TWI541249B - 5-position modified pyrimidines and their use - Google Patents

Description

5-position modified pyrimidines and their use Related application

The present application claims priority to U.S. Provisional Application No. 61/323,145, filed on Apr. 12, 2010, which is hereby incorporated by reference.

The present invention relates to the field of nucleic acid chemistry, and more particularly to 5-position modified uridine nucleosides and their phosphite and triphosphate derivatives. The invention also relates to methods of making and using the same. The invention encompasses the use of a modified nucleoside as a partial oligonucleotide or aptamer.

The following description provides general information about the content of the case, however, the information referred to or the published publications cited therein are not prior art to the present case.

To date, considerable attention has been paid to the development of modified nucleosides as therapeutic, diagnostic, and incorporation into oligonucleotides. For example, modified nucleosides such as AZT, ddI, d4T, and others have been used to treat AIDS. 5-Trifluoromethyl-2'-deoxyuridine nucleoside is active against herpes keratitis and 5-iodo-1-(2-deoxy-2-fluoro-bD-arabinose furan) cells Pyrimidine is active against CMV, VZV, HSV-1, HSV-2, and EBV (textbook for drug design and development, Povl Krogsgaard-Larsen and Hans Bundgaard, Eds., Harwood Academic Publishers, 1991, Ch. 15).

Modified nucleosides are useful for diagnostic applications. In these applications, nucleosides are incorporated into the determined positions in the DNA, and various diagnostic methods can be used to determine the position of the modified nucleoside. These methods include isotope markers, fluorescent markers, biotinylation, and strand breaks. An example of a strand break is that a nucleoside is reacted with hydrazine to produce a urea nucleoside, which is then reacted with piperidine to cause a strand break (Maxam-Gilbert's method).

Modified nucleosides are also incorporated into the oligonucleotide. In many methods, oligonucleotides can be used for therapy. In vivo, antisense oligonucleotides can bind to specific genetic coding regions to prevent protein expression or to prevent many cellular functions. In addition, methods such as the Systemic Coordination Index Derivative Derivative (SELEX) program or the systematic evolution of coordinators for exponential enrichment allow selective binding to oligonucleotides of the target molecule (referred to as "aptamers" ") can be identified or manufactured. The SELEX program is described in U.S. Patent No. 5,270,163, the disclosure of which is incorporated herein by reference.

The SELEX method involves selecting oligonucleotides from a candidate mixture to actually achieve the desired binding affinity and selectivity. Starting from a random mixture of oligonucleotides, the method comprises separating the unbound, oligonucleotides that are in contact with the target, self-binding (or interaction) with the target, in favor of binding (or interaction). Oligonucleotides, isolated oligonucleotide-target pairs, proliferating from oligonucleotide-target pairs of isolated oligonucleotides to generate a ligand-enriched oligonucleotide mixture, followed by repeated binding, Separation, division, and proliferation through the desired number of cycles.

Modified nucleosides can be incorporated into antisense oligonucleotides, ribonuclease, and oligonucleotides used or recognized by the SELEX program. Such nucleosides can provide for in vivo and in vitro oligonucleotides to stabilize internal and external nucleases, alter the charge, hydrophilicity or lipophilicity of the molecule, and/or provide different three dimensional structures.

Previously known nucleoside modifications include 2'-position sugar modification, 5-position pyrimidine modification, 8-position oxime modification, exocyclic amine modification, 4-thiouracil nucleoside substitution, 5-bromine or 5-iodine- Substitution of uracil, backbone modification and methylation. Modifications also include 3' and 5' modifications, such as capping. PCT WO 91/14696, incorporated herein by reference, describes a method of chemically modifying an antisense oligonucleotide to enhance entry into cells.

The modification of pyrimidine nucleosides via a palladium coupling reaction is described in U.S. Patent Nos. 5,428,149, 5,591,843, 5,633,361, 5,719,273, and 5,945,527, the disclosures of which are incorporated herein by reference. In some embodiments, the nucleophile and carbon monoxide are coupled to a pyrimidine nucleoside having a leaving group at the 5-position of the pyrimidine ring, preferably forming an ester and a guanamine derivative.

Several methods have been used to render oligonucleotides resistant to decomposition by exo-nucleases. PCT WO 90/15065 describes a phosphite, a phosphorous monosulfonate and/or a diphosphoryl disulfonic acid linked by two or more linkages to the 5' and/or 3' end of the oligonucleotide. A method of making an oligonucleotide resistant to an exonuclease by salt. PCT WO 91/06629 discloses oligonucleotides having one or more phosphodiester linkages between adjacent nucleosides that are replaced by acetal/ketal linkages capable of binding RNA or DNA.

It would be beneficial to provide new nucleosides for treatment and diagnosis and to incorporate them into oligonucleotides. When incorporated into an oligonucleotide, it would be beneficial to provide a new oligonucleotide that exhibits a different high affinity for binding to the target molecule, and/or compared to a naturally occurring nucleoside The preparer, which shows enhanced nuclease and endonuclease resistant oligonucleotides. It is also useful to provide modified nucleotide modifications that provide additional biological activity in addition to endonucleases and exonuclease resistance.

Brief description of the invention

Provided herein is a 5-position modified uridine nucleoside of the formula:

Wherein R is selected from the group consisting of -(CH ₂ ) _n -R ^X1 ; R ^X1 is selected from the group consisting of

* indicates the position at which the R ^X1 group is attached to the (CH ₂ ) _n linking group

Wherein R ^X4 is selected from the group consisting of branched or straight chain lower alkyl (C1-C20); halogen (F, Cl, Br, I); nitrile (CN); boric acid (BO ₂ H ₂ ); carboxylic acid (COOH) Carboxylate (COOR ^X2 ); primary guanamine (CONH ₂ ); secondary guanamine (CONHR ^X2 ); tertiary guanamine (CONR ^X2 R ^X3 ); sulfonamide (SO ₂ NH ₂ ); N-alkane a group consisting of sulfoximine (SONHR ^X2 ); wherein R ^X2 and R ^X3 are independently selected from branched or straight chain lower alkyl (C1-C20); phenyl (C ₆ H ₅ ); ^X4 substituted benzene ring (R ^X4 C ₆ H ₄ ) wherein R ^X4 is as defined above; carboxylic acid (COOH); carboxylate (COOR ^X5 ) wherein R ^X5 is a branched or straight chain lower alkyl group ( C1-C20); and a cycloalkyl group wherein R ^X2 = R ^X3 = (CH ₂ ) n; wherein n = 0-10; wherein X is selected from, but not limited to, -H, -OH a group consisting of -OMe, -O-allyl, -F, -OEt, -OPr, -OCH ₂ CH ₂ OCH ₃ and -azido; wherein R' is selected from, but not limited to, - a group consisting of OAc; -OBz; and -OSiMe ₂ tBu; wherein R" is selected from, but not limited to, H, DMT, and triphosphate (-P(O)(OH)-OP(O)(OH) group or the salt thereof _{-OP (O) (OH) 2} ) consisting of; And in Can be substituted with carbocyclic analogues, α-raceomere, epimerose, such as acacia, xylose or lyxose, pyranose, furanose, sedoheptulose, acyclic Analogs and nucleoside analogs of nucleobases, such as methyl ribosides.

Included are 3'-phospholipids and 5'-triphosphate derivatives or salts thereof of the compound of the formula:

All of them are as defined above.

Prepared using standard synthetic or enzymatic methods, the compounds herein can be incorporated into oligonucleotides or aptamers.

Also provided herein are methods for making the compounds disclosed herein as well as compounds made by the methods.

In a specific embodiment, there is provided a process for the preparation of a C-5 modified amine carbonyl pyrimidine, which comprises: a 5-position modified pyrimidine with a trifluoroethoxycarbonyl group and an amine in the presence of a base Reaction; and isolation of the C-5 modified amine carbonyl pyrimidine.

In another embodiment, there is provided a process for the preparation of a C-5 modified aminocarbonylpyrimidine 3'-phosphamidite, the process comprising: modifying a C-5 modified amine carbonyl in the presence of a base Pyrimidine is reacted with cyanoethyldiisopropyl-chlorophosphoramine; and the 3'-phosphite is isolated.

In yet another embodiment, a method of preparing a 5-5-triphosphate of a C-5 modified aminocarbonylpyrimidine is provided, the method comprising:

a) a C-5 modified amine carbonyl pyrimidine having the formula:

Wherein R and X are as defined above, reacting with an acid anhydride in the presence of a base, followed by cleavage of the 5'-DMT group with an acid to form a 3'-acetate having the following structure:

b) performing a Ludwig-Eckstein reaction on the 3'-acetate of step a) followed by anion exchange chromatography;

c) 5'-triphosphate or a salt thereof which is isolated from a C-5 modified aminocarbonylpyrimidine having the following structure:

Detailed description of the invention

A detailed description will now be made with reference to representative embodiments of the invention. While the invention has been described in terms of the specific embodiments illustrated, it is understood that the invention is not limited to the specific embodiments. On the contrary, the invention is intended to cover all modifications, modifications, and equivalents

It will be apparent to those skilled in the art that many methods and materials that are similar or equivalent to those disclosed herein can be used herein and are within the scope of the invention. The disclosure is not limited by the methods and materials described.

The technical and scientific terms used herein are the same as those of those skilled in the art to which the invention pertains, unless otherwise defined. Although the invention may be operated or tested using any methods, devices, and materials that are similar or equivalent to those disclosed herein, the preferred methods, devices, and materials are described below.

All of the publications, published patent documents, and patent applications cited herein are hereby incorporated by reference in their entireties in the entireties in All of the publications, published patent documents, and patent applications cited herein are hereby incorporated by reference in their entirety in their entireties in the the the the the the the the

The singular articles "a", "the" and "the" are intended to mean the meaning of the singular and "the" and "the" Therefore, "an aptamer" includes a mixture of aptamers and the like.

As used herein, the word "about" refers to an unimportant modification or a numerical change, and thus the basic function of the object associated with the value does not change.

The term "each" as used herein in reference to a plurality of items refers to at least two of the plurality. Not all of the items need to meet the additional restrictions.

The articles "including", "comprising", "including", and any variations thereof are used in the non-closed usage, and the process, method, process limitation, or composition of matter included Components, and may include those not explicitly recited, or by processes, methods, process limitations, or substance compositions.

The term "nucleotide" as used herein, refers to a ribonucleotide or deoxyribonucleotide, or a modified form thereof, and analogs thereof. The types of nucleotides are steroids (eg, adenine, hypoxanthine, and derivatives and analogs thereof), and pyrimidines (eg, cytosine, uracil, thymine, and derivatives and analogs thereof). .

Compound

In a specific embodiment, the present disclosure provides a compound of the formula:

Wherein R is selected from the group consisting of -(CH ₂ ) _n -R ^X1 ; R ^X1 is selected from the group consisting of

* indicates the position at which the R ^X1 group is attached to the (CH ₂ ) _n linking group

Wherein R ^X4 is selected from, but not limited to, branched or linear lower alkyl (C1-C20); halogen (F, Cl, Br, I); nitrile (CN); boric acid (BO ₂ H ₂ ); carboxylic acid (of COOH); carboxylic acid ester (COOR ^X2); a Amides (CONH _2); two Amides (CONHR ^X2); three Amides (CONR ^X2 R ^X3); Amides sulfonamide (SO ₂ NH ₂ a group consisting of N-alkylsulfonamide (SONHR ^X2 ); wherein R ^X2 , R ^X3 are independently selected from, but not limited to, branched or straight chain lower alkyl (C1-C20); Phenyl (C ₆ H ₅ ); R ^X4 substituted benzene ring (R ^X4 C ₆ H ₄ ), wherein R ^X4 is as defined above; carboxylic acid (COOH); carboxylate (COOR ^X5 ), wherein R ^X5 is a branched or straight chain lower alkyl group (C1-C20); and a cycloalkyl group wherein R ^X2 = R ^X3 = (CH ₂ ) _n ; wherein n = 0-10; wherein X is selected from , but not limited to, a group consisting of -H, -OH, -OMe, -O-allyl, -F, -OEt, -OPr, -OCH ₂ CH ₂ OCH ₃ and -azido; wherein R ' is selected from the group consisting of, but not limited to, -OH; -OAc; -OBz; -C(O)CH ₂ OCH ₃ and -OSiMe ₂ tBu; wherein R" is selected from, but not limited to, H 4,4'-dimethoxytrityl , Groups, or salts of he (DMT) and triphosphate (-P (O) (OH) -OP (O) (OH) -OP (O) (OH) 2) consisting of; and wherein Can be substituted with carbocyclic analogues, α-raceomere, epimerose, such as acacia, xylose or lyxose, pyranose, furanose, sedoheptulose, acyclic Analogs and nucleoside analogs of nucleobases, such as methyl ribosides.

In another embodiment, the present disclosure provides a compound of the formula or a salt thereof:

Wherein R, R" and X are as defined above. The compound of the formula is a formula for the incorporation of a modified nucleoside into an oligonucleotide by chemical synthesis.

In yet another embodiment, the present disclosure provides a compound of the formula or a salt thereof:

Wherein R, R' and X are as defined above. The compounds of this formula are of a general formula for the incorporation of modified nucleosides into oligonucleotides by enzyme synthesis.

As used herein, the term "C-5 modified carboquinolidine nucleoside" or "C-5 modified amine carbonyluridine nucleoside" means having a carboxy group at the C-5 position of the uracil nucleoside. Indoleamine (-C(O)NH-) modified uridine nucleoside, including, but not limited to, the above part (R). Examples of C-5 modified carbamazepine uridine nucleosides include U.S. Patent Nos. 5,719,273 and 5,945,527, and U.S. Provisional No. 5, 2010, entitled "Nuclease-Resistant Oligonucleotides" Application No. 61/422,957 (the '957 application). Representative C-5 modified pyrimidines include: 5-(N-benzylcarbamoylamine)-2'-deoxyuracil (BndU), 5-(N-benzylcarbendazimamine) -2'-O-methyluracil nucleoside, 5-(N-benzylcarbamoylamine)-2'-fluorouracil, 5-(N-isobutylcarboxyguanamine)-2 '-Deoxyuridine nucleoside (iBudU), 5-(N-isobutylcarbophthalamide)-2'-O-methyluridine, 5-(N-isobutylcarboxyguanamine)- 2'-fluorouracil nucleoside, 5-(N-tryptamine carboxamide)-2'-deoxyuridine (TrpdU), 5-(N-tryptamine carboxamide)-2' -O-methyluridine nucleoside, 5-(N-tryptamine carboxamide)-2'-fluorouracil nucleoside, 5-(N-[1-(3-trimethylammonium)propyl) Carboxylamidine)-2'-deoxyuridine nucleoside chloride, 5-(N-naphthylmethylcarboxamide)-2'-deoxyuracil (NapdU), 5-(N- Naphthylmethylcarboxamide)-2'-O-methyluridine, 5-(N-naphthylmethylcarboxamide)-2'-fluorouracil or 5-(N- [1-(2,3-Dihydroxypropyl)]carboxamide)-2'-deoxyuridine nucleoside).

Specific examples of C-5 modified amine carbonyluridines described herein for illustrative purposes include the following compounds, as well as the 5'-triphosphate and 3'-phosphoramine of the compound and salts thereof. The synthesis of these is described in Examples 1-5.

5-(4-fluorobenzylamine carbonyl)-2'-deoxyuridine nucleoside,

5-(( R )-2-furanmethylmethylamine carbonyl)-2'-deoxyuridine nucleoside,

5-(( S )-2-furanmethylmethylamine carbonyl)-2'-deoxyuridine nucleoside,

5-(2-(4-morpholinyl)ethylaminecarbonyl)-2'-deoxyuridine nucleoside;

5-(2-(1-(3-Ethyl-benzimidazolyl))ethylaminecarbonyl)-2'-deoxyuridine nucleoside.

The chemical modification of the C-5 modified uridine nucleosides described herein can also be combined with 2'-position saccharide modification, exocyclic amine modification, and substitution of 4-thiouracil nucleoside, alone or in any combination.

Salt

The corresponding salt of the compound, for example, a pharmaceutically acceptable salt, may be prepared, purified, and/or treated as convenient or desirable. Examples of pharmaceutically acceptable salts are as described in Berge et al., "Pharmaceutically Acceptable Salts" (1977) J. Pharm. Sci. 66:1-19.

For example, it may be (, such as -COOH may be -COO ^-) if the compound is anionic, or has a functional group of an anionic, a salt may be formed with a suitable cation. Examples of suitable inorganic cations include, but are not limited to, alkali metal ions such as Na ⁺ and K ⁺ , alkaline earth metal cations such as Ca ²⁺ and Mg ²⁺ , and other cations such as Al ⁺³ . Examples of suitable organic cations include, but are not limited to, ammonium ions (ie, NH ₄ ⁺ ) and substituted ammonium ions (eg, NH ₃ R ^x+ , NH ₂ R ^x ₂ ⁺ , NHR ^x ₃ ⁺ , NR ^x ₄ ⁺ ). Some suitable substituted ammonium ions are obtained from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, Phenylbenzylamine, choline, melomin and trimethylolamine, and amino acids such as lysine and arginine. An example of a common quaternary ammonium ion is N(CH ₃ ) ₄ ⁺ .

If the compound is cationic or has a functional group which may be cationic (for example, -NH ₂ may be -NH ₃ ⁺ ), a salt may be formed with a suitable anion. Examples of suitable inorganic anions include, but are not limited to, those derived from the following inorganic acids: hydrochloric acid, hydrobromic acid, hydroiodic acid, sulfuric acid, sulfurous acid, nitric acid, nitrous acid, phosphoric acid, and phosphorous acid.

Examples of suitable organic anions include, but are not limited to, those derived from the following organic acids: 2-acetoxybenzoic acid, acetic acid, ascorbic acid, aspartic acid, benzoic acid, camphorsulfonic acid, cinnamic acid, citric acid, Ethylenediaminetetraacetic acid, ethanedisulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxy maleic acid, hydroxynaphthoic acid, isethionic acid, lactic acid , lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, galactonic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, benzenesulfonic acid, Acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, p-aminobenzenesulfonic acid, tartaric acid, toluenesulfonic acid, and valeric acid. Examples of suitable polyorganic anions include, but are not limited to, those derived from the following polybasic organic acids: tannic acid, carboxymethyl cellulose.

Unless otherwise stated, the specific compounds mentioned also include the salt forms thereof.

Oligonucleotide preparation

In one aspect, the present disclosure provides a method of preparing a modified oligonucleotide using the modified nucleoside described herein, alone or in combination with other modified nucleosides and/or naturally occurring nucleosides. . The automated synthesis of oligodeoxynucleosides is a routine procedure in many laboratories (see, eg, Matteucci, MD and Caruthers, MH, (1990) J. Am. Chem. Soc., 103: 3185-3191, by reference It is incorporated herein). The synthesis of oligoribonucleosides is also well known (see, for example, Scaringe, SA, et al. , Nuc; eic Acids Res. 18: 5433-5441 (1990), which is incorporated herein by reference). As indicated above, phosphites are used to incorporate modified nucleosides into oligonucleotides by chemical synthesis, and triphosphates are used to incorporate modified nucleosides into oligonucleides by enzyme synthesis. Glycosylate. (See, eg, Vaught, JV, et al. (2010) J. Am. Chem. Soc. , 132, 4141-4151; Gait, MJ "Practical Oligonucleotide Synthesis Method" (1984) IRL Press (Oxford, UK) Herdewijn, P. "Oligonucleotide Synthesis" (2005) (Humana Press, Totowa, NJ (each of which is incorporated herein in its entirety by reference).

As used herein, when used in an oligonucleotide, the terms "modification,""modified," and any variations thereof, mean the four constituent nucleotide bases of an oligonucleotide (ie, A, G, T). At least one of /U, and C) is an analog or ester of a naturally occurring nucleotide. In some embodiments, the modified nucleotides exhibit nuclease resistance of the oligonucleotide. Other modifications may include backbone modifications, methylation, atypical base pairing combinations, such as isobase isocytidine and isoguanidine, and the like. Modifications may also include 3' and 5' modifications, such as capping. Other modifications may include the substitution of one or more naturally occurring nucleotides with an analog, such as, for example, an uncharged linker (eg, methylphosphonate, phosphotriester, decylamine phosphate). , urethane, etc.) and those with a charged linker (eg, phosphorothioate, dithiophosphate, etc.), with an intercalator (eg, acridine, psoralen, etc.), containing a chelating agent ( For example, metals, radioactive metals, boron, oxidized metals, etc., those containing alkylating agents, and those having modified moieties (eg, alpha-rotating isomeric nucleic acids, etc.). In addition, a hydroxyl group normally present on a sugar of a nucleotide may be replaced by a phosphonic acid group or a phosphate group; protected by a standard protecting group; or activated to make additional linkages to additional nucleotides or to a solid support body. The OH groups at the 5 ' and 3 ' ends may be phosphorylated, or may be an amine, having an organic capping moiety of from about 1 to about 20 carbons, and in one embodiment, from about 10 to about 80 kDa. The diol (PEG) polymer, in another embodiment, is a PEG-substituted polymer of from about 20 to about 60 kDa or other hydrophilic or hydrophobic bio or synthetic polymer.

Polynucleotides may also contain similar forms of conventional ribose or deoxyribose, including 2'-O-methyl, 2'-O-allyl, 2'-O-ethyl, 2'-O-propyl. Base, 2'-O-CH ₂ CH ₂ OCH ₃ , 2'-fluoro- or 2'-azido group, carbocyclic sugar analog, α-raceomere, isomeric sugar, such as gum arabic , xylose or sucrose, pyranose, furanose, sedoheptulose, acyclic analogs, and nucleoside analogs of nucleobases, such as methyl ribosides. As indicated above, one or more phosphodiester linkages can be replaced by other selective linking groups. These selective linking groups include, wherein the phosphate is P(O)S ("thioester"), P(S)S ("dithioester"), (O)NR ^x ₂ ("guanamine" Specific examples of the replacement of ester "), P(O)R ^x , P(O)OR ^x , CO or CH ₂ ("methylal"), wherein each R ^x or R ^x ' is independently H or substituted Or an unsubstituted alkyl group (C1-C20) optionally containing an ether (-O-) linkage, an aryl group, an alkenyl group, a cycloalkyl group, a cycloalkenyl group or an aralkyl group. Not all linkages in a polynucleotide must be identical. Substitution in the form of analogs of sugars, terpenes, and pyrimidines can be advantageous in designing the final product, for example, the selective backbone can be similar to the polyamine backbone.

If the modification is present, the nucleotide structure can be modified before or after the polymer combination. The nucleotide sequence can be interrupted by a non-nucleotide component. The polynucleotide may be further modified by, for example, bonding to a labeling component after polymerization.

As used herein, "nucleic acid", "oligonucleotide" and "polynucleotide" are used to denote a polymer of nucleotides and include DNA, RNA, DNA/RNA hybrids, and such nucleic acids, oligonucleosides. Modifications of acids and polynucleotides, including various entities or moieties attached to any position of the nucleotide unit. The terms "polynucleotide", "oligonucleotide" and "nucleic acid" include double- or single-stranded molecules as well as triple helix molecules. Nucleic acids, oligonucleotides, and polynucleotides are terms with a broader meaning than the term aptamer. Therefore, nucleic acids, oligonucleotides, and polynucleotides include nucleotide polymers that are aptamers. However, nucleic acids, oligonucleotides, and polynucleotides are not limited to aptamers.

As used herein, when referring to a modification of a nucleic acid, "at least one nucleotide" means one, several or all nucleotides in a nucleic acid that represent any or all of any or all of the nucleic acids present. A, C, T, G or U may or may not be modified.

In other aspects, the present disclosure provides a method of preparing an aptamer and an enzyme-linked immuno aptamer using the modified nucleoside described herein, alone or in combination with other modified nucleosides and/or naturally occurring nucleosides ( SOMAmer) (described below). In a specific embodiment, aptamers and enzyme-linked immunoaptamers are prepared using the conventional SELEX or modified SELEX procedures described below.

As used herein, "nucleic acid ligand", "aptamer", "enzyme aptamer" and "colon" refer to a non-naturally occurring nucleic acid that has a desired effect on a target molecule. Desirable effects include, but are not limited to, binding to a target, catalytically altering the target, reacting with the target to modify or alter the functional activity of the target or target, and covalently attaching to the target (eg, suicidal) Inhibitors), as well as promoting the reaction between the target and other molecules. In a specific embodiment, the reaction molecule specifically binds to the target molecule, and the target molecule is a three-dimensional chemical structure rather than a mechanism different from the Watson/Crick base pairing or triple helix configuration. A polynucleotide that binds to a nucleic acid ligand, wherein the aptamer is not a nucleic acid having a known physiological function of binding by a label molecule. An aptamer for a particular target comprises a nucleic acid identified by a mixture of candidate nucleic acids, wherein the aptamer is a ligand for the target, and the method of identification comprises: (a) contacting the candidate mixture with the target, wherein Other nucleic acids in the candidate mixture that are separable from the remainder of the candidate mixture, the nucleic acid has enhanced affinity for the target; (b) separates the nucleic acid with enhanced affinity from the remainder of the candidate mixture; and (c) A nucleic acid having enhanced affinity is amplified to generate a ligand-enriched nucleic acid mixture, thereby identifying an aptamer of the target molecule. It is understood that the affinity effect is different in degree; however, in this context, the "specific binding affinity" of the aptamer for its target represents compared to other non-standards in the mixture or sample. The binding of the target component, the aptamer typically binds to its target with a higher degree of affinity. An "aptamer", "enzyme aptamer" or "nucleic acid ligand" is a collection of copies of one or a series of nucleic acid molecules having a particular nucleotide sequence. Aptamers can include any suitable number of nucleotides. "Plural aptamer" refers to a collection of more than one such molecule. Different aptamers can have the same or different numbers of nucleotides. The aptamer can be DNA or RNA and can be single-stranded, double-stranded, or contain double-stranded or triple-stranded regions.

As used herein, an "enzyme-linked immunosuppressant" or a modified aptamer with a low off-rate indicates that the aptamer is characterized by an improved dissociation rate. Enzyme-linked immunoaptamers can be produced using the SELEX method described in U.S. Patent Publication No. 20090004667, entitled "Method for Producing Aptamers Using Improved Dissociation Rate".

As used herein, "protein" is synonymous with "peptide", "polypeptide" or "peptide fragment". A "purified" multipeptide, protein, peptide or peptide fragment is substantially free of cellular material or other protein contamination from cells, tissues, or cell-free sources that acquire amino acid sequences, or substantially free of chemical synthesis Chemical precursors or other chemicals used.

SELEX method

The terms "SELEX" and "SELEX program" as used interchangeably herein generally refer to (1) selecting a nucleic acid that interacts with a target molecule in a desired manner, such as binding to a protein with high affinity, and (2) an amplification station. A combination of selected nucleic acids. The SELEX program can be used to identify aptamers with high affinity for specific target molecules or biomarkers.

SELEX typically includes preparing a mixture of candidate nucleic acids, binding a candidate mixture to a desired target molecule to form an affinity complex, isolating an affinity complex from an unbound candidate nucleic acid, isolating the self-affinity complex, and isolating the nucleic acid, purifying the nucleic acid. And identify specific aptamer sequences. This procedure can include multiple rounds to further increase the affinity of the selected aptamer. The amplification step can be included in one or more time points in this procedure. See, for example, U.S. Patent No. 5,475,096 entitled "Nucleic Acid Complexes". The SELEX program can be used to generate aptamers that covalently bind to their targets as well as aptamers that non-covalently bind to their targets. See, for example, U.S. Patent No. 5,705,337, entitled "Systematic Evolution of Nucleotide Coordination by Exponential Enrichment: Chemical SELEX".

The SELEX program can be used to identify high affinity aptamers containing modified nucleotides that confer improved characteristics to the aptamer, such as, for example, improved in vivo stability or improved delivery characteristics. Examples of such modifications include chemical substitution of ribose and/or phosphate and/or base positions. The aptamer identified by the SELEX program contains modified nucleotides, which are described in U.S. Patent No. 5,660,985, the disclosure of which is incorporated herein by reference. Chemically modified at the 5'- and 2'-positions of pyrimidines. U.S. Patent No. 5,580,737, as above described highly specific aptamers, which contain one or more of the 2'-amino (2'-NH _2), 2'- fluoro (2'-F), and / or 2'-O-methyl (2'-OMe) is a modified nucleotide. See also U.S. Patent Application Publication No. 20090098549, entitled "SELEX &PHOTOSELEX", which describes the extended physical and chemical properties of nucleic acid libraries and their use in SELEX and PHOTOSELEX.

SELEX can also be used to identify aptamers with the desired dissociation rate characteristics. See U.S. Patent Publication No. 2009 0004 667, the disclosure of which is incorporated herein by reference in its entirety in its entirety in its entirety in Improved SELEX method. It describes a method for producing aptamers and photoaptamers that dissociate from their respective target molecules at a lower off-rate. The method comprises contacting a candidate mixture with a target molecule, allowing the nucleic acid-target complex to occur, and a low dissociation rate enrichment procedure, wherein the nucleic acid-target complex with high dissociation rate dissociates and no longer Formed, while the complex with low dissociation rate remains intact. In addition, the method includes the use of a modified nucleotide to produce an aptamer having improved dissociation rate performance when manufacturing a candidate nucleic acid mixture (see U.S. Patent Application Serial No. 20090098549, entitled "SELEX and photo SELEX"). (See also U.S. Patent No. 7,855,054 and U.S. Patent Application Serial No. 20070166740). Each of these applications is hereby incorporated by reference in its entirety.

As used herein, "target" or "target molecule" or "target" refers to any compound on which the nucleic acid can act in the desired manner. Target molecules can be proteins, peptides, nucleic acids, carbohydrates, lipids, glycans, glycoproteins, hormones, receptors, antigens, antibodies, viruses, pathogens, toxic substances, metabolites, transition state analogs, cofactors, Inhibitors, drugs, dyes, nutrients, growth factors, cells, tissues, any part or fragment of any of the foregoing, without limitation. Virtually any chemical or biological agonist can be a suitable target. Molecules of any size can be used as targets. Targets can also be modified in a specific manner to enhance the likelihood and intensity of interaction between the target and the nucleic acid. The target may also include any small change in a particular compound or molecule, such as in the case of a protein, such as small changes in the amino acid sequence, disulfide bond formation, saccharification, lipidation, acetylation, phosphorylation, or Any other regulation or modification, such as engagement with a labeling component, does not substantially alter the identity of the molecule. A "target molecule" or "target" is a collection of copies of one or a series of molecular or multi-molecular structures that can bind to an aptamer. A plural "target molecule" or "target" means more than one collection of molecules. The target in the specific example of the SELEX program of U.S. Patent No. 6,376,190, entitled "The modified SELEX program without purification of the protein" is the peptide.

Chemical synthesis

Methods of chemically synthesizing compounds provided herein are described herein. These and/or other conventional methods may utilize modifications and/or adaptations to known methods to facilitate the synthesis of additional compounds provided herein.

Referring to Scheme 1, in one method, the modified aminocarbonylpyrimidine at position C-5 herein is prepared by reacting a 5-position modified pyrimidine with a trifluoroethoxycarbonyl group and an amine in the presence of a base; And prepared separately from the C-5 modified aminocarbonylpyrimidine.

In some embodiments, the trifluoroethoxycarbonylpyrimidine is selected from the group consisting of, but not limited to, compounds having the structure:

Wherein X is selected from the group consisting of, but not limited to, -H, -OH, -OMe, -O-allyl, -F, -OEt, -OPr, -OCH ₂ CH ₂ OCH ₃ and -azido. Group, and where Can be substituted with carbocyclic analogues, α-raceomere, epimerose, such as acacia, xylose or lyxose, pyranose, furanose, sedoheptulose, acyclic Analogs and nucleoside analogs of nucleobases, such as methyl ribosides. In some embodiments, the amine is selected from the group consisting of, but not limited to, a compound having the formula RNH ₂ wherein R is selected from the group consisting of -(CH ₂ ) _n -R ^X1 ; R ^X1 Is selected from the group consisting of:

Wherein R ^X4 is selected from the group consisting of branched or straight chain lower alkyl (C1-C20); halogen (F, Cl, Br, I); nitrile (CN); boric acid (BO ₂ H ₂ ); carboxylic acid (COOH) Carboxylate (COOR ^X2 ); primary guanamine (CONH ₂ ); secondary guanamine (CONHR ^X2 ); tertiary guanamine (CONR ^X2 R ^X3 ); sulfonamide (SO ₂ NH ₂ ); N-alkane a group consisting of sulfoximine (SONHR ^X2 ); wherein R ^X2 and R ^X3 are independently selected from branched or straight chain lower alkyl (C1-C20); phenyl (C ₆ H ₅ ); ^X4 substituted benzene ring (R ^X4 C ₆ H ₄ ) wherein R ^X4 is as defined above; carboxylic acid (COOH); carboxylate (COOR ^X5 ) wherein R ^X5 is a branched or straight chain lower alkyl group ( C1-C20); and a cycloalkyl group wherein R ^X2 = R ^X3 = (CH ₂ ) _n ; and wherein n = 0-10.

In a particular embodiment, the amine is selected from the group consisting of:

In some embodiments, the base is selected from the group consisting of tertiary amines consisting of triethylamine, diisopropylamine, and the like.

Referring to Scheme 1, there is also provided a method for synthesizing a 3'-phosphamidamine of a C-5 modified aminocarbonylpyrimidine, comprising: modifying the C-5 modified aminocarbonylpyrimidine and cyanide in the presence of a base Ethyl diisopropyl-chlorophosphoramidite reaction; and isolation of the 3'-phosphite. In some embodiments, the C-5 modified amine carbonyl pyrimidine has the structure:

Wherein R and X are as defined above. In some embodiments, the base is selected from the group consisting of tertiary amines consisting of triethylamine, diisopropylamine, and the like.

Referring again to Scheme 1, there is also provided a method for the synthesis of a 5-5-triphosphate of a C-5 modified aminocarbonylpyrimidine comprising:

a) reacting a C-5 modified amine carbonyl pyrimidine having the formula: in the presence of a base with an anhydride:

Wherein R and X are as defined above, followed by the use of an acid to cleave the 5'-DMT group to form a 3'-acetate having the structure:

b) performing a Ludwig-Eckstein reaction on the 3'-acetate of step a) followed by anion exchange chromatography;

c) 5'-triphosphate or a salt thereof which is isolated from a C-5 modified aminocarbonylpyrimidine having the following structure:

The base used is selected from the group consisting of, but not limited to, a tertiary amine. In some embodiments, the base is pyridine. The acid in step a is selected from the group consisting of, but not limited to, dichloroacetic acid, trichloroacetic acid, and 1,1,1,3,3,3-hexafluoro-2-propanol.

In an alternative method, the trifluoroethoxycarbonylpyrimidine has the following structure:

Referring to Scheme 2, the compound is formed by reacting Compound (7) of Scheme 2 with carbon monoxide and trifluoroethanol in the presence of a palladium catalyst and a base. The base is selected from the group consisting of, but not limited to, a tertiary amine selected from triethylamine.

Included herein are compounds prepared by the various methods described above.

The following examples are for illustrative purposes only and are not intended to limit the scope of the invention as defined by the appended claims. All of the embodiments described herein are practiced using standard techniques that are known and commonly employed by those skilled in the art. Common molecular biotechnology described in the following examples can be in accordance with standard laboratory manuals, such as Sambrook et al. Molecular Cloning: A Laboratory Manual, 3rd. ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001).

The modified nucleosides described in Examples 1-3 and 5 were produced in the following general manner. The nomenclature used herein is based on the system described by Matsuda et al., Nucleic Acids Research 1997, 25: 2784-2791.

Example 1 5'- O Synthesis of -DMT-dU-5-carboxyguanamine (3a-e)

5'- O -Dimethoxytrityl-5-(4-fluorobenzylaminocarbonyl)-2'-deoxyuridine (3a) . By Matsuda et al. (Nomura, Y.; Ueno, Y.; Matsuda, A. Nucleic Acids Research 1997 , 25: 2784-2791; Ito, T., Ueno, Y.; Matsuda, A. Nucleic Acids Research 2003 , The method of 31:2514-2523) was used to prepare the starting material, 5'- O -dimethoxytrityl-5-trifluoroethoxycarbonyl-2'-deoxyuridine ( 1 ). Heating ( 1 ) (9.85 g, 15 mmol), 4-fluorobenzylamine ( 2a ) (2.25 g, 18 mmol, 1.3 eq.), triethylamine (60 ° C) at an inert atmosphere. A solution of 4.2 mL, 30 mmol) and anhydrous acetonitrile (30 mL) was continued for 2-24 hours. The quantitative conversion of ( 1 ) to indoleamine ( 3a ) was confirmed by thin layer column chromatography (tank 60, 5% methanol/dichloromethane) or HPLC. The reaction mixture was concentrated in vacuo and purified by flash chromatography (Still, WC; Kahn, M.; Mitra, A. J. Org. Chem. 1978 , 43: 2923) using 0-3% methanol. The residue was purified by washing with 1% triethylamine / 99% ethyl acetate. The fractions containing the pure product are combined and evaporated. A trace of residual solvent was removed by co-evaporation with dry acetonitrile, followed by drying under high vacuum to afford ( 3a ) (6.57 g, 64% yield) as a white solid. ¹ H-NMR (300 MHz, CD ₃ CN) δ 2.20-2.40 (2H, m), 3.28 (2H, d, J = 4.3 Hz), 3.76 (6H, s), 4.01 (1H, dd, J = 3.8 , 4.2 Hz), 4.26-4.30 (1H, m), 4.48 (2H, bd, J = 6.1 Hz), 6.11 (1H, t, J = 6.5 Hz), 6.85-7.46 (13H, m), 7.03-7.36 (4H, m), 8.58 (1H, s), 9.01 (1H, t, J = 6.1 Hz). MS ( m/z ) for C ₃₈ H ₃₆ FN ₃ O ₈ , calculated 681.25; ] ^- .

5'- O -Dimethoxytrityl-5-(( R )-2-furanmethylmethylaminecarbonyl)-2'-deoxyuridine (3b) . Using (R) -2- methyl-furan-methylamine (2b), as prepared in (3a) was prepared as the compound (3b) and isolated to give a white solid (9.3 g, 94% yield). The eluate for column chromatography was 1% triethylamine / 4% methanol / 95% ethyl acetate. ¹ H-NMR (CD ₃ CN) δ 1.51-1.57 (1H, m), 1.84-1.94 (3H, m), 2.18-2.38 (2H, m), 3.25-3.52 (4H, m overlap), 3.66-3.93 (3H, m overlap), 3.78 (6H, s), 3.97-4.02 (1H, m), 4.24-4.29 (1H, m), 6.12 (1H, t, J = 6.5), 6.86-7.47 (13H, m ), 8.54 (1H, s) , 8.83 (1H, bs) .MS (m / z) for _{C 36 H 39 N 3 O 9} , calc. 657.27; Found 656.5 [MH] ^-.

5'- O -Dimethoxytrityl-5-(( S )-2-furanmethylmethylaminecarbonyl)-2'- deoxyuridine (3c) . Using (S) -2- methyl-furan-methylamine (2c), as prepared in (3b) was prepared like Compound (3c) and isolated to give a white solid (9.9 g, 99% yield). ¹ H-NMR (CD ₃ CN) δ 1.50-1.59 (1H, m), 1.84-1.95 (3H, m), 2.18-2.40 (2H, m), 3.24-3.50 (4H, m overlap), 3.69-3.97 (3H, m overlap), 3.78 (6H, s), 3.98-4.02 (1H, m), 4.25-4.30 (1H, m), 6.14 (1H, t, J = 6.5), 6.87-7.47 (13H, m .), 8.54 (1H, s ), 8.84 (1H, bs) MS (m / z) for _{C 36 H 39 N 3 O 9} , calc. 657.27; Found 656.5 [MH] ^-.

5'- O - dimethoxytrityl-5- (2- (4-morpholinyl) ethanamine carbonyl) -2'-oxo-uracil nucleosides to (3d). Using (2-(4-morpholinyl)-ethylamine ( 2d ), Compound ( 3d ) was obtained as obtained ( 3a ), and was isolated to afford white solid (8.2 g, 80% yield). The eluate for column chromatography was 5% methanol/2% triethylamine / 93% dichloromethane. ¹ H-NMR (CD ₃ CN) δ 2.21-2.39 (2H, m), 2.39-2.41 (4H, m), 2.48 (2H, t, J = 6.2 Hz), 3.27-3.29 (2H, m), 3.41 (2H, dt, J = 5.8, 6.2 Hz), 3.61-3.64 (4H, m), 3.78 (6H, s), 3.98-4.02 (1H, m), 4.25-4.30 (1H, m), 6.10 (1H) , t, J = 6.4), 6.86-7.47 (13H, m), 8.55 (1H, s), 8.79 (1H, bt, J ~ 6 Hz). MS( m/z ) for C ₃₇ H ₄₂ N ₄ O ₉ , calculated value 686.30; experimental value 685.7 [MH] ^- .

5'- O -Dimethoxytrityl-5-(2-(N-benzimidazolyl)ethylaminecarbonyl) -2'-deoxyuridine (3e) . Compound ( 3e ) was prepared as in the preparation of ( 3a ) using N-benzimidazolyl-2-ethylamine ( 2e ) (CAS RN64928-88-7). The eluent for column chromatography was 2% methanol / 1% triethylamine / 97% dichloromethane. The isolated pure product was a tan solid (8.2 g, 74.5% yield). ¹ H-NMR (CD ₃ CN) δ 2.20-2.36 (2H, m), 3.27-3.29 (2H, m), 3.60 (2H, q, J = 6.5 Hz), 3.758 (3H, s), 3.762 (3H) , s), 3.97 (2H, t, J = 6.5 Hz), 3.98-4.02 (1H, m), 4.27-4.30 (1H, m), 6.09 (1H, t, J = 6.5 Hz), 6.86-7.48 ( 13H,m), 6.91-7.10(4H,m), 8.52(1H,s), 8.76(1H,t, J =6.1 Hz).MS( m/z ) for C ₄₀ H ₃₉ N ₅ O ₉ , Value 733.27; experimental value 732.0 [MH] ^- .

Example 2 5'- O Synthesis of -DMT-nucleoside CE-phosphoramidite (4a-4e)

5'- O -Dimethoxytrityl-5-(4-fluorobenzylaminecarbonyl)-3'- O -[(2- cyanoethyl)(N,N-diisopropylamino) Phosphyl]-2'-deoxyuridine (4a) . The solution of DMT-protected nucleoside ( 3a ) (4.00 g, 5.9 mmol) in dry dichloromethane (40 mL) was cooled to approximately -10 ° C under dry argon. Diisopropylethylamine (3.1 mL, 17.6 mmol, 3 eq.) was added followed by 2-cyanoethyldiisopropylphosphonite (1.7 mL, 7.7 mmol, 1.3 eq.). The solution was stirred for one hour, and the completion of the reaction was confirmed by thin-layer column chromatography (gelatin 60, ethyl acetate /hexane). The reaction mixture was partitioned between ice cold 2% sodium bicarbonate (200 mL) and ethyl acetate (200 mL). The organic layer was washed with brine, dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by flash column chromatography using mobile phase of 1% triethylamine / 99% ethyl acetate. The fractions containing the pure product were combined and evaporated in vacuo (<30 ° C). A trace of residual solvent was removed by co-evaporation with anhydrous acetonitrile, followed by drying under high vacuum to afford ( 4a ) (4.10 g, 80% yield) as a white solid foam. ¹ H-NMR (CD ₃ CN, two isomers) δ 1.02-1.16 (12H, m), 2.27-2.57 (2H, m), 2.51/2.62 (2H, 2t, J = 6.0/6.0 Hz), 3.25-3.37 (2H, m), 3.50-3.79 (4H, m overlap), 3.738 (3H, s), 3.742 (3H, s), 4.13/4.16 (1H, 2q, J = 3.5/3.7 Hz), 4.37 -4.43(1H,m),4.44-4.47(2H,m),6.09/6.10(1H,2t, J =6.4/7.1 Hz),6.83-7.44(13H,m),7.01-7.30(4H,m) , 8.58/8.60 (1H, 2s), 8.98 (1H, b, J ~ 5.5 Hz), 9.24 (1H, bs). ³¹ P-NMR (CD ₃ CN) δ 148.01(s),148.06(s). ¹⁹ _{. F-NMR (CD 3 CN} ) MS (m / z) for _{C 47 H 53 FN 5 O 9} P, calcd δ -117.65 (m) 881.36; Found 880.3 [MH] ^-.

5'- O -Dimethoxytrityl-5-(( R )-2-furanmethylmethylaminecarbonyl)-3'- O -[(2-cyanoethyl)(N,N-diiso) Propylamino)phosphino]-2'-deoxyuridine (4b) . Compound ( 4b ) was prepared as in the preparation of ( 4a ). A 1:1 mixture of non-imaged isophosphites was isolated as a white solid foam (3.15 g, 62% yield). The eluate for column chromatography was 1% triethylamine / 20% hexane / 79% ethyl acetate. ¹ H-NMR (CD ₃ CN, two isomers) δ 1.14-1.27 (12H, m), 1.51-1.59 (1H, m), 1.86-1.94 (3H, m), 2.27-2.59 (2H, m ), 2.54/2.65 (2H, 2t, J = 6.0/5.7 Hz), 3.27-3.38 (2H, m), 3.44-3.97 (9H, m overlap), 3.782 (3H, s), 3.786 (3H, s) , 4.14.18 (1H, m), 4.39-4.48 (1H, m), 6.11/6.13 (1H, 2t, J = 5.6/6.1 Hz), 6.96-7.47 (13H, m), 8.58/8.60 (1H, 2s), 8.75 (1 H, bt, J ~ 5.4 Hz), 9.36 (1H, bs). ³¹ P-NMR (CD ₃ CN) δ 148.09(s), 148.13(s). MS( m/z ) _{C 45 H 56 N 5 O 10} P, calc. 857.38; Found 856.6 [MH] ^-.

5'- O -Dimethoxytrityl-5-(( S )-2-furanmethylmethylamine carbonyl)-3'- O -[(2-cyanoethyl)(N,N-diiso) Propylamino)phosphino]-2'-deoxyuridine (4c) . Compound ( 4c ) was prepared as in the preparation of ( 4b ). A 1:1 mixture of non-imaged isophosphites was isolated as a white solid foam (3.74 g, 74% yield). ¹ H-NMR (CD ₃ CN, two isomers) δ 1.14-1.27 (12H, m), 1.51-1.59 (1H, m), 1.86-1.94 (3H, m), 2.28-2.51 (2H, m ), 2.53/2.65 (2H, 2t, J = 6.0/6.0 Hz), 3.25-3.41 (2H, m), 3.44-4.14 (9H, m overlap), 3.783 (3H, s), 3.786 (3H, s) , 4.24.19 (1H, m), 4.40-4.49 (1H, m), 6.11/6.13 (1H, 2t, J = 6.3/6.3 Hz), 6.86-7.48 (13H, m), 8.58/8.60 (1H, 2s), 8.75 (1 H, bt, J ~ 5.4 Hz), 9.36 (1H, bs). ³¹ P-NMR (CD ₃ CN) δ 148.09(s), 148.13(s). MS( m/z ) C ₄₅ H ₅₆ N ₅ O ₁₀ P, calc. 857.38; found: 856.5 [MH] ^- .

5'- O -Dimethoxytrityl-5-(2-(4-morpholinyl)ethylaminecarbonyl)-3'- O - [(2-cyanoethyl)(N,N-diiso) propyl amino) phosphino] -2'-deoxy-uracil nucleoside (4d). The compound ( 4d ) was prepared as in the preparation of ( 4a ), except that the eluent from column chromatography used for purification was 1% triethylamine / 5% absolute ethanol / 94% ethyl acetate. A 1:1 mixture of non-imaged isophosphites was isolated as a white solid foam (3.9 g, 75% yield). ¹ H-NMR (CD ₃ CN, two isomers) δ 1.04-1.19 (12H, m), 2.28-2.59 (2H, m), 2.43-2.47 (6H, m overlap), 2.53/2.64 (2H, 2t, J = 6.2/6.2 Hz), 3.27-3.76 (8H, m overlap), 3.61-3.65 (4H, m), 3.78 (3H, s), 3.789 (3H, s), 4.12-4.19 (1H, m ), 4.39-4.49 (1H, m), 6.11/6.13 (1H, 2t, J = 5.2//5.2), 6.86-7.48 (13H, m), 8.58/8.60 (1H, 2s), 8.78 (1H, bt) , J ~ 5.3 Hz), 9.78 (1H, bs). ³¹ P-NMR (CD ₃ CN) δ 148.08(s), 148.11(s). MS( m/z ) for C ₄₆ H ₅₉ N ₆ O ₁₀ P , calculated as 886.4; experimental value 885.7 [MH] ^- .

5'- O -Dimethoxytrityl-5-(2-(N-benzimidazolyl)ethylaminecarbonyl) -3'- O -[(2-cyanoethyl)(N,N-di Isopropylamino)phosphino]-2'-deoxyuridine (4e) . The compound ( 4e ) was prepared as in the preparation of ( 4a ), except that the eluent from column chromatography used for purification was 1% triethylamine/10% absolute ethanol/89% ethyl acetate. A 1:1 mixture of mirror image isophosphazene was isolated as a white solid foam (1.6 g, 31% yield). ¹ H-NMR (CD ₃ CN, two isomers) δ 1.03-1.18 (12H, m), 2.27-2.57 (2H, m), 2.52/2.63 (2H, 2t, J = 6.0/6.0), 3.27 -3.37(2H,m), 3.49-3.80(6H,m overlap),3.732(3H,s),3.735/3.738(3H,2s),4.00(2H,bt, J ~6.0 Hz),4.12-4.18( 1H, m), 4.30-4.47 (1H, m), 6.08/6.10 (1H, 2t, J = 6.3/6.3 Hz), 6.85-7.48 (13H, m), 6.93-7.09 (4H, m), 8.57/ 8.60 (1H, 2s), 8.82/8.83 (1H, 2bt, J ~ 4.3/4.3 Hz), 9.48 (1H, bs). ³¹ P-NMR (CD ₃ CN) δ 148.07(s), 148.10(s).

Example 3 3'- O Synthesis of acetyl-nucleoside (5a-5e)

5-(4-Fluorobenzylaminecarbonyl)-3'- O -ethinyl-2'-deoxyuridine ( 5a) .

The nucleoside ( 3a ) (3.00 g, 4.4 mmol) was dissolved in a solution of anhydrous pyridine (30 mL) and anhydride (3 mL). Solution was stirred overnight and concentrated in vacuo to give 3'- O - acetylsalicylic - nucleosides. The residual solvent was removed by co-evaporation with anhydrous toluene (10 mL). The residue was dissolved in dry dichloromethane (10 mL) andEtOAcEtOAc The red solution was stirred overnight and the product crystallized during stirring. The slurry was cooled to -20 ° C, filtered, and washed with diethyl ether. The residue was dried in vacuo to give a white solid ( 5a ) (1.10 g, 59% yield). ¹ H-NMR (CD ₃ CN) δ 2.07 (3H, s), 2.33-2.38 (1H, m), 2.50-2.52 (1H, m), 3.63-3.64 (2H, m), 4.10 (1H, bdd, J = 3.1, 5.1 Hz), 4.46 (2H, d, J = 6.0 Hz), 5.19-5.26 (2 H, m overlap), 6.15 (1H, t, J = 7.0 Hz), 7.15 (2H, tt, J =2.2, 9.0 Hz), 7.31-7.38 (2H, m), 8.79 (1H, s), 9.14 (1H, bt, J = 6.1 Hz), 11.95 (1H, bs). ¹⁹ F-NMR (CD ₃ CN ) δ -116.02 (tt, J = 5.5,9.0 Hz)) MS (m / z) for _{C 19 H 20 FN 3 O 7} , Calcd 421.13; Found 419.8 [MH] ^-..

5 - ((R) -2- furylmethyl methylaminocarbonyl) -3'- O - acetyl-2'-deoxy pyrimidine nucleoside urea (5b).

By the method of (4b) Preparation of Compound (5b), which is prepared by the Department of (5a) and by precipitation of a mixture of dichloromethane and ethyl acetate were obtained as a white solid was isolated (1.27 g, 73% yield). ¹ H-NMR (CDCl ₃ ) δ 1.57-2.02 (4H, m), 2.12 (3H, s), 2.46-2.50 (2H, m), 3.03 (1H, bs), 3.43-3.64 (2H, m), 3.75-3.97 (2H, m), 3.78-4.10 (3H.m), 4.20-4.21 (1H, m), 5.40-5.42 (1H, m), 6.35 (1H, dd, J = 6.5, 7.7 Hz), 8.91 (1H, t, J = 5.5 Hz), 9.17 (1H, s), 9.44 (1H, bs). MS ( m/z ) for C ₁₇ H ₂₃ N ₃ O ₈ , calc. 397.15; MH] ^- .

5 - ((S) -2- furylmethyl methylaminocarbonyl) -3'- O - acetyl-2'-deoxy pyrimidine nucleoside urea (5c).

The compound ( 5c ) was obtained from ( 4c ), which was obtained by the method of the preparation of ( 5a ) and by isolation of a mixture of dichloromethane and diethyl ether to give a slightly orange solid (1.35 g, 77% yield). . ¹ H-NMR (CDCl ₃ ) δ 1.57-2.03 (4H, m), 2.12 (3H, s), 2.47-2.51 (2H, m), 2.98 (1H, bs), 3.40-3.68 (2H, m), 3.78-3.95 (2H, m), 3.90-4.12 (3H.m), 4.20-4.21 (1H, m), 5.39-5.42 (1H, m), 6.33 (1H, dd, J = 6.7, 7.4 Hz), 8.90 (1H, t, J = 5.5 Hz), 9.15 (1H, s), 9.37 (1H, bs). MS ( m/z ) for C ₁₇ H ₂₃ N ₃ O ₈ , calc. 397.15; MH] ^- .

5-(2-(4-morpholino)ethylaminecarbonyl)-3'- O -ethinyl-2'-deoxyuridine (5d) .

The nucleoside ( 3d ) (1.00 g, 1.37 mmol) was dissolved in a solution of anhydrous pyridine (10 mL) and anhydride (1 mL). The solution was stirred overnight and concentrated in vacuo to give 3'-O-ethyl- s- s. The residual solvent was removed by co-evaporation with anhydrous toluene (10 mL). The residue was dissolved in 1,1,1,3,3,3-hexafluoro-2-propanol (20 mL) (Leonard, NJ Tetrahedron Letters , 1995 , 36:7833) and heated at about 50 ° C for 3 hour. The completion of the cleavage of the DMT group was confirmed by tlc. The red solution mixture was quenched by pouring into stirred methanol (200 mL). The resulting yellow solution was concentrated in vacuo and the residue was crystalljjjjjjj The product was crystallized upon cooling and the resulting slurry was aged at -20 ° C, then filtered and washed with ethyl acetate. 3'-O-Ethyl-nucleoside ( 5d ) was isolated as a white solid (0.46 g, 79% yield). ¹ H-NMR (DMSO-d6) δ 2.07 (3H, s), 2.32-2.45 (7H, m), 2.49-2.52 (1H, m), 3.33-3.40 (2H, m), 3.57 (4H, t , J = 4.5 Hz), 3.60-3.63 (2H, m), 4.09 (1H, bdd, J = 3.2, 5.2 Hz), 5.17-5.25 (2H, m), 6.14 (1H, t, J = 7.0 Hz) , 8.74 (1H, s), 8.89 (1H, bt, J = 5.4 Hz), 11.90 (1H, bs). MS ( m/z ) for C ₁₈ H ₂₆ N ₄ O ₈ , 422.18; [MH] ^- .

5-(2-(1-(3-Ethyl-benzimidazolyl))ethylaminecarbonyl)-3'- O -ethinyl -2'-deoxyuridine (5e) .

The compound ( 5e ) was prepared as in the preparation of ( 5d ) except that the product was directly crystallized except that the DMT-fragmentation reaction was poured into methanol. The diacetyl nucleoside ( 5e ) isolated by filtration was a white solid (0.55 g, 78% yield). ^{1 H-NMR (DMSO-d6} ) δ 2.07 (3H, s), 2.30-2.37 (1H, m), 2.49-2.52 (1H, m), 2.63 (3H, s) 3.33 (1H, bs), 3.55- 3.64 (4H, m overlap), 3.99 (2H, t, J = 6.4 Hz), 4.09 (1H, bdd, J = 2.3, 5.2 Hz), 5.15-5.25 (2H, m), 6.13 (1H, dd, J = 6.3, 7.6 Hz), 7.11 (1H, ddd, J = 1.2, 7.6, 7.9 Hz), 7.22 (1H, ddd, J = 1.2, 7.6, 7.9 Hz), 7.33 (1H, dd , J = 0.8, 7.9) Hz), 8.02 (1H, dd, J = 0.8, 8.0 Hz), 8.05 (1H, bs), 8.83 (1H, bt), 8.71 (1H, s), 11.87 (1H, bs). MS ( m/z For C ₂₃ H ₂₅ N ₅ O ₉ , 515.17; found: 513.9 [MH] ^- .

Example 4 3'- O Selective Synthesis of Ethyl-Nucleoside (5a-5d)

3'-O-ethylidene-nucleoside ( 5a-d ) can also be derived from the starting material 3'- O -ethinyl-5'-O-dimethoxytrityl-5-iodo-2'- Deoxyuridine nucleosides (7) are synthesized by a selective route (Scheme 2) (Vaught, JD, Bock, C., Carter, J., Fitzwater, T., Otis, M., Schneider, D., Rolando, J., Waugh, S., Wilcox, SK, Eaton, BE J. Am. Chem. Soc. 2010 , 132 , 4141-4151). Briefly, referring to Scheme 2, the trifluoroethoxycarbonylation of palladium(II)-catalyzed iodide produces an activated ester intermediate ( 8 ). Condensation of ( 8 ) with amines ( 2a-d ) (1.3 equivalents, triethylamine (3 equivalents), acetonitrile, 60-70 ° C, 2-24 hours) followed by cleavage of 5'- O- DMT-protection (3% trichloroacetic acid/dichloromethane or 1,1,1,3,3,3-hexafluoro-2-propanol, room temperature) to give ( 5a-d ), which is via an intermediate product ( 3a- d ) (Process 1) The products obtained are the same.

3'- O - acetylsalicylic -5'- O - dimethoxytrityl-5- (2,2,2-trifluoro-ethoxycarbonyl) -2'-deoxy-uracil nucleoside (8 ). Filling a 500 mL thick-walled glass pressure reactor with argon and filling with 3'- O -acetamido-5'- O -dimethoxytrityl-5-iodo-2'-deoxyuracil nucleoside (7) (15.9 g, 22.8 mmol), anhydrous acetonitrile (200 mL), triethylamine (7.6 mL, 54.7 mmol), and 2,2,2-trifluoroethanol (16.4 mL, 228 mmol). The resulting solution was vigorously stirred and degassed by evacuating to <100 mmHg for 2 minutes. The flask was filled with argon and bis(benzonitrile)dichloropalladium(II) (175 mg, 0.46 mmol) was added. The resulting yellow solution was again degassed, followed by carbon monoxide (99.9%) from the gas manifold (note that this is a toxic gas). When the reaction mixture was stirred vigorously, the pressure was maintained at 1-10 psi of CO and heated at 60-65 °C for 12 hours. The cooled reaction mixture was filtered (note! this was a toxic gas) to remove the black precipitate and concentrate in vacuo. The orange residue was separated with dichloromethane (120 mL) and 10% sodium bicarbonate (80 mL). The organic layer was washed with water (40 mL) and dried over sodium sulfate. This crude product can be used or further purified by silica gel flash column chromatography eluting with 30% hexane / 1% triethylamine / 69% ethyl acetate to give a colorless solid foam (8) (12.7 g, 80% yield). ¹ H-NMR (CD ₃ CN) δ 2.03 (3H, s), 2.37-2.56 (2H, m), 3.36-3.38 (2H, m), 3.78 (6H, s), 4.15-4.19 (1H, m ), 4.37-4.55 (2H, m), 5.21-5.26 (1H, m), 6.09 (1H, t, J = 6.1 Hz), 6.84-7.46 (13H, m), 8.53 (1H, s). ¹⁹ F _{. -NMR (CD 3 CN) δ} -74.07 (t, J = 8.8 Hz) MS (m / z) for _{C 35 H 33 F 3 N 2} O 10, calc. 698.21; Found 697.4 [MH] ^-.

Example 5 Nucleoside 5'- O - Synthesis of triphosphate

5-(4-Fluorobenzylaminecarbonyl)-2'-deoxyuridine nucleoside-5'- O -triphosphate ( para -triethylammonium salt) ( 6a ). By (500 μmol-grade (5x)) Ludwig and Eckstein (Ludwig, J. and Eckstein, FJ Org. Chem. 1989 , 54: 631) from 3'-O-ethinyl-nucleoside ( 5a ) Synthesis of triphosphate ( 6a ). After aminolysis and evaporation, the crude triphosphate product is purified by anion exchange column chromatography as described in the general procedure (below).

General procedure for anion exchange HPLC purification of nucleoside triphosphates . The nucleoside triphosphate was purified by anion exchange column chromatography using an HPLC column packed with a Source Q resin (GE Healthcare) mounted on a preparative HPLC system at 278 nm. The linear elution gradient used two buffers (buffer A: 10 mM triethylamine bicarbonate/10% acetonitrile, and buffer B: 1 M triethylamine bicarbonate/10% acetonitrile) during the elution process. At room temperature, the gradient was shifted from low buffer B content to high buffer B. The desired product is generally the final material eluted from the column and is the widest peak spanning the residence time of about ten to twelve minutes (earlier elution products include various reaction by-products, most importantly nucleosides) Diphosphate). Several fractions were collected during the product stripping process. Each fraction was analyzed by reverse phase HPLC (Waters 2795 HPLC with a Waters symmetrical column (PN: WAT054215)). The liquid fraction containing the pure product (common >90%) was evaporated in a Genevac VC 3000D evaporator to produce a light tan resin. The heavy components in deionized water were used together for the final analysis. Product quantification was performed using analysis of a Hewlett Packard 8452A diode array mass spectrometer at 278 nm for detection. The yield was calculated from the equation A = εCL, where A is the UV absorbance, ε is the estimated extinction coefficient, and L is the optical path (1 cm).

The crude product ( 6a ) was dissolved in about 5 mL of Buffer A (Table 1: Preparative-High Performance Liquid Chromatography, Condition 1). A filtered aliquot of each of the filtered injections containing 1 mL of this solution was injected into a Waters 625 HPLC with a 486 detector (Mobile) with a Resource Q 6 mL column (GE Healthcare product code: 17-1179-01) The phase gradient was 0%-100% buffer B and eluted at 12 mL/min in 50 minutes). For ( 6a ) [ε _estimated 13,700 cm ^-1 M ^-1 ], the isolated purified product was 130 μmol (26% yield). ¹ H-NMR (D ₂ O) δ 1.15 (27H, t, J = 7.3 Hz), 2.32 - 2.37 (2H, m), 3.07 (18H, q, J = 7.3 Hz), 4.06-4.17 (3H, m Overlap), 4.42 (2H, bd, J ~ 0.7 Hz), 4.49-4.53 (1H, m), 4.70 (>7H, bs, HOD), 6.12 (1H, t, J = 6.8 Hz), 6.96-7.26 ( 4H, m), 8.45 (1H, s). ¹⁹ F-NMR (D ₂ O) δ -116.18 (m). ³¹ P-NMR (D ₂ O) δ -10.58 (d, J = 20 Hz), - 11.45 (d, J = 20 Hz), -23.29 (t, J = 20 Hz). MS ( m / z ) for C ₁₇ H ₂₁ FN ₃ O ₁₅ P ₃ , calculated 619.02; experimental value 618.0 [MH] ^- .

Table 1. Preparation - High Performance Liquid Chromatography, Condition 1

5-(( R )-2-furanmethylmethylamine carbonyl)-2'-deoxyuridine nucleoside-5'- O - triphosphate (para-triethylammonium salt) (6b) . The triphosphate ( 6b ) is synthesized from 3'- O -ethinyl-nucleoside ( 5b ) as in the manufacture of ( 6a ). The crude product ( 6b ) was used in a single injection of the Waters 2767 preparation system using a Waters 2489 detector using Waters AP-5 (Waters PN) filled with 196 mL of Source 15Q resin (GE Healthcare product code: 17-0947-05). :WAT023331, 50 mm x 100 mm) column for purification. The same buffer as above was used, but the elution gradient was modified to 25% to 80% buffer B and eluted at 50 mL/min in 90 minutes (Table 2: preparative-high performance liquid chromatography) , condition 2). A second purification was performed in a C18 HPLC column to remove residual impurities (Table 4: preparative-high performance liquid chromatography, condition 4). For ( 6b ) [ε _estimated 10,200 cm ^-1 M ^-1 ], the isolated product was isolated in 325 μmol (65% yield). ¹ H-NMR (D ₂ O) δ 1.17 (27H, t, J = 7.3 Hz), 1.49-1.63 (1H, m), 1.77-2.02 (3H, m), 2.34-2.39 (2H, m), 2.85 -3.83 (5H, m overlap), 3.08 (18H, q, J = 7.3 Hz), 4.01-4.19 (3H, m overlap), 4.52-4.56 (1H, m), 4.70 (>7H, bs, HOD), 6.15 (1H, t, J = 6.8 Hz), 8.48 (1H, s). ³¹ P-NMR (D ₂ O) δ -10.50 (d, J = 20 Hz), -11.51 (d, J = 20 Hz) ., -23.25 (t, J = 20 Hz) MS (m / z) for _{C 15 H 24 FN 3 O 16} P 3, calc. 595.04; Found 594.1 [MH] ^-.

Table 2. Preparation - High Performance Liquid Chromatography, Condition 2

5-(( S )-2-furanmethylmethylaminecarbonyl)-2'-deoxyuridine nucleoside-5'- O - triphosphate ( para -triethylammonium salt) (6c) . The triphosphate ( 6c ) is synthesized from 3'- O -ethinyl-nucleoside ( 5c ) as in the manufacture of ( 6a ). The crude product ( 6c ) was used in a single injection of the Waters 2767 preparation system using a Waters 2489 detector using Waters AP-5 (Waters PN) filled with 196 mL of Source 15Q resin (GE Healthcare product code: 17-0947-05). :WAT023331, 50 mm x 100 mm) column for purification. The same buffer as above was used, but the elution gradient was modified to 25% to 80% buffer B and eluted at 50 mL/min in 90 minutes (Table 2: preparative-high performance liquid chromatography) , condition 2). A second purification was performed in a C18 HPLC column to remove residual impurities (Table 4: preparative-high performance liquid chromatography, condition 4). For ( 6c ) [ε _estimated 10,200 cm ^-1 M ^-1 ], the isolated product was isolated in 255 μmol (51% yield). ¹ H-NMR (D ₂ O) δ 1.17 (27H, t, J = 7.3 Hz), 1.49-1.63 (1H, m), 1.78-2.01 (3H, m), 2.34-2.39 (2H, m), 2.85 -3.82 (5H, m overlap), 3.09 (18H, q, J = 7.3 Hz), 4.01-4.19 (3H, m overlap), 4.52-4.56 (1H, m), 4.70 (>7H, bs, HOD), 6.15 (1H, t , J = 6.7 Hz), 8.48 (1H, s). ³¹ P-NMR (D ₂ O) δ - 10.60 (d, J = 20 Hz), -11.42 (d, J = 20 Hz) ., -23.25 (t, J = 20 Hz) MS (m / z) for _{C 15 H 24 FN 3 O 16} P 3, calc. 595.04; Found 594.1 [MH] ^-.

5-(2-(4-morpholinyl)ethylaminecarbonyl)-2'-deoxyuridine nucleoside-5'- O - triphosphate ( bis -triethylammonium salt) (6d) . The triphosphate ( 6d ) is synthesized from 3'- O -ethinyl-nucleoside ( 5d ) as produced ( 6a ). The crude product ( 6d ) was purified using the same equipment and buffer as the user (6a), but the gradient was modified to flow 15% to 60% of buffer B during the 50 minute elution to enhance the product. Resolution (Table 3: preparative-high performance liquid chromatography, condition 3). For ( 6d ) [ε _estimated 10,200 cm ^-1 M ^-1 ], the isolated product was isolated in 54 μmol (11% yield). ¹ H-NMR (D ₂ O) δ 1.17 (18H, t, J = 7.3 Hz), 2.37-2.41 (2H, m), 2.91-2.98 (2H, m), 3.09 (12H, q, J = 7.3 Hz ), 3.20-3.27 (4H, m), 3.87-3.90 (4H, m), 3.63-3.68 (2H, m), 4.10-4.18 (3H, m overlap), 4.56-4.60 (1H, m), 4.70 ( >7H, bs, HOD), 6.15 (1H, bt, J = 6.3 Hz), 8.48 (1H, s). ³¹ P-NMR (D ₂ O) δ-9.99 (d, J = 21 Hz), -11.90 (d, J = 20 Hz) , - 23.19 (t, J = 20 Hz) MS (m / z) for _{C 16 H 27 N 4 O 16} P 3, calc 624.06;. Found 623.1 [MH] ^-.

Table 3. Preparation - High Performance Liquid Chromatography, Condition 3

Table 4. Preparation - High Performance Liquid Chromatography, Condition 4

5-(2-(N-Benzimidazolyl)ethylaminecarbonyl)-2'-deoxyuridine nucleoside-5'- O -triphosphate ( bis -triethylammonium salt) (6e) . The triphosphate ( 6e ) is synthesized from 3'- O -ethinyl-nucleoside ( 5e ) as in the manufacture of ( 6a ). The crude product ( 6e ) was purified using the same equipment and buffer as the user ( 6a ), but the gradient was modified to flow 15% to 60% of buffer B during the 50 minute elution period to enhance the product. Resolution (Table 3. Preparation - High Performance Liquid Chromatography, Condition 3). For ( 6e ) [ε _estimated 13,700 cm ^-1 M ^-1 ], the isolated product was isolated in 101 μmol (20% yield). ¹ H-NMR (D ₂ O) δ 1.17 (18H, t, J = 7.3 Hz), 2.17-2.36 (2H, m), 3.09 (12H, q, J = 7.3 Hz), 3.60-3.73 (2H, m ), 4.01 (2H, t, J = 5.4 Hz), 4.03-4.15 (3H, m), 4.45-4.50 (1H, m), 4.70 (>7H, bs, HOD), 6.04 (1H, t, J = 6.6 Hz), 6.95-7.12 (4H, m), 8.02 (1H, s). ³¹ P-NMR (D ₂ O) δ - 10.35 (d, J = 20 Hz), -11.40 (d, J = 20 Hz) .), - 23.23 (t, J = 20 Hz) MS (m / z) for _{C 19 H 24 N 5 O 16} P 3, calc. 671.04; Found 670.1 [MH] ^-.

The foregoing specific embodiments and examples are merely examples. No particular embodiment, embodiment, or element of a particular embodiment or embodiment is considered to be a critical, required or essential element or feature of the scope of the application. In addition, no elements described herein are required to be used in the scope of the appended claims unless the element is "required" or "critical". Various changes, modifications, substitutions and other changes can be made in the specific embodiments disclosed herein without departing from the scope of the invention. The description, including the examples, is intended to be illustrative and not limiting, and all such modifications and substitutions are considered to be included within the scope of the invention. Therefore, the invention is to be determined by the appended claims and the equivalents thereof For example, any of the steps recited in the method or program claim can be performed in any feasible order, and are not limited to the order presented by the specific embodiments, embodiments, or claims.

Claims (13)

a compound selected from the group consisting of a compound having the following structure or a salt thereof: Wherein R is selected from the group consisting of -(CH ₂ ) _n -R ^X1 ; R ^X1 is selected from a group consisting of; * indicates the position at which the R ^X1 group is attached to the (CH ₂ ) _n linking group; wherein R ^X4 is F; n = 0-10; X is selected from -H, -OH, -OMe a group consisting of -O-allyl, -F, -OEt, -OPr, -OCH ₂ CH ₂ OCH ₃ and -azido; R' is selected from -H, -Ac, -Bz, - a group consisting of P[N(iPr) ₂ ](OCH ₂ CH ₂ CN) and -SiMe ₂ tBu; and R" is selected from H, 4,4'-dimethoxytrityl (DMT) And a group consisting of trisodium phosphate (-P(O)(OH)-OP(O)(OH)-OP(O)(OH) ₂ ) or a salt thereof. A compound according to claim 1 which has the following structure or a salt thereof: A compound of the first aspect of the patent application, which has the following structure: A compound of claim 1 or 3, which is incorporated as part of an oligonucleotide. The compound of claim 4, wherein the oligonucleotide is selected from the group consisting of ribonucleic acid or deoxyribonucleic acid. A compound of claim 1 or 3, which is incorporated as part of an aptamer. A compound according to claim 6 wherein the system is selected from the group consisting of ribonucleic acid or deoxyribonucleic acid. An oligonucleotide or aptamer comprising at least one modified nucleotide having the following structure: R is selected from the group consisting of -(CH ₂ ) _n -R ^X1 ; R ^X1 is selected from a group consisting of; * indicates the position at which the R ^X1 group is attached to the (CH ₂ ) _n linking group; R ^X4 is F; n = 0-10; and X is selected from -H, -OH, -OMe , -O- allyl, -F, -OEt, -OPr, -OCH 2 CH 2 OCH 3 and - the group consisting of azido group. An oligonucleotide or aptamer according to claim 8 which comprises at least one modified nucleotide independently selected from the following structures: An oligonucleotide or aptamer as claimed in claim 8 or 9, wherein the oligonucleotide or system is selected from the group consisting of ribonucleic acid or deoxyribonucleic acid. A process for the preparation of a compound according to any one of claims 1 to 7 which comprises reacting a pyrimidine with a trifluoroethoxycarbonyl group at a 5-position in the presence of a tertiary amine base with an amine; The C-5 modified amine carbonyl pyrimidine is isolated; wherein the trifluoroethoxycarbonylpyrimidine has the following structure: Wherein X is selected from the group consisting of -H, -OH, -OMe, -O-allyl, -F, -OEt, -OPr, -OCH ₂ CH ₂ OCH ₃ and -azido; The amine is selected from the group consisting of RNH ₂ , wherein R is selected from the group consisting of -(CH ₂ ) _n -R ^X1 ; R ^X1 is selected from Group consisting of; * indicates the position at which the R ^X1 group is attached to the (CH ₂ ) _n linking group; R ^x4 is F; and n = 0-10. The method of claim 11, wherein the amine is selected from the group consisting of The group formed. The method of claim 11, wherein the base is selected from the group consisting of triethylamine or diisopropylamine.