proteins. Antisense oligonucleotides form a subclass of inhibitory nucleic acid therapeutic compounds, although compounds commonly assigned to this class do not always act in a true "antisense" manner. In addition, of these two categories of therapeutic oligonucleotides, it should be noted that it is also possible that therapeutic nucleic acid compounds interact directly with the target proteins to a large extent in the same way as traditional therapeutic drugs do. True antisense interactions involve the hybridization of complementary oligonucleotides (hence, the term "antisense") to their selected nucleic acid target (e.g., viral RNA or other unwanted genetic messages) in a sequence-specific manner so that the The complex that forms, either alone or in combination with other reagents (for example enzymes such as RNase) can no longer function as a template for the translation of genetic information into proteins. Other inhibitory oligonucleotides have sequences that are not necessarily complementary to the target sequence but which, like the antisense oligonucleotides, have the potential to interfere with the expression (e.g., replication and / translation) of unwanted genetic material. An antisense oligonucleotide can be designed to interfere with the expression of foreign genes (eg, viral genes such as HIV) or with the aberrant expression of endogenous genes (eg, a normal gene that is aberrantly expressed as a mutated oncogene). These unwanted genetic messages are involved in many morbid states, which include viral infections and carcinomas. Inhibitory oligonucleotides raise the possibility of therapeutic suppression of a disease state in the early stage of replication and expression, instead of attacking the resulting protein in the later stage of disease progression, in the way that traditional medicines do. Oligonucleotides used in gene therapy are designed to provide an oligonucleotide, or synthetic gene, that has a desired effect that would otherwise be absent or damaged in a patient. Each gene normally present in the human body is responsible for the production of a particular protein that contributes both to the structure and to the functioning of the body. If this gene is defective or is not present, protein synthesis will fail or will not exist, and will result in a genetic deformity or disease. The incorporation of therapeutic nucleic acid compounds into the genetic material of the cells of a patient can be carried out through a vehicle such as a retrovirus, whereby the production of the necessary protein is allowed. Regardless of whether the therapeutic nucleic acid compounds are designed for gene therapy, antisense therapy or any other situation in which it is desired to alter the proteins at the genetic or other level, the design of these synthetic oligonucleotides is key to the level of success that can be obtained. Importantly, these oligonucleotides must usually be modified in a manner that provides the oligonucleotide with resistance to nucleases so that they are able to survive in the presence of the various nucleases that are endogenous to the human body or the body of the animals. The same is true for oligonucleotide probes used in the analysis of serum samples, because the same exogenous nucleases present in the human body can degrade unmodified therapeutic oligonucleotides that are also present in human serum and can degrade unmodified oligonucleotide probes in these samples as well. In particular, unmodified (or "wild-type") oligonucleotides are susceptible to degradation by nucleases at their 3 'and 5' positions of the internucleotide linkages that bind the individual nucleoside units in the completed oligonucleotide. Accordingly, attempts to impart nuclease resistance to the therapeutic oligonucleotides have been directed to the modification of these internucleotide linkages, and have been successful primarily with respect to the modification of the "non-bridge" oxygen atoms in the naturally occurring phosphodiester linkage (eg, oligonucleotides modified in phosphorothioate having an oxygen that does not form a single bridge, substituted with a sulfur atom (U.S. Patent No. 3,846,402), and modified oligonucleotides in the phosphorodithioate having Oxygen that do not form a bridge substituted with sulfur atoms (U.S. Patent No. 5,218,103) However, it is known that sulfur-containing oligonucleotides such as these bind to proteins, resulting in a non-specific level of activity that may not be acceptable In addition, the oligonucleotides modified in the phosphorothioate part are particularly susceptible to degradation by nuclease at the 3 'position in the modified internucleotide linkages, especially by nucleases that leave a subsequent breakdown to 5'-phosphate of the internucleotide link, due to the fact that only one of the atoms "that do not bridge "Oxygen in the phosphodiester bond is modified. There are several methods currently available for the synthesis of oligonucleotides that can be used to generate oligonucleotides having modified basic structures or skeletons. These methods involve either the solution or the solid phase synthesis. The more traditional solution-based synthesis approach requires relatively small amounts of synthon or mononucleotide synthase reagents and can provide significant amounts of the desired final product. Nevertheless, the solution synthesis has the disadvantage that it requires isolation and tedious purification of the intermediate product after each addition of a mononucleotide subunit. As a result, the solution-based phosphotriester chemistry is not suitable for the practical synthesis of larger oligonucleotides (ie, greater than 6 bases in length) that are required for use in nucleic acid therapeutics. In the case of solid phase synthesis, the complete reaction sequence is carried out on a solid support with the mononucleotide subunits which are added sequentially to form a growing chain attached to one end of the polymeric support. Therefore, the solid phase method allows easy separation of the reagents, and the only real drawback of this method is that it requires an excess of mononucleotide synthon reagents (several times the amount required for solution synthesis) as well as other expensive reagents. It would be desirable to have a modified oligonucleotide not containing sulfur of a length that is suitable for use as a therapeutic nucleic acid compound or as a diagnostic probe and having a sufficient number of modified bonds to impart nuclease resistance to the modified oligonucleotide. It would be further desirable to have a polymer supported method for the synthesis of such modified oligonucleotides that do not contain sulfur.

A modification that does not contain sulfur involves the substitution of a P-C bond instead of a P-0 bond at the 3 'position of an unmodified phosphodiester bond to provide a modified internucleotide bond at the 3' carbon. The monomeric 3'-methylene phosphate nucleotides required as intermediates for solution-based preparations of this modified phosphodiester linkage have been prepared by the use of solution chemistry. See, for example, Albrecht et al., Ttrahedron, 40, 79-85 (1984), - Albrecht et al. , J. Arper. Chem. Soc., 92, 5511-5513 (1970); Mor et al. , GBF Monogr. Ser., Chem. Syn. Mol. Biol. , 8, 107-113 (1987). Traditional phosphodiester methods of phase-in-solution synthesis have resulted in the incorporation of these monomeric modified oligonucleotide subunits into fully modified ribonucleotide 3'-methylene phosphonate dimers and trimers. Jones et al., Amer. Chem. Soc., 92, 5510-5511 (1970, analog incorporated in a dimer); Mazur et al., Tetrahedron, 40 (20), 3949-3956 (1984) (analog incorporated in a trimer). In addition, Morr et al. , GBF Monogr. Ser., Chem. Syn. Mol. Biol. , supra. , has reported the synthesis of a modified 3'-methylene deoxyribonucleotide phosphonate dimer from the same monomeric 3'-methylene phosphonate nucleosides with subsequent incorporation of the modified dimer (containing a unique modified internucleotide linkage between the two monomer subunits) in a larger oligonucleotide. Heinemann, et al. , Nucleic Acids Res., 19, 427 (1991). However, these methods are too laborious to be susceptible to large-scale production of modified oligonucleotides. The multiple modifications of the 3 'carbon necessary to impart nuclease resistance to an oligonucleotide have not been reported in deoxy oligonucleotides greater than a trimer, due to the inherent limitations of phosphotriester chemistry. In addition, the solution phase methodologies of the prior art can not be applied to faster and more efficient polymer supported methodologies of oligonucleotide synthesis because the phosphonate synthons used in the phosphotriester method do not have sufficient coupling efficiencies to work effectively outside the solution phase. Therefore, it is an object of the present invention monomeric oligonucleotide intermediates useful in the synthesis supported by polymers of oligonucleotides modified at the 3 'carbon. It is a further object of the present invention to provide a method supported by polymers for the synthesis of oligonucleotides having multiple 3 'carbon modifications.

It is a further object of the present invention to provide oligonucleotides having at least one modification at 3 'carbon useful in nucleic acid therapeutics and / or in nucleic acid diagnostics.

BRIEF DESCRIPTION OF THE INVENTION The present invention provides nuclease-resistant modified 3 'carbon oligonucleotides which can be used in the field of nucleic acid therapeutics and diagnostics. The modified oligonucleotides of the present invention have at least one modified internucleotide linkage in which the divalent oxygen moiety at the 3 'position of the internucleotide linkage is replaced by a tetravalent carbon moiety. A method and intermediates for making the modified oligonucleotides of the present invention are also provided.

The present invention provides nuclease-resistant modified 3 'carbon oligonucleotides useful in the therapeutics and diagnosis of nucleic acid. According to the present invention, the divalent oxygen moiety in the 3 'position of one or more internucleotide bonds of the oligonucleotides is replaced by a tetravalent carbon moiety, therefore generate two additional substituent groups at the 3 'position. A method for manufacturing these modified oligonucleotides is also contemplated by the present invention. The novel monomeric nucleoside and nucleotide intermediates useful in the manufacture of the 3'-carbon modified oligonucleotides are also contemplated within the scope of the present invention. The oligonucleotides modified at the 3 'carbon of the present invention have at least one modified internucleotide linkage in which the P-C bond is substituted by the P-0 bond at the 3' position of the naturally occurring phosphodiester linkage. Unlike the P-0 bond in the 3 'position of a phosphodiester bond that occurs naturally, the P-C bond of the modified bond at the 3 'carbon can not be broken or separated by nucleases under normal physiological conditions. This PC bond is obtained by substituting a 3'-methylene (referred to as -CH2-), 3 '-hydroxymethylene (designated -CHOH-) or another modified carbon function (designated -CXY-) by a 3'-oxygen in the bond internucleotide phosphodiester. In order to assist in the understanding of the present invention, the following terms, as used herein, have the definitions indicated below. "Oligonucleotide" refers to a polymer or at least two nucleoside units, wherein each of the individual nucleoside units is covalently linked to at least one different nucleoside unit through a single phosphorus moiety. In the case of naturally occurring oligonucleotides, the covalent bond between the nucleoside units is a phosphodiester linkage. However, the term "oligonucleotide", as used herein, includes oligonucleotides that are modified (as compared to naturally occurring oligonucleotides) with respect to any one or more of the following: (1) the phosphotriester linkage between the nucleoside units; (2) the individual nucleoside units themselves; and / or (3) the ribose or sugar portion of the nucleoside units. Unless otherwise specified, the term "base" or "nucleobase" refers to a purine or pyrimidine, such as adenine, guanine, cytosine, thymidine and uracil, as well as to modified forms of these bases, such as -methylcytosine and 5-propinylpyrimidines. "Nucleoside" refers to an individual monomeric nucleoside unit consisting of a base covalently attached at the 1 'position of a 5 carbon sugar. The sugar of 5 carbons will usually be a naturally occurring sugar such as deoxyribose, ribose or arabinose, but it can be any sugar of 5 carbons or a modified form thereof, which includes, but is not limited to 2 ' -fluoro-2'-deoxyribose or even carbocyclic sugars in which a carbon function is replaced by the oxygen atom in the sugar ring (ie, a 6-carbon analogue). Usually, the base will be attached to the sugar portion in conventional positions such as N9 of adenine, guanine or other purines, or NI of cytosine, thymine, uracil and other pyrimidines. "Nucleotide" refers to a monomeric nucleoside unit that further has a phosphorus moiety covalently attached to the sugar moiety of the nucleoside at the 3 'or 5' position of the sugar. A "modified internucleotide link" refers to a modification in the phosphodiester linkage linking individual nucleoside units to naturally occurring oligonucleotides. The term "modified oligonucleotide" refers specifically to an oligonucleotide having at least one modified nucleotide linkage. The term "partially modified nucleotide" means a modified oligonucleotide in which at least one, but less than all of the internucleotide linkages is modified. The term "completely modified oligonucleotide" means a modified oligonucleotide in which all of the internucleotide linkages are modified. The term "internucleotide bond at carbon 3 '" or "bond at carbon 3'" or "bond modified at carbon 3 '" means an internucleotide linkage in which the divalent oxygen potion at the 3' position of a bond internucleotide phosphodiester is replaced by a portion of tetravalent carbon. The term "internucleotide linkage 3 '-methylene" or "3'-methylene linkage" or "3'-methylene linkage" means an internucleotide linkage at the 3'-carbon, where the tetravalent carbon atom at the 3'-position of the 3 'carbon bond is covalently bound, independently, to two hydrogen atoms. The term "3'-hydroxymethylene internucleotide linkage" or "3'-hydroxymethylene linkage" or "3'-hydroxymethylene modified linkage" means a 3'-hydroxymethylene internucleotide linkage in which the tetravalent carbon atom at the 3'-position of the linkage in carbon 3 'it is covalently linked, independently, to both a hydrogen atom and a hydroxyl group.

The term "modified oligonucleotide at carbon 3 '" refers to an oligonucleotide having at least one bond at the 3' carbon. The term "3'-methylene modified oligonucleotide" refers to an oligonucleotide having at least one 3'-methylene linkage. The term "3'-hydroxymethylene modified oligonucleotide" refers to an oligonucleotide having at least one 3'-hydroxymethylene linkage. "Target sequence" refers to the nucleotide sequence at which an oligonucleotide or a modified oligonucleotide is designed to hybridize. In the case of inhibitory oligonucleotides, the "target sequence" can be, but is not necessarily limited to, messenger RNA that occurs naturally for a viral protein, a cancer-related protein or other proteins involved in morbid states. Specifically, oligonucleotides modified at the 3 'carbon of the present invention have at least one modified nucleotide bond at the 3' carbon, as shown below.

In this structure, Cl and C2 represent the 3 'and 5' positions, respectively, of the nucleoside units which are bound in the oligonucleotide through a modified internucleotide bond in the carbon 3 'of the present invention. The modified internucleotide bond in carbon 3 'can be described more fully with reference to the following structure, which shows the individual nucleoside units that surround this particular link in greater detail: With reference to this oligonucleotide structure, B is a purine or pyrimidine base, usually adenine, guanine, cytosine or thymine (in the case of DNA) or uracil (in the case of RNA). Z is a hydrogen atom (-H-), wherein B is a terminal base of the oligonucleotide or the phosphorus atom in the following internucleotide linkage of the oligonucleotide. R4 is usually a hydrogen atom (-H-) (in the case of DNA) or a hydroxyl portion (-0H-) (in the case of RNA, or in the case of an oligonucleotide having arabinose units in the backbone) , but may be other atoms or portions, such as fluorine (-F-) where other 5-carbon sugars are used in the oligonucleotide backbone. R5 represents a suitable counter-ion to the oxygen atom bound by a bond, with a negative charge, in the internucleotide linkage. The preferred R5 function in the bond at the 3 'carbon varies according to the particular application selected for use in the modified oligonucleotides at the 3' carbon of the present invention and will be apparent to a person skilled in the art from the teachings described in the present. For example, it is usually preferred that R5 is a suitable counterion such as sodium, ammonium or alkylammonium, as these types of portions tend to be less disruptive to the natural structure of the oligonucleotide and is the most common counterion in the wild-type oligonucleotides that they come in the form of salts. However, during the chemical synthesis of oligonucleotides, R5 may also be a suitable protecting group for the internucleotide link during the time when it is subjected to relatively rigid chemical conditions. The function 3 ', designated -CXY-, is a carbon function in which X and Y are combinations of monovalent ligands that are generally designated as those that: (1) cause minimal disruption of the structure of the internucleotide linkage; or in some cases, (2) provide, directly or indirectly (i.e., through derivatization) a tag or other means to identify or target the modified oligonucleotide. In the first case, the -CXY- function of the 3'-modified linkage must be small enough to allow the resulting 3'-carbon modified oligonucleotides to efficiently mimic nucleic acids that occur naturally, for example in its ability to hybridize strongly with its desired objective. In this regard, the preferred X and Y portions include hydrogen (-H-) and fluorine atoms (-F-), and hydroxyl groups (-OH-). Again, it may be preferred that both portions, X and Y, are hydrogen atoms (ie, the bond at the 3 'carbon is a 3'-methylene bond) for the reason that the hydrogen atoms are expected to cause less amount of disruption with respect to the natural structure of the oligonucleotide. However, in some cases, for example when recognition by an RNAse H of a nucleic acid duplex containing the 3 'carbon modification is required, it may be desirable to incorporate a hydrophilic function, such as a hydroxyl group (-0H -) or a fluorine atom (-F-) at the 3 'carbon of the 3'-modified internucleotide linkage to provide a more effective substitution for electronegative 3' oxygen that occurs naturally in the internucleotide phosphodiester linkages. This is because modifications imparting a hydrophilic character to the 3 'carbon function (-CXY-) are expected to be more effective in mimicking wild-type oligonucleotides than more hydrophobic modifications, such as the two hydrogen atoms in the modification 3 '-methylene (-CH2-). Therefore, a preferred modification in the 3 'carbon also includes 3'-hydroxymethylene linkages, wherein one of X or Y is a hydroxyl group. In this case, oligonucleotides containing multiple modified bonds can be expected to show more effective hybridization around the internucleotide linkage, allowed by a similar solution structure for these nucleic acid analogues in relation to their naturally occurring counterparts.

During the chemical synthesis of oligonucleotides, the efficiencies of the coupling reactions for each of the nucleoside units that is added to the growing oligonucleotide greatly affects the overall efficiency of the reaction. For example, the theoretical yield of 18 units synthesized by the base sequence addition with an efficiency of 95% for each coupling reaction is only 42%. The theoretical yield for the same 18 units derived from sequential coupling reactions with 90% efficiency is just 17%. Even 6 units manufactured using coupling reactions that have 95% efficiency will only have a theoretical yield of 74%. Due to the separation of the growing oligonucleotide product from the subsequent background material to each coupling reaction which is very laborious, time-consuming and inefficient in the case of solution-based synthesis, these older methods of oligonucleotide synthesis do not they can be used effectively to generate modified oligonucleotides of the lengths that are generally required for diagnostic and therapeutic purposes. In addition, the time-consuming nature of solution-based syntheses significantly increases the cost of the final product, making this method of synthesis little feasible for commercial production of oligonucleotides. The only exception, of course, is the case of a shorter oligonucleotide, usually a dimer, that can be designed to interact directly with a protein. The time-consuming nature of solution-based synthesis could be a critical factor in making these oligonucleotides shorter. The present invention additionally provides a fast and efficient, polymer-supported method for making oligonucleotides containing the modified bond in the 3 'carbon described in the above. This rapid and automated method can be adapted to manufacture modified oligonucleotides at 3 'carbon of lengths comparable to that of unmodified oligonucleotides manufactured by traditional polymer-supported techniques. This is important, because oligonucleotides of about 10-12 base pairs or greater usually required for use as sequence-specific probes for simple genomes such as E. coli. The upper limit of approximately 60 nucleotide bases is set for isothermal processes because the melting temperatures (Tm)) of longer oligonucleotide products converge towards the same value at, or at approximately the same point. On the other hand, the antisense oligonucleotides must be effective at physiological temperatures, and usually have 15 to 25 oligonucleotides in length. Generally, longer antisense oligonucleotides within this range are desirable because they are less likely to occur because of opportunities in large genomes. For example, an oligonucleotide of 17 units may be unique to a mammalian genome. On the other hand, if an antisense oligonucleotide is very long (i.e., substantially longer than 25 nucleotides), it can non-specifically hybridize to other sequences that are not the target sequence. This type of non-specific hybridization is inevitable because the physiological body temperature of the patient can not be adjusted to increase the restraint capacity. The method of the present invention requires the synthesis of many en-route nucleoside intermediates to obtain the final modified oligonucleotide product at the desired 3 'carbon. The key monomeric intermediates include nucleoside substrates, 3'-alkenyl nucleosides, 3'-aldehyde nucleosides and nucleotide synthons. Briefly, an aldehyde nucleoside is coupled to an aldehyde protected nucleotide synthon to form a modified internucleotide linkage at the 3 'carbon in the final coupling step of the polymer supported synthesis. A second synthon, specifically a nucleotide synthon protected in hydroxy is also provided. This second synthon can be used to generate a modified bond at the 3 'carbon in solution or to provide a polymer supported synthesis during the 3' unmodified position. In these structures, which are shown below, the nucleoside and nucleotide intermediates of the present invention are shown as deoxyribonucleosides and deoxyribonucleotides, although it should be understood and appreciated that nucleosides containing other sugars, such as ribose, can also be manufactured as intermediates for a modified oligonucleotide, without departing from the teachings herein. Usually, it will be preferred to use commercially available nucleosides as starting material in the multi-step synthesis process of the present invention. It is preferred to initiate synthesis through the formation of a nucleoside substrate by first protecting the 5 'position of the ribose or deoxyribose ring of the commercially available nucleoside unit and subsequently derivatizing the 3' position of the ring to form a suitable reactive group, such as phenylthiocarbonate, to generate the nucleoside substrate. A deoxyribonucleoside substrate is shown below, wherein B is the base, R is a protecting group at the 5 'position and R1 is a reactive group at the 3' position of the nucleoside.

Nucleoside substrate In Table I, examples are provided, based on the chemical formula for the nucleoside substrate shown above, to demonstrate the sequence of steps for preparing a nucleoside substrate from commercially available thymidine nucleosides ("T"), a pyrimidine, compounds 1-3) and M6-benzoyl-2'-deoxyadenosine ("ABz", a purine protected at the base, compounds 4-6).

Table 1 Compound R Base R1 1 HTH 2 tBuMe2Si TH 3 tBu e2Si TC (S) OPh 4 H ABz H 5 tBuMe2Si ABZ H 6 tBuMe2Si ABZ C (S) OPh Compound 1 in Table I represents the non-derivatized form of a nucleoside reagent of commercially available thymidine, while compound 4 represents the protected amino form of the commercially available 2'-deoxyadenosine reagent. Compounds 2 and 5, respectively, represent the same thymidine and N ^-benzoyl-2'-deoxyadenosine nucleoside reagents that have been protected by derivatization of the 5'-hydroxyl group with a t-butyldimethylsilyl function before conversion of the group 3 '-hydroxyl to the desired thionocarbonate (compounds 3 and 6). Protective groups in addition to t-butyldimethylsilyl (eg, dihydropyrosyl) can also be used to protect the 5'-hydroxyl group from the sugar moiety of the individual nucleoside units. The preferred protecting group will be apparent to a person ordinarily skilled in the art, when taking into account factors such as the particular nucleoside to be derivatized and compatibility with other chemical methods as well as other practical and commercial considerations. The second key intermediate is a nucleoside analog having a double bond carbon function at the 3 'position of the sugar ring, referred to as the 3'-alkenyl nucleoside. This second intermediate is prepared from the nucleoside substrate described above. Specifically, these 3'-alkenyl nucleoside intermediates can be prepared from the corresponding thionocarbonates, such as compounds 3 and 6 in Table I. The 3'-alkenyl nucleosides and have the general formula shown below. 3 '-alkenyl nucleoside An alkylation reaction is required to generate the desired 3'-carbon derivatized nucleosides from the appropriate nucleoside substrate (eg, from thionocarbonates). Usually, a cyano (.CN) radical has been used as the alkylating reagent to obtain the alkylation in nucleoside chemistry. However, based on the conditions of the reaction and the nucleoside substrate to be used, this type of alkylating reagent can generate optically impure racemic mixtures of the resultant carbon 3 'derivatized nucleosides. Racemic mixtures are not useful for generating oligonucleotides due to the wrong stereochemistry at the carbon 3 'bond which would result in an incorrect internucleotide linkage, and therefore would avoid the proper helical structure that is needed for biological activity of the oligonucleotide. However, it has surprisingly been found that an alkylating reagent containing tin of the ethylene type, having the general formula of carbon-carbon double bond shown below, can be used to generate an optically pure 3'-alkenyl nucleoside at from a nucleoside substrate.

R »jSn-« ww ^^ Z ~ ^ a Alkylating reagent containing tin of the ethylene type R2 and R3 of the alkylating reagent can be any organic group that does not interfere with the reactivity of the alkylating reagent or with the stereochemistry of the reaction product. However, it is preferred that R2 is an ethyl carboxylate group (C02Et) and R3 a butyl group, as set forth in the preferred tributyltin ethyl acrylate in Table II, below. It is further preferred that the alkylating reagent be in cis form.

Table II Compound R3 R2 Regiochemistry 7 Bu C02Et C? S 8 Bu co2Et trans It is considered that the tin portion of the alkylating reagent is capable of promoting radical chemistry. Because the tin alkylating reagent is more bulky than the prior art cyano radical of the alkylating reagents, the tin alkylating reagent is considered to take advantage of the steric hindrance to force the entire substitution to take place from one side of ribose or deoxyribose (or other sugar), from the nucleoside ring, and therefore generates an optically pure intermediate. In other words, the newly formed carbon-carbon bond at the 3 'position of the resulting 3' nucleoside-alkenyl bond will be completely on one side of the ribose or deoxyribose ring. Table III provides examples to demonstrate that the resulting 3'-alkenyl nucleosides generated from contacting the appropriate nucleoside substrate with the preferred cis-t-butyltin ethyl acrylate alkylating reagent, described above.

Table III Compound R Base R2 9 tBuMe2Si T C02Et 10 tBuMe2Si ABz C02Et Tin alkylating reagents of this type can be derived from modifications to described procedures for non-nucleotide synthesis, as described by Bald in, et al., J. "Chem. Soc. Chem. Car., 133-134 (1984 ) and Baldwin, et al., J. Chem. Soc. Chem. Comm., 682-684 (1985), and these methods can be adapted to the chemistry of nucleosides, as described herein. adapting other additional derivatives of this general structure that have been reported are effective in the transfer of vinyl groups, Crips and Flynn, Tetrahedron Letters, 31, 1347-1350 (1990), Flynn, et al., Nucleosides and Nacleotid.es, 10, 763-779 (1991). Compounds such as number 9 and 10, shown in Table III, are suitable intermediates for the synthesis of modified oligonucleotides because they can be effectively converted to a third key intermediate, specifically, 3 '-aldehyde nucleoside derivatives and equivalents having the chemical formula shown below and exemplified in Table IV. 3 'nucleoside-aldehyde Table IV Compound R B R4 11 tBuMe2Si T H These nucleoside aldehydes serve as direct intermediates, or substrates, for the synthesis of modified oligonucleotides in the 3 'carbon of the present invention. The basic reaction to generate these 3'-carbon modified oligonucleotides is through a novel coupling reaction of the 3'-aldehyde nucleoside intermediate described in the above with coupling reagents having the general formula shown in the following, in presence of a basic catalyst.

Coupling Reagent In the case of oligonucleotide synthesis, R5 is a protecting group and R6 will usually be a nucleotide component. In other words, the coupling reagent will be nucleotide synthon (intermediary) as described more fully below. In those situations where Rs is not a nucleotide component, R6 will usually be a useful portion in the end modification of an oligonucleotide. Compounds 12, 13 and 14 shown in Table V, below, are specific examples of the coupling reagent used to incorporate the 3'-aldehyde nucleoside precursors into oligonucleotides. In this table, compound 12 is commercially available dimethyl phosphite. Compounds 13 and 14 are 3 '-hydroxy protected nucleotide synthons or intermediates.

Table V Compound R5 R6 12 Me Me 13 Me 3 '-Q- tBuMe2SiT 14 Me 3' -Q-DMTrT The reaction of the coupling reagent with 3'-aldehyde nucleoside intermediates results in the formation of a modified nucleotide having the general formula shown below.

Modified Nucleotide Table VI below shows that the modified nucleotides (compound 15) generated from 3'-aldehyde deoxyribonucleoside of tert-butyldimethylsilyl-protected thymidine and coupling reagent 13.

Table VI Compound R CXY B R4 R5 R6 15 tBuMß2Si X-OH, YH TH Me 3'-fl-tBuMe2SiT XH, Y-OH The coupling reaction between the 3'-aldehyde intermediate and 3-protected nucleotide synthons or intermediates -hydroxy (e.g., coupling reagents 13 and 14 described above) is directly applicable for solution-based synthesis of shorter oligonucleotides containing the modified 3'-carbon linkage of the present invention. Additionally, dialkyl phosphites such as dimethyl phosphite (compound 12) can be coupled to the 3'-aldehyde nucleoside intermediate to generate 3'-nucleoside monophosphates and dinucleoside 3'-monophosphates. However, for the polymer-supported synthesis of internucleotide bonds modified at the 3 'carbon consecutive, a 3'-aldehyde protected nucleotide synton or intermediate is required. This synton or 3'-aldehyde-protected nucleotide intermediate is obtained by first protecting the aldehyde function of a 3'-aldehyde nucleoside with a protecting group that can be removed by using relatively moderate conditions, similar to the cleavage of the dimethoxytrityl group in the conventional synthesis of oligonucleotides with trichloroacetic acid. Examples of this type of protecting group include the diacetal function and the N, -diphenylimidazolidine function shown below.

Nucleoside / Nucleotide N., ü-diphenyl imidazolidine Protected Aldehyde Other protecting groups suitable for the protection of the aldehyde function of the 3'-aldehyde nucleoside will be apparent to those skilled in the art after the teachings of the present invention and include recognized acetals, oxathiolanes and other aldehyde protecting groups. Table VII below shows the variations of the protected nucleoside of N., N-diphenylimidazolidino aldehyde.

Table VII Compound R B 16 tBuMe2Si T 17 H T 18 HP (0) OMe T Compounds 16 and 18 have additional groups (R) at the 5 'position. In the case of the aldehyde-protected nucleoside of compound 16, deprotection of the 5 'position provides a free 5'-hydroxyl (compound 17) that can be further derivatized to a nucleoside alkyl phosphite (compound 18). This synthon or nucleotide intermediate protected from aldehyde can be used in the synthesis cycle for the gradual construction of the internucleotide bonds modified at the 3 'carbon on a solid support. Due to the active phosphorus moiety in the 3 'protected nucleotide synton or intermediate-aldehyde which binds to the 5' position, the polymer supported synthesis of the modified oligonucleotide must proceed in a 5'-3 '- direction, in contrast to conventional synthesis methods for the preparation of unmodified oligonucleotides. Although the unconventional 5'-3 'synthesis direction is known in the context of supported synthesis in unmodified oligonucleotide polymers, synthesis in that direction is remarkably slow and, therefore, is not favored in a commercial environment. However, it has surprisingly been found that the aldehyde-protected nucleotide synthons or intermediates of the present invention allow rapid synthesis of the modified 3'-carbon bonds in a 5'-3 direction. Typically, oligonucleotide polymer supported synthesis is initiated through a nucleoside that has been bound to a solid support as a starting point. The method of the present invention is not different in this respect. Specifically, the nucleoside analogs containing the protected aldehyde function, but not protected at the 5 'position, such as compound 17, are first attached to a suitable solid support via the derivatization of the unprotected 5' position, according to the established synthesis methods. A preferred solid support is a controlled pore glass, but it will be appreciated that other solid supports are known in the art and will be suitable for synthesis according to the method of the present invention. The binding of the first nucleoside to the solid support can be carried out by using conventional succinyl or sarcosinyl linkers, but it is not limited to these reactions. The resulting properly derivatized solid support is then used to initiate the 5'-3 'solid phase synthesis of modified oligonucleotides at 3' carbon by using conventional DNA / RNA synthesizers according to the following method. Initially, the support is treated with acid which breaks down the aldehydic protective group of the initially bound nucleoside. The free aldehyde of this linked nucleoside is then coupled to the 3'-aldehyde protected nucleotide synthon or intermediate, such as compound 18, to generate a hydroxymethylene modified dimer oligonucleotide on the solid support. After several steps of washing the supported oligonucleotide in the polymer, the support can be treated again with acid to regenerate the aldehyde function in what is now a growing modified oligonucleotide chain. The repetition of the coupling step lengthens the oligonucleotide chain, one base at a time. However, it should be understood that many different modifications to this cycle can be made according to the requirements of a particular oligonucleotide product to be synthesized. (For example, the synthesis of a partially modified oligonucleotide). These modifications are summarized in the following. In the conventional synthesis of oligonucleotides, a "finishing" or ending step is necessary to avoid an undesired additional coupling of erroneous sequences. For the synthesis method of the present invention, the finishing of the resin from the undesired collateral reaction of the erroneous sequences can be carried out by the use of dimethyl phosphite (compound 12), which will end the chain in growth and will prevent the union of the erroneous sequences. The coupling reaction described herein is not limited to the coupling of the 3'-aldehyde protected nucleotide synthons or intermediates, such as compound 18. For example a 3'-hydroxy protected nucleoside, such as compound 14, is also can be coupled as the terminal step in a series of coupling reactions to leave a protected 3 '-hydroxyl at the 3' end of an oligonucleotide to which, among other things, it tends to facilitate the subsequent purification of the oligonucleotide by high performance liquid chromatography (CLAP). Alternatively, compound 12 can also be coupled to the 3 'end portion. Similarly, other synthons or phosphite intermediates can be attached to the 3 'end portion of the oligonucleotide. For example, the portion R6 of the coupling reagent shown in Table V can be, but is not limited to, molecules such as 3'-dideoxynucleosides or cholesterol, or some fluorescent molecule such as fluorescein or any molecule compatible with this chemistry.

The coupling reaction of the present invention generates a free hydroxyl group on the 3 'carbon of the modified oligonucleotide. Therefore, when 3 '-hydroxymethylene linkages are desired, no further modification of the 3-carbon is necessary. However, when other bonds modified at the 3 'carbon are desired, the hydroxyl function may be modified, either during or after the synthesis of the oligonucleotide. For example, the hydroxyl group can be substituted with a hydrogen atom (in the case of 3'-methylene bonds) or a fluorine atom or another atom or group. If it is desired to have a modified oligonucleotide that exclusively contains 3'-modified bonds (ie, an oligonucleotide completely modified at the 3-carbon position) the substitution of the free hydroxyl groups can be carried out in a single step after the construction of the desired oligonucleotide sequence. However, if different inter-nucleotide links are to be incorporated, such as unmodified links (phosphodiester) in a partially modified oligonucleotide product, the substitution of the free hydroxyl group in the 3'-carbon modified bonds must be carried out during the synthesis cycle after the construction of each modified bond in the 3 'carbon. The synthesis method of the present invention is compatible with the current methods of the state of the art for synthesis supported on automated oligonucleotide polymer. Therefore, the synthesis of a partially modified oligonucleotide, consisting of bonds modified at the 3 'carbon and unmodified bonds (phosphodiester), can be carried out easily, provided that the phosphodiester synthesis is carried out in the same 5 '-3' direction unconventional for the phosphodiester bonds as used for the incorporation of modified nucleosides in the 3 'carbon. For example, suppose a synthesis of phosphonate which is first initiated as described above for the incorporation of bonds modified at the 3 'carbon, the desired number of bonds modified at the 3' carbon are first synthesized, then the last 3 The aldehyde generated in the chain is coupled to a 5 '-alkyl-3'-dimethoxytritylated phosphite such as compound 14 above. After this coupling reaction, the free hydroxymethylene phosphonate can be modified as described above or can be protected by "finishing" with, for example, acetic anhydride. The final acid deprotection of the 3 'terminal dimethoxytrityl function provides a 3"-hydroxyl group that can be used to synthesize phosphodiester bonds through subsequent couplings with suitable 5'-phosphoramidites If a return to the phosphonate chemistry is desired, the terminal 3'-hydroxyl group can be reacted with a synthon or 5'-phosphoramidite intermediate containing a protected aldehyde function such as compound 19.

Synth or Intermediate 5'-phosphoramidite Table VIII Compound R5 R7 B 19 CH3 (CH3) 2CH T In this manner, oligonucleotide analogs can be constructed with at least one modified phosphonate linkage at any desired position. In a similar way, this chemical process can also be combined with other phosphoramidite-based technologies such as phosphorothioates, phosphorodithioates, methylphosphonothioates and methylphosphonates.

It should also be understood that the function R5 is not necessarily restricted to the methyl group (Me or CH3). In this case, any protective group compatible with chemistry can be used. Alternatively, other biologically desirable portions can be attached to the modified oligonucleotide through atoms that do not form bridges of the modified internucleotide linkage by synthesis of the appropriate phosphite / phosphoramidite (compounds 12, 14, 18, 19, in which R5 is the group of interest) . The modified oligonucleotides can be deprotected according to established protocols for unmodified oligonucleotides. For example, when the methyl protection is used in the phosphorus moiety, the protected oligonucleotide is first treated with thiophenol, or with a recognized equivalent, to eliminate the methyl function. After this reaction, the oligonucleotide is treated for an appropriate time with ammonia to separate the oligonucleotide product from the solid support and to remove any base protecting group, and the purification is performed according to standard recognized methods such as CLAP or electrophoresis in polyacrylamide gel. The following examples are provided to aid in the understanding of the present invention, the true scope of which is set forth in the appended claims. It should be understood that modifications can be made to the procedures that are established, without departing from the spirit of the invention. Unless otherwise specified, solvents and reagents were obtained from commercial sources. 3'-Q-tert-butyldimethylsilylimide is prepared from the reaction of commercially available 5'-Q- (4,4'-dimethoxytrityl) thymidine with tert-butyldimethylsilyl chloride for the removal of the trityl group with p-toluenesulfonic acid. 3 '-0- (4,4'-dimethoxytrityl) thymidine is prepared from the reaction of 5'-Q-tert-butyldimethylsilyl thymidine (prepared as in Example 1) with 4,4'-dimethoxytriyl chloride followed by removal of the silyl group with tetrabutylamine fluoride.

Synthesis of 5'-O-tert-butyldimethylsilyl thymidine (Compound 2) '-Q_-tert-butyldimethylsilylthymidine is prepared (compound 2, table I) when dissolving thymidine (25 g, 0.10 mole), 4-N, -dimethylaminopyridine (3.15 g, 0.026 mole) and triethylamine (13.5 g, 18.6 ml, 0.13 mole) in 200 ml of U, U -dimethylformamide dried under a stream of argon. 19.4 g (0.13 mol) of tert-butyldimethylchlorosilane are added to the dissolved mixture, and the whole mixture is stirred for 6 hours before the evaporation of the solvent. The resulting gum is dissolved in 30 ml of ethyl acetate and the solution is extracted with 10 ml of a saturated solution of sodium bicarbonate, 30 ml of water and 30 ml of brine. After drying the organic layer, the residue is purified by flash chromatography using dichloromethane / ethanol (50/1) as eluent. Evaporation of the product containing the fractions gives 31.5 g of 5'-O-tert-butyldimethylsilylthymidine as a white foam (86% yield).

Synthesis of 5'-O-er-butyldimethylsilyl- '-O-phenoxythiocarbonylthymidine (Compound 1) '-Q_-tert-Butyldimethylsilyl-3'-0_-phenoxythiocarbonyl-thymidine (compound 3, Table I) is prepared by dissolving 7.29 g (20.4 mmol) of 5 '-Q-tert-butyldimethylsilyl thymidine from Example 1 in 100 ml of dry dichloromethane and subsequently by adding 3.89 g (31.8 mmoles) of 4-N / N-dimethylaminopyridine and 3.58 g (20.74 mmoles) of phenyl chlorothionoformate, followed by stirring at room temperature for 4 hours, at which time 10 ml are added of water and the organic layer is extracted with water. The organic layer is evaporated and the residue is purified by chromatography using 2% ethanol in dichloromethane as eluent. Evaporation of the solvent from the fractions containing the product provides 7.9 g of the protected nucleoside 5'-Q-tert-butyldimethylsilyl-3'-Q-phenoxythiocarbonylthymidine as a white foam (79% yield).

EXAMPLE 3 Synthesis of N6-benzoyl-5 '-O-tert-butyldimethylsilyl-deoxyadenosine (Compound 5) 6-Benzoyl-5 '-Q-tert-butyldimethylsilyl-2'-deoxyadenosine (compound 5, table I) is prepared by a procedure similar to that used to prepare 5'-β-tert-butyldimethylsilyl thymidine, as described in the example 1, except that N6-benzoyl-2'-deoxyadenosine (10 g, 26.9 mmol), 4-NlJ-dimethylaminopyridine (0.86 g, 7.0 mmol), triethylamine (3.6 g, 4.96 mL, 35.4 mmol) and tert-butyldimethylchlorosilane are dissolved. (5.33 g, 35.4 mmoles) in 100 ml of dry N, l-dimethylformamide. The reaction is allowed to stir for 2 hours before being treated as described in Example 1. Chromatography is performed by using iichloromethane / ethanol (20/1) as eluent, to provide 9.6 g of 6-benzoyl- 5'-Q-tert-butyldimethylsilyl-2'-deoxyadenosine as a white foam (73% yield).

Synthesis of N6-benzoyl-5 '-O-er-butyldimethylsily 1-2' -deoxy- '-Q-phenoxythiocarbonyladenosine (Compound 6) N6-Benzoyl-5'-Q-tert-butyldimethylsilyl-2'-deoxy-3'-Q-phenoxycarbonyladenosine is prepared by a procedure similar to that used for 5'-Q-tert-butyldimethylsilyl-3'-Q-phenoxythiocarbonyl-thymidine, as described in example 2, except that 0.5 g (1.0 mmol) of ü6-benzoyl-5 '-Q-tert-butyldimethylsilyl-2'-deoxyadenosine is dissolved for Example 3, 4-M, N-dimethylaminopyridine (0.26 g) , 2.1 mmol) and phenylchlorothionoformate (0.24 g, 1.4 mmol) in 20 ml of dry dichloromethane. In this case, the reaction is stirred for 16 hours before being treated in the usual manner. Chromatography is carried out by using ethyl acetate in toluene as eluent to give 0.39 g of the protected nucleoside N6-benzoyl-5 '-Q-tert-butyldimethylsilyl-2'-deoxy-3' -Q-phenoxythiocarbonyladenosine ( compound 6, 61% yield).

Synthesis of ethyl-3-tribultilestannil-2-propenoate (Compounds 7 and 8) Ethyl-3-tributylstannil-2-propenoate is prepared (cis and trans forms), compounds 7 and 8, table II) for use as an alkylating reagent when mixing ethyl propiolate (57.18 g, 0.58 mmol), tributyltin hydride (140 g, 129 ml, 0.48 moles) and azo-Jbis-iso-butyronitrile ( 1 g, 0.007 moles) in a one liter round bottom flask. This mixture is frozen, pumped-heated and degassed 3 times and then stirred in an oil bath at 80 ° C for 4 hours (great care is taken due to the potentially explosive nature of the reaction), after which time the reaction it is removed from the oil bath and allowed to cool to room temperature. The cooled mixture is coevaporated with 100 ml of ethyl acetate and the resulting residue is purified by column chromatography using hexane / dichloromethane (99/1) as eluent. Evaporation of the fractions containing the product provides 73.5 g of cis-ethyl-3-tributylstannil-2-propenoate as alkylating reagent (compound 7, 32% yield) and 66.8 g trans-ethyl-3-tributylstannil-2-propenoate alkylating agent (compound 8, 29% yield).

EXAMPLE 6 Synthesis of 5 '-O-tert-butyldimethylsilyl-' -deoxy-1-ethyl-rilyl-idine (compound 9) '-Q-tert-Butyldimethylsilyl-3 '-deoxy-3'-methylacrylthymidine is prepared by suspending 5'-tert-butyldimethylsilyl-3'-Q-phenoxythiocarbonyl-thymidine (1.4 g, 2.8 mmol), hexamethyldimin (0.46 g) , 1.4 mmol) in CIS-ethyl-3-tributylstannil-2-propenoate (3.06 g, 7.9 mmol) in a 100 ml round bottom flask and then adding 0.14 g (1 mmol) of azo-Jbis-iscfoutironitrile to the solution. The mixture is frozen-pumped-heated and degassed three times and then placed in an oil bath at 87 ° C for 2 days. Approximately every 12 hours additional aliquots of 0.14 g of azo-Jbis-iso-butyronitrile are added. (0.14 g) to the reaction mixture. After an incubation period of two days, the reaction is allowed to cool and the solvent is removed by evaporation. Chromatography of the residue with dichloromethane / ethanol (100/1) gives, after evaporation of the appropriate fractions, 0.90 g of 5'-Q-tert-butyldimethylsilyl-3'-deoxy-3'-ethylacrylthymidine. (compound 9, table III) as a white foam (72% yield).

Synthesis of N6-benzoyl-5'-O-tert-butyldimethylsilyl-2 '. '- dideoxy-3'-ethylacryliladenosine (Compound 10) N6-benzoyl-5 '-O-tert-butyldimethylsilyl-2', 3'-dideoxy-3'-ethylacrylalylinosine (compound 10, Table III) is prepared by dissolving E6-benzoyl-5'-Q-tert-butyldimethylsilyl-2 '-deoxy-3' -Q-phenoxythiocarbonyladenosine (50 mg, 0.08 mmol), cls-ethyl-3-tributylstannil-2-propenoate (92 mg, 0.236 mmol) and hexamethylitesite (13 mg, 0.04 mmol) in 2 ml of toluene in a 40 ml Schlenk tube, and subsequently 15 mg (0.11 mmol) of azo-Jbis-iscteutyronitrile are added to the mixture. The resulting mixture is frozen-pumped-heated and degassed 3 times, and then heated to 87 ° C in an oil bath for 3 days. Approximately every 12 hours 15 mg of fresh azo-Jbis-iscbutyronitrile is added to the reaction. After the incubation period of 3 days, the reaction is cooled, the solvent is evaporated and the residue is chromatographed with 1% ethanol with dichloromethane as eluent. A yield of 19 mg of M6-benzoyl-5 '-Q-tert-butyldimethylsilyl-2', 3'-dideoxy-3'-ethylacryloyladenosine is obtained as a white solid after evaporation of the appropriate fractions (42% yield). ).

EXAMPLE 8 Synthesis of 5'-O-er-butyldimethylsilyl- '-deoxy-3'-formylimidine (Compound 11 '-O-tert-Butyldimethylsilyl-3'-deoxy-3'-formylimidine (compound 11, Table VII) is prepared by dissolving 5'-Q-tert-butyldimethylsilyl-3'-deoxy-3'-methylacrylthymidine (1 g) , 2.3 mmol) and 4-methylmorpholine-U-oxide (0.52 g, 4.4 mmol) in a solution of 40 ml of acetone and 4 ml of water to give a light pale yellow solution. Subsequently aqueous osmium tetroxide (11.2 ml, 0.050 g / ml, 2.2 mmol) is added and the resulting light yellow mixture is stirred for 2 hours. After this time, 1.17 g of sodium periodate (5.47 mmol) are added to the reaction, and stirring is continued for an additional 1 hour. The stirred mixture is then treated with 0.60 g of sodium bisulfite and the reaction is stirred for an additional 1 hour. At this time, the solvent is evaporated and the residue is redissolved in 30 ml of ethyl acetate. The solution containing redissolved residue is washed with water (3 x 20 ml) and then dried over anhydrous sodium sulfate. Evaporation of the solvent affords 0.59 g of 5'-Q-tert-butyldimethylsilyl-3 '-deoxy-3'-formylimidine as a crude red foam which is generally used without further purification (71% yield). ai £ MELQ_i Synthesis of 3 '-Q-tert-butyldimethylsilylthymidine-5'-O-methylf sphyte (Compound 1.1) 3 '-Q-tert-Butyldimethylsilylthymidine-5'-Q-methylphosphite (compound 13, Table VIII) is prepared by first dissolving 3' -Q-tert-butyldi-ethyl silylthymidine (1.3 g), 3.6 mmol) in dry dichloromethane (15 ml) and triethylamine (2 ml) and to this solution is added chloride, U-diisopropylmethylphosphonamide (1.28 ml, 6.6 mmoles). This mixture is stirred for 15 minutes before its dilution in ethyl acetate (50 ml). The organic layer is washed with a saturated solution of sodium bicarbonate (50 ml) and water (50 ml) and the solvent is evaporated. This residue is redissolved in acetonitrile (15 ml) and water (0.53 ml, 29 mmol) and a tetrazole solution (0.5 M, 7.3 ml, 3.6 mmol) are added to the solution. The reaction is stirred for 20 minutes before the dilution of the mixture in dichloromethane (50 ml). This organic solution is washed with a saturated solution of sodium bicarbonate (50 ml), a solution of 10% sodium carbonate (50 ml) and brine (50 ml) and then dried over anhydrous sodium sulfate. Filtration and evaporation of the solvent provide a gum which is purified on a chromatographic column. The column is eluted first with chloroform, then with 2% ethanol in chloroform before elution of the product with 4% ethanol in chloroform. The product 3'-Q-tert-butyldimethylsilylthymidine-5'-Q-methylphosphite is obtained as a gum after evaporation of the solvent (1.1 g, 69%). _?to Synthesis of 3 '-O- (4,4'-dimethoxytrityl) thymidine-5'-O-methylphosphite (Compound 14) 3 '-__- (4,4'-dimethoxytrityl) thymidine-5'-Q-methylphosphite (compound 14, table VIII) is prepared by co-evaporation twice from 3' -Q- (4,4'-dimethoxytrityl) thymidine ( 0.85 g, 1.56 mmoles) with dry pyridine (20 ml) and dissolved in dry dichloromethane (8.5 ml) and triethylamine (0.68 ml). The solution is cooled to -78 ° C by using a dry ice / acetone bath and to these is added U, U-diisopropylmethylphosphonamide chloride (0.49 ml, 2.52 mmoles). This mixture is stirred for 20 minutes before its elution and ethyl acetate (50 ml). The organic layer is washed with a saturated solution of sodium bicarbonate (50 ml) and water (50 ml) and the solvent is evaporated. This residue is redissolved in acetonitrile (15 ml) and water (0.25 ml, 13.9 mmol) and a tetrazole solution (0.5 M, 5.1 ml, 2.6 mmol) are added to the acetonitrile mixture. The reaction is stirred for 20 minutes before the dilution of the mixture in dichloromethane (50 ml). This organic solution is washed with a saturated solution of sodium bicarbonate (50 ml), a solution of 10% sodium bicarbonate (50 ml) and brine (50 ml), and then dried over anhydrous sodium sulfate. Filtration and evaporation of the solvent provide a gum which is dissolved in ethyl acetate (10 ml) for precipitation of the product in heptane (500 ml). The product, 3 '-Q- (4,4'-dimethoxytrityl) thymidinyl-5'-Q-methylphosphite (compound 14, Table VIII) is obtained as a white solid after filtration and drying overnight under vacuum, with a yield of 0.75 g, or 77%.

Synthesis of 5'-O-er-butyldimethylsilyl- '-dsoxy-1-methoxyphosphonylhydroxymethylthimidinyl- (' -5 ') -' -O-tert-butyldimethylsilylthymidine (compound 15) 5'-Q-tert-butyldimethylsily-3 'is prepared -deoxy-3 '-methoxyphosphonylhydroxymethylthymidinyl- (3' -5 ') -3'-Q-tert-butyldimethylsilylthymidine (compound 15, table IX) by first dissolving 5'-Q-tert-butyldimethylsilyl-3' -deoxy-3 ' crude formylimidine (compound 11, 0.78 g, 2.11 mmol, obtained from 1 g of compound 9, according to example 8) in dry pyridine (14 ml). This solution is mixed with a solution of 3 '-Q-tert-butyldimethylsilylthymidine-5'-Q-methyl-H-phosphonate (1.91 g, 4.4 mmol) in benzene (20 ml) and to the mixture is added 1,8-diazabicyclo [5.4.0] undec-7-ene (DBU, 165 μl, 1.1 mmol). The mixture is stirred for 30 minutes before the evaporation of the solvent and the chromatographic purification of the residue. Elution of the product is carried out by using 5% ethanol in dichloromethane, as eluent to provide the desired product (compound 15, 1.02 g, 56% calculated from compound 9) as a whitish foam. fiIBJELO_-2.

Synthesis of 5'-O-tert-butyldimethylsilyl-3'-deoxy- '- (2-N-diphenylimidazolidino) -thymidine (Compound 16) '-Q-tert-Butyldimethylsilyl-3 '-deoxy-3' - (2-U / N-diphenylimidazolidino) -thymidine (compound 16, Table X) is prepared by dissolving 5'-Q-tert-butyldimethylsilyl-3 ' -deoxy-3 '-formiltimidine (1.3 g, 3.5 mmol) in 30 ml of benzene and then add 1.5 g (7.0 mmol) of 1,2-dianylinethane to the solution. The resulting mixture is stirred overnight at 60 ° C. At this time, 20 ml of a saturated aqueous solution of sodium bicarbonate are added and the solution is extracted with ethyl acetate (3 x 30 ml). After drying over anhydrous sodium sulfate, the organic layer is evaporated and the residue is chromatographed with ethanol / triethanolamine / dichloromethane (1/2/97) as eluent. A yield of 1.25 g of 5 '-Q-tert-butyldimethylsilyl-3' -deoxy-3 '- (2-U / N-diphenylimidazolidino) -thymidine is obtained as a light brown solid after evaporation of the appropriate fractions ( 63% yield). .eleven Synthesis of '-deoxy-' - (2-N. -diphenylimidazolidino) -thymidine (Compound 17) 3 '-deoxy-3' - (2 -M, H-diphenylimidazolidino) -thymidine (compound 17, Table X) is prepared by dissolving 5 * -Q-tert-butyldimethylsilyl-3 '-deoxy-3"- (2- N, β-J-diphenylimidazolidino) -thymidine (0.8 g, 1.4 mmol) in 50 mL of THF, stir and then add tetrabutylammonium chloride (1 M solution in THF, 8.3 mL, 8.3 mmol) to the stirred solution. The mixture is stirred for 30 minutes, triethylamine (2 ml) is added before evaporation of the solvent, the residue is purified by flash chromatography using ethanol / triethylamine / dichloromethane. (2/2/96) as eluent. The fractions containing the product that are evaporated and the residue is precipitated in pentane to provide 0.63 g of 3'-deoxy-3 '- (2-U, U-diphenylimidazolidino) -thymidine as a light brown solid (99% yield) ).

Synthesis of '-deoxy-3' - (2-N. N-diphenylimidazole idino) - thymidine-5'-Q-methylphosphite (Compound IB) The synthesis of 3 '-deoxy-3' - (2-N- / N-diphenylimidazolidino) -thymidin-5'-Q-methylphosphite (compound 18, Table X) is prepared by co-evaporation twice of 3 '-deoxy-3 '- (2-N, N-diphenylimidazolidino) -thymidine (200 mg, 0.45 mmol) with dry pyridine (20 ml) and dissolved in dry dichloromethane (5 ml) and triethylamine (0.17 ml). The solution is cooled to -78 ° C by using a dry ice / acetone bath and to this is added N., N.-diisopropylmethylphosphonamide chloride (0.14 ml, 0.72 mmol). This mixture is stirred for 20 minutes when an additional portion of N / N-diisopropylmethylphosphonamide chloride (0.02 ml, 0.01 mmol) is added. After an additional 15 minutes, the mixture is diluted in ethyl acetate (50 ml). The organic layer is washed with a saturated solution of sodium bicarbonate (50 ml) and water (50 ml), and then the solvent is evaporated. This residue is redissolved in acetonitrile (5 ml) and then in water (0.16 ml, 2.9 mmol) and a tetrazole solution (0.5 M, 1.0 ml, 0.5 mmol) is added to the acetonitrile mixture. The reaction is stirred for 20 minutes before the dilution of the mixture in dichloromethane (50 ml). This organic solution is washed with a saturated solution of sodium bicarbonate (50 ml), a solution of 10% sodium carbonate (50 ml) and brine (50 ml) and then dried over anhydrous sodium sulfate. Filtration and evaporation of the solvent gives a gum which is dissolved in ethyl acetate (3 ml). Pentane (400 ml) is added to precipitate the product. The resulting supernatant is decanted and the solid is washed with pentane before being allowed to dry overnight under vacuum to generate 180 mg of the product, which represents a 76% yield.

EXAMPLE 15 3 '-deoxy-3' - (2-N-diphenylimidazolidin) -thymidine-51-N. N -diisopropyl-Q-methylphos-.oamidite (Compound 19) 3 '-deoxy-3' - (2-N,.,. Diphenylimidazolidin) -thymidine-5'H, N-diisopropyl-Q-methylphosphoramidite (compound 19, Table X) is prepared by co-evaporation 2 times of 3 '- deoxy-3 '- (2-JJ / N-diphenylimidazolidin) -thymidine (200 mg, 0.45 mmol) with dry pyridine (20 ml) and then dissolving the resulting solid in dry dichloromethane (5 ml) and triethylamine (0.17 ml) . To this solution is added, at room temperature, U, U-disopropylmethylphosphonamide chloride (0.12 ml, 0.62 mmol). After 15 minutes, the mixture is diluted in ethyl acetate (50 ml). The organic solution is washed with a saturated solution of sodium bicarbonate (50 ml), a solution of 10% sodium bicarbonate (50 ml) and brine (50 ml) and subsequently dried over anhydrous sodium sulfate. Filtration and evaporation of the solvent provide a gum which is dissolved in ethyl acetate (3 ml). Pentane (400 ml) is added to precipitate the product. The resulting supernatant is decanted and the solid is washed with pentane before allowing to dry overnight under vacuum (yield 160 mg, 59%).

EXAMPLE 16 Synthesis of oligonucleotide analogs A. 3 '-deoxy-3' - (2-N-diphenylimidazolidin) -nucleoside binding to solid supports The derivatized nucleosides are attached to an appropriate solid support by means of the 5'-hydroxyl portion so that the synthesis of analogues proceeds in a 5'-3 '- direction. For example: 3 '-deoxy-3' - (2-N / U-diphenylimidazolidin) -thymidine (200 mg, 0.43 mmol) and 4-N, H-dimethylaminopyridine (78.5 mg, 0.65 mmol) is coevaporated with dry pyridine (2 x 20 ml). The obtained residue is dissolved in dry pyridine (20 ml) and succinic anhydride (34.4 mg, 0.34 mmol) is added to the solution. This reaction mixture is allowed to stir overnight. After this time, the solvent is evaporated and the residue coevaporated with toluene before its dissolution in dichloromethane (50 ml). The organic layer is washed twice with water (20 ml) and dried over anhydrous sodium sulfate. Filtration provides a clear solution which is concentrated to approximately 10 ml for precipitation of the product in hexane / ether (1/1, 50 ml). The product 3'-deoxy-3 '- (2-H, N-diphenylimidazolidin) -thymidine-51-Q-succinate (195 mg, 80%) is isolated by filtration and used without further purification. The succinylated nucleoside (240 mg, 0.43 mmol) is dissolved in a mixture of dry pyridine (5 ml) and dry dioxane (5 ml). To the solution is added 1,3-dicyclohexylcarbodiimide (177 mg, 0.86 mmol) and 4-nitrophenol (60 mg, 0.43 mmol), and the reaction mixture is stirred overnight. At this point, the precipitate is filtered and the solvent is evaporated. The residue is coevaporated twice with toluene (10 ml) and crude 3 '-deoxy-3' - (2-N, N-diphenylimidazolidin) -thymidine-5 '-Q-succinate- (4-nitrophenyl) ester. subsequently used to derive controlled pore glass. Controlled pore glass derivatized with long chain alkylamine (89 μmol / g) is suspended in dry tetrahydrofuran (THF, 5 ml) and to this is added 3'-deoxy-3 '- (2-N, N-diphenylimidazolidin ester) ) -thymidine-5'-Q-succinate-4-nitrophenyl crude as a solution in THF (3 ml) and triethylamine (2.3 ml). This mixture is stirred overnight and the derivatized support is isolated by filtration. The support is washed with N, N-dimethylformamide (3 x 10 ml), dioxane (3 x 10 ml), methanol (5 x 10 ml) and ether (3 x 10 ml). The finishing or finishing of the resin is carried out through the controlled pore glass suspension in dry pyridine and treatment with acetic anhydride (0.5 ml). This mixture is stirred for 30 minutes. The fully functionalized support is then filtered and washed with methanol (6 x 10 ml) and ether (3 x 10 ml) before drying.

B. Synthesis of oligonucleotide phosphonates The synthesis of oligonucleotide analogs is carried out by using an Applied Biosystems 394 DNA / RNA synthesizer. By using this instrument, analogues containing uniform or intermittent phosphonate functions can be synthesized by using the reagents described in the above.

C. Synthesis of an oligonucleotide completely In the simplest case, the solid support modified with 3 '-deoxy-3' - (2-N, N-diphenylimidazolidin) -nucleoside is deblocked in the presence of acid to provide the 3'-nucleoside nucleoside bound to the resin. After washing the resin to remove the acid, coupling is carried out in the presence of 3'-modified-5'-phosphite such as compound 18 (0.1 M in benzene) using DBU (53 mM in pyridine) as activator . The synthesis proceeds in a 5 '-3' direction. After the additional washing steps, the cycle is repeated using monomeric building blocks such as compound 18 to generate an appropriate sequence. A 3'-Q-dimethoxytritylated phosphite such as 14 (0.1M in benzene) is coupled as the final (3'-terminal) nucleotide in the sequence. For example, the automated stages require synthesizing six uniform phosphonate hydroxymethylene linkages with thymidine as the nucleobase (at a scale of approximately 0.5 μM) and was as follows: Stage. ReactiYQ / SQlYente Time / minute 1 3% trichloroacetic acid 3.0% in dichloromethane 2 dichloromethane 0.3 3 pyridine / benzene (1/1) 0.3 4 compound 18 (0.1 M in 2.0 benzene) plus DBU * (54 mM in pyridine) 5 pyridine / benzene (l / l) 0.3 6 dichloromethane 0.3 7 Steps 1 to 6 are repeated for each addition of compound 18 (in this case, four additional times). For the terminal nucleotide unit, steps 1 to 6 are repeated with the proviso that compound 14 (0.1 M in benzene) replaces compound 18 in the coupling step (step 4). A completely modified heptamer, named T * t * T * T * T * T * T (* indicates the position of a modified 3 '-hydroxymethylene nucleotide linkage), containing six consecutive nucleotide phosphonate 3'-hydroxymethylene bonds is synthesized according to this procedure, resulting in a 71% yield (31P NRM (D20); d 19-20 ppm). The modified oligonucleotide is purified by standard CLAP techniques (eg, Evaluating and Isolating Synthetic Oligonucleotide.), Bulletin of user 13, (Applied Biosystems Inc. 1987) using triethyl as a protecting group and then detritilating according to the procedure of Wiesler et al., Synthesis and Purification of Phosphorodithioate DNA, by Protocole for Oligonucleotides and Analogs; Synthesis and Properties, 191-206 (Ed. Agrawal, Humana Press 1993).

D. Synthesis of a partially modified oligonucleotide This example demonstrates the use of the chemistry used to generate the phosphonate link together with conventional methods of oligonucleotide chemistry such as phosphoramidine chemistry. An example is shown for the synthesis of modified oligonucleotide tt * TTTTTTTTTTTT (* again indicates the position of the 3'-modified internucleotide linkage -hydroxymethylene). However, to the extent that the synthesis of a completely modified oligonucleotide, as demonstrated in example 16C, the synthesis proceeds in a 5'-3 '- direction. In this example, the commercially available 3'-dimethoxytritylated support is detritylated using standard machine protocols and coupled with 3 '-deoxy-3' - (2-N / N-diphenylimidazolidin) -thymidine-5'-N N-diisopropyl-Q-methylphosphoramidite (compound 19, 0.1 M in acetonitrile) in the presence of tetrazole (0.5 M in acetonitrile) for 5 minutes. After the coupling, standard finishing and oxidation protocols are applied. After the synthesis of this dinucleotide unit, the protocols indicated in section 8 of the previous cycle for uniform oxymethylene phosphonate are applied, to generate a single hydroxymethylene phosphonate bond with a 3'-dimethoxytrityl terminal function. At this point, the solid support is treated with the machine finish solutions for 5 minutes. The remainder of the sequence is completed by using commercially available 5'-Q-cyanoethyl-N, N-diisopropylphosphoramidites (in this example, thymidines) by using standard machine protocols for these phosphoramidites.

A 14-unit oligotimidylate contains a phosphonate hydroxymethylene linkage (18% yield: 31P NMR (D20), 19-20 ppm hydroxymethylene phosphonate, 0 ppm phosphodiester).

E. Measurement of the derivatized CPG charge capacity The charge of the modified deoxyribonucleoside is determined by performing a single coupling cycle. In this case, the resin is treated with trichloroacetic acid, washed with solvent and coupled with 3'-Q- (4,4'-dimethoxytrityl) thymidine-5'-Q-methylphosphite (compound 14, 0.1 M in benzene) in the presence of DBU (53 mM in pyridine). The quantification of the release of the dimethoxytrityl cation by treatment of the solid support with trichloroacetic acid in methylene chloride is then used to estimate the modified nucleosides that are charged to CPG. The trityl fraction is collected in a 10 ml volumetric flask and a bright orange trityl cation solution is diluted to 10 ml with 0.1 M p-toluenesulfonic acid. 200 μl of this solution in 800 μl of 0.1 M p-toluenesulfonic acid in Acetonitrile provides an absorbance reading from which the load capacity can be calculated as follows: Load (μmol / g) = A498 x 1000 x (dilution factor) x V E x (amount of CPG in g) where: A498 = absorbance 498 nm of the solution V = total volume of the concentrated solution E = coefficient of extinction of the dimethoxytrityl cation (7 x 104 liter / mol) In this way, charges of modified nucleoside of 4-10 μmol / g are obtained for the derivatized solid supports.

F. Deprotection of the internucleotide phosphonate triesters and their breaking of the support To 1 μmol of CPG oligonucleotide synthesis (dried in argon) is added 1 ml of a 1 M solution (in dimethylformamide) of disodium 2-carbamoyl-2-cyanoethylene-l, 1-dithiolate. The deprotection is allowed to proceed overnight (16 hours) at room temperature. At the end, CPG is washed three times with water, followed by three times with acetone. The CPG-bound phosphodiester is subsequently dried under argon and separated from the support by the addition of 2 ml of 30% NH40H. The separation is complete after 3 hours at room temperature. Vacuum drying provides a vacuum that is quantifiable by dissolving in 1 ml of water and measuring the absorbance at 260 lambda. Disodium 2-carbamoyl-2-cyanoethylene-l, 1-dithiolate is prepared according to published procedures (Dahl, et al., Acta Chemica, 44, 639-641 (Scandinavica, 1990) .Sodium is carefully dissolved (4.6 g. 0.2 mmol) in ethanol (125 ml) A suspension of cyanoacetamide (16.8 g, 0.2 mmol) in ethanol (50 ml) is added dropwise to this solution, then carbon disulfide (12.2 ml, 0.2 mmol) is added. to the resulting suspension, and the mixture is allowed to stir for 1 hour, then an additional portion of sodium (4.6 g, 0.2 moles) is added as a solution in ethanol (125 ml). It is refrigerated overnight to allow the product to precipitate.The solid is filtered and subsequently washed with ethanol.This crude material is redissolved in 80% aqueous methanol (100 ml) and ethanol is added to the solution ( 300 ml) The product is allowed to crystallize at 4 ° C overnight before collection to be filtered by filtration (yield, 36.4 g, 70%).

EXAMPLE 17 Resistance to nuclease of olisonupl F > ? t or modified This example demonstrates nuclease resistance of the modified olignucleotides of the present invention, as compared to unmodified (wild-type) oligonucleotides, when contacted with a 3'-exonuclease and a 5'-exonuclease. The 3'-hydroxymethylene heptamer of Example 16C is used as a modified oligonucleotide in the representative 3 'carbon. A corresponding unmodified 7-unit oligonucleotide, identical in sequence but containing only unmodified phosphodiester bonds, is synthesized in an automated synthesizer according to standard synthetic, polymer-supported techniques, for use as a control in the following procedures. The 3'-methylene modified oligonucleotide is analyzed for its sutibility to nuclease digestion according to two different methods. In the first method, both the modified oligonucleotide and the unmodified control are labeled with radioactive phosphorus (32P), at their respective 5 'ends and then their stability is determined first by contacting them with oligonucleotides with 3' viper venom phosphodiesterase. -exonuclease and then by measuring the size of the resulting oligonucleotide product or products by using polyacrylamide gel electrophoresis (PAGE) according to standard or conventional techniques. In the second method, the modified oligonucleotide and its unmodified counterpart are analyzed and degradation is determined in the presence of phosphodiesterase of bovine spleen 5'-exonuclease by the nuclease, via high performance liquid chromatography (CLAP).

A. Resistance to viper venom phosphodiesterase Both the modified oligonucleotide and its phosphodiester (control) counterpart are labeled by the use of? -32P-ATP and polynucleotide kinase according to standard techniques known in the art. The excess of? -32P-ATP is separated from the oligonucleotides by gel filtration through G-50 Sephadex * 01 (Pharmacia). Radioactive oligonucleotide solutions containing 3.4 pmoles of oligonucleotide are treated with 0, 0.3 x 10"5, 0.6 x 10 -5, 1.2 x 10'5, 2.4 x 10'5 and 4.8 x 10" 5 units of viper venom phosphodiesterase (Boehringer Mannheim, Germany), respectively, and the resulting solutions they are incubated at 37 ° C for 30 minutes. After incubation, an equal volume of 90% formamide containing bromophenol blue, xylene cyanol and 25 mM EDTA is added to each oligonucleotide solution and the mixtures are heated at 90 ° C for 5 minutes. Subsequently the samples are analyzed by separation in a 20% denaturing polyacrylamide gel. With the exception of the reaction with 0 units, the phosphodiester oligonucleotides (unmodified) are degraded in all actions. The degradation was complete in all reactions using 1.2 x 10"5 or greater enzyme units, In contrast, no significant degradation of the modified oligonucleotide (hydroxymethylene phosphonate) was observed in any reaction.

B. Resistance to bovine spleen phosphodiesterase A 250 μl unit of a suspension of phosphodiesterase from bovine spleen exonuclease (Boehringer Mannheim) is added to each oligonucleotide solution of interest (0.5 units of A260), and the solution is added to a total volume of 300 μl with Tris buffer. EDTA (0.1 M). After 30 minutes of incubation at room temperature, the reactions are analyzed by CLAP using 50 mM sodium phosphate, pH 6, as the aqueous phase, with a gradient of 0-30% acetonitrile for 25 minutes. The products are identified by comparison to chromatographies of standard samples. Under these conditions, the phosphodiester oligonucleotide (unmodified) is degraded by 80% of the enzyme while, for the modified oligonucleotide, about 16% of the breakdown product is observed. It is noted that in relation to this date, the best method known to the applicant to carry out the aforementioned invention, is that which is clear from the present description of the invention. Having described the invention as above, property is claimed as contained in the following:

Claims (19)

REVINDICACIQNES

1. A modified oligonucleotide of at least 10 bases, characterized in that it has at least two internucleotide bonds modified at the 3 'carbon of the structure: wherein R5 is a counterion and X and Y are selected from the group consisting of hydrogen (-H-), fluorine (-F-) or hydroxyl (-0H-) moieties.

2. The modified oligonucleotide according to claim 1, characterized in that the modified oligonucleotide is between 10 and 60 pairs in length.

3. The modified oligonucleotide according to claim 2, characterized in that the modified oligonucleotide has at least three internucleotide bonds at the 3 'carbon.

4. The modified oligonucleotide according to claim 3, characterized in that the internucleotide bonds modified at the 3 'carbon are at the 3' end of the modified oligonucleotide.

5. The modified oligonucleotide according to claim 1, characterized in that all of the internucleotide bonds are internucleotide bonds modified at the 3 'carbon.

6. The modified oligonucleotide according to claim 1, characterized in that X and Y are hydrogen atoms.

7. The modified oligonucleotide according to claim 6, characterized in that the modified oligonucleotide is between 12 and 60 bases in length.

8. The modified oligonucleotide according to claim 7, characterized in that the modified oligonucleotide has at least three 3'-methylene modified internucleotide bonds.

9. The modified oligonucleotide according to claim 8, characterized in that all of the internucleotide linkages are 3'-methylene modified internucleotide linkages.

10. The modified oligonucleotide according to claim 1, characterized in that X and Y are independently a hydrogen atom and a hydroxyl group.

11. The modified oligonucleotide according to claim 10, characterized in that the oligonucleotide is between 12 and 60 pairs in length.

12. The modified oligonucleotide according to claim 11, characterized in that the modified oligonucleotide has at least three 3'-hydroxymethylene modified internucleotide bonds.

13. The modified oligonucleotide according to claim 12, characterized in that all of the internucleotide linkages are internucleotide modified 3'-hydroxymethylene.

14. A method for preparing a 3'-alkenyl nucleoside, characterized in that it comprises alkylating a nucleoside substrate with an alkylating reagent of the structure: in which R3 and R4 are organic groups.

15. The method according to claim 14, characterized in that R3 of the alkylating reagent is an ethyl carboxylate group (C02Et) and R4 of the alkylating reagent is a butyl group.

16. A method for preparing a 3'-protected aldehyde protected nucleotide synthon or intermediary (intermediary) characterized in that it comprises the steps of: (a) alkylating a nucleoside substrate to generate a 3'-alkenyl nucleoside; (b) removing the alkenyl function of the 3'-alkenyl nucleoside to generate a 3'-aldehyde nucleoside; (c) protecting the 3 'portion of an aldehyde on the 3'-aldehyde nucleoside; and (d) derivatizing the hydroxyl group at the 5 'position of the protected 3' -aldehyde nucleoside to generate a phosphite function at the 5 'position.

17. A method for synthesizing a modified oligonucleotide characterized in that it comprises coupling a 3 '-aldehyde nucleoside with a synton or 3'-aldehyde protected nucleotide intermediate.

18. The method according to claim 17, characterized in that the synton or 3'-aldehyde protected nucleotide intermediate has the structure: wherein Ph is a phenyl group, R is a protecting group and B is a base selected from the group consisting of adenine, thymidine, guanine, cytosine and uracil.

19. A protected nucleoside of N / N-diphenylimidazolidino aldehyde of the structure: wherein Ph is a phenyl group, R is a protecting group and B is a base selected from the group consisting of adenine, thymidine, guanine, cytosine and uracil.