HK1235792A1 - 5-position modified pyrimidines and their use - Google Patents

Description

5-position modified pyrimidines and their use

The present application is a divisional application of chinese patent application 201180028946.3 entitled "5-modified pyrimidines and their uses" filed on 12.4.2011.

RELATED APPLICATIONS

This application claims the benefit of U.S. provisional application 61/323,145 filed on 12.4.2010, which is incorporated herein by reference in its entirety.

Technical Field

The present disclosure relates to the field of nucleic acid chemistry, in particular to uridine and its phosphoramidite and triphosphate derivatives modified at the 5-position. The disclosure also relates to methods of making and using the same. The present disclosure includes the use of modified nucleosides as part of oligonucleotides or aptamers.

Background

The following description provides a summary of information relevant to the present disclosure and is not an admission: any information provided herein or any publications cited is prior art to the present disclosure.

There has been considerable interest in the development of modified nucleosides as therapeutic agents, diagnostic agents, and for incorporation into oligonucleotides. For example, modified nucleosides (such as AZT, ddI, d4T, and the like) have been used to treat AIDS. 5-trifluoromethyl-2' -deoxyuridine is effective against herpetic keratitis, and 5-iodo-1- (2-deoxy-2-fluoro-b-D-arabinofuranosyl) cytosine has activity against CMV, VZV, HSV-1, HSV-2 and EBV (A Textbook of Drug Design and Development, eds. PovlKrogsgaard-Larsen and Hans Bundgaard, Harwood Academic Publishers,1991, Chapter 15).

Modified nucleosides have shown utility in diagnostic applications. In these applications, the nucleoside is incorporated into the DNA at a detectable location and the location of the modified nucleoside is detected using various diagnostic methods. These methods include: radiolabelling, fluorescent labelling, biotinylation and strand cleavage. One example of strand cleavage includes: the nucleoside is reacted with hydrazine to produce a urea nucleoside, which is then reacted with piperidine to cause chain cleavage (Maxam-Gilbert method).

Modified nucleosides have also been incorporated into oligonucleotides. There are several ways in which oligonucleotides can be used as therapeutic agents. Antisense oligonucleotides can bind to certain genetically encoded regions in an organism to prevent expression of proteins, or to block various cellular functions. Furthermore, a method of systematic evolution of ligands, known as the SELEX method or exponential enrichment, enables the person skilled in the art to identify and prepare oligonucleotides (called "aptamers") that selectively bind to a target molecule. The SELEX process is described in U.S. patent No. 5,270,163, the contents of which are incorporated herein by reference.

The SELEX method includes: oligonucleotides are selected from a mixture of candidates to achieve essentially any desired binding affinity and selectivity criteria. Starting from a random mixture of oligonucleotides, the method comprises: contacting the mixture with the target under conditions conducive to binding (or interaction), separating unbound oligonucleotides from oligonucleotides that have bound (or interacted) with the target molecule, dissociating oligonucleotide-target pairs, amplifying the oligonucleotides dissociated from the oligonucleotide-target pairs to produce a mixture of ligand-enriched oligonucleotides, and then repeating the binding, separating, dissociating, and amplifying steps through a plurality of cycles as desired.

Modified nucleosides can be incorporated into antisense oligonucleotides, ribozymes, and oligonucleotides used in or identified by the SELEX method. These nucleosides can confer in vivo and in vitro stability of the oligonucleotide to endonucleases and exonucleases, alter the charge, hydrophilicity or lipophilicity of the molecule, and/or provide differences in three-dimensional structure.

Modifications of nucleosides that have been described previously include: sugar modifications at the 2' -position, pyrimidine modifications at the 5-position, purine modifications at the 8-position, modifications at exocyclic amines, substitution of 4-thiouridine, substitution of 5-bromo or 5-iodo-uracil, backbone modifications, and methylation. Modifications also include 3 'and 5' modifications such as capping. PCT WO 91/14696 (incorporated herein by reference) describes methods of chemically modifying antisense oligonucleotides to enhance entry into cells.

U.S. Pat. Nos. 5,428,149, 5,591,843, 5,633,361, 5,719,273 and 5,945,527, which are incorporated herein by reference in their entirety, describe the modification of pyrimidine nucleosides by a palladium coupling reaction. In some embodiments, a nucleophile and carbon monoxide are coupled to a pyrimidine nucleoside (which contains a leaving group at the 5-position of the pyrimidine ring), preferably to form ester and amide derivatives.

Various methods have been used to provide oligonucleotides that are resistant to exonuclease degradation. PCT WO 90/15065 describes a method for preparing exonuclease resistant oligonucleotides, wherein 2 or more phosphoramidite, phosphorothioate and/or phosphorodithioate linkages are incorporated at the 5 'and/or 3' end of the oligonucleotide. PCT WO 91/06629 discloses oligonucleotides having one or more phosphodiester linkages between adjacent nucleosides, which phosphodiester linkages are replaced by the formation of acetal/ketal type linkages capable of binding RNA or DNA.

Advantageously, novel nucleosides for therapeutic and diagnostic use and for inclusion in oligonucleotides are provided. When incorporated into oligonucleotides, it is advantageous to provide new oligonucleotides that: which exhibit a different high affinity for binding to a target molecule and/or exhibit increased resistance to exonucleases and endonucleases compared to oligonucleotides prepared from naturally occurring nucleosides. It would also be useful to provide nucleotides with modifications that would confer biological activities other than or in addition to endonuclease and exonuclease resistance.

Disclosure of Invention

The present disclosure provides 5-position modified uridine having the general formula:

wherein

R is selected from- (CH)₂)_n-R^X1；

R^X1Selected from:

H*CH₃

^*represents said R^X1Radical and (CH)₂)_nAttachment point of linker

Wherein

R^X4Selected from: branched or straight chain lower alkyl (C1-C20); halogen (F, Cl, Br, I); nitrile (CN); boric acid (BO)₂H₂) (ii) a Carboxylic acid (COOH); carboxylic acid ester (COOR)^X2) (ii) a Primary amide (CONH)₂) (ii) a Secondary amides (CONHR)^X2) (ii) a Tertiary amides (CONR)^X2R^X3) (ii) a Sulfonamides (SO)₂NH₂) (ii) a N-alkyl Sulfonamides (SONHR)^X2)；

Wherein

R^X2、R^X3Independently selected from: branched or straight chain lower alkyl (C1-C20); phenyl (C)₆H₅)；R^X4Substituted benzene ring (R)^X4C₆H₄) Wherein R is^X4As defined above; carboxylic acid (COOH); carboxylic acid ester (COOR)^X5) Wherein R is^X5Is branched or straight chain lower alkyl (C1-C20); and cycloalkyl, wherein R^X2＝R^X3＝(CH₂)_n；

Wherein n is 0 to 10;

wherein

X is selected from the group including, but not limited to: -H, -OH, -OMe, -O-allyl, -F, -OEt, -OPr, -OCH₂CH₂OCH₃And-an azido group;

wherein

R' is selected from the group including, but not limited to: -OAc, -OBz and-OSiMe₂tBu；

Wherein

R' is selected from the group including, but not limited to: H. DMT and triphosphate (-P (O) (OH) -O-P (O) (OH)₂) Or a salt thereof; and is

Wherein

Can be replaced by carbocyclic sugar analogs, α -anomeric sugars, epimeric sugars such as arabinose, xylose or lyxose, pyranoses, furanoses, sedoheptulose, acyclic analogs and abasic nucleoside analogs such as methyl nucleoside.

The present disclosure includes 3 '-phosphoramidite and 5' -triphosphate derivatives or salts thereof of said compounds having the general formulae:

wherein all parts are as defined above.

The compounds of the present disclosure may be incorporated into oligonucleotides or aptamers using standard synthetic or enzymatic methods for preparing these compounds.

The present disclosure also provides methods of making the compounds of the present disclosure and compounds made by the methods.

In one embodiment, a process for preparing a C-5 modified aminocarbonyl pyrimidine is provided, the process comprising: reacting a pyrimidine modified at the 5-position with a trifluoroethoxycarbonyl group with an amine in the presence of a base; and isolating the C-5 modified aminocarbonylpyrimidine.

In another embodiment, a process for preparing a 3' -phosphoramidite of a C-5 modified aminocarbonyl pyrimidine is provided, the process comprising: reacting the C-5 modified aminocarbonylpyrimidine with cyanoethyldiisopropylphosphoramidite in the presence of a base; and isolating the 3' -phosphoramidite.

In another embodiment, a process for preparing a 5' -triphosphate of a C-5 modified aminocarbonyl pyrimidine is provided, the process comprising:

a) reacting a C-5 modified aminocarbonylpyrimidine having the formula:

wherein R and X are as defined above,

the 5 '-DMT group is subsequently cleaved with an acid to form a 3' -acetate having the structure:

b) subjecting the 3' -acetate of step a) to a Ludwig-Eckstein reaction followed by anion exchange chromatography; and

c) isolating a 5' -triphosphate of a C-5 modified aminocarbonyl pyrimidine having the structure:

Detailed Description

Reference will now be made in detail to the representative embodiments of the present invention. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that they are not intended to limit the invention to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents, which may be included within the scope of the invention as defined by the appended claims.

Those skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which can be used in the practice of the present disclosure and are within the scope thereof. The present disclosure is in no way limited to the methods and materials described.

Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the present invention, the preferred methods, devices, and materials are now described.

All publications, published patent documents and patent applications cited in this disclosure are indicative of the level of skill in the art to which this disclosure pertains. All publications, published patent documents and patent applications cited herein are incorporated by reference to the same extent as if each individual publication, published patent document or patent application were specifically and individually indicated to be incorporated by reference.

As used in this disclosure, including the appended claims, the singular forms "a," "an," and "the" include plural referents and are used interchangeably with "at least one" and "one or more" unless the content clearly dictates otherwise. Thus, reference to "aptamers" also includes mixtures of aptamers and the like.

The term "about" as used herein represents slight numerical modification or variation such that the essential function of the item with which these numbers are associated is not changed.

When used to refer to a plurality of items, the term "each" is intended to mean at least 2 of the items. It is not necessarily required that all items forming the plurality satisfy the additional restrictions concerning.

As used herein, the terms "comprises," "comprising," "includes," "including," "contains," "containing," and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, article of manufacture, or composition of matter that comprises, includes, or contains an element or a list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, article of manufacture, or composition of matter.

The term "nucleotide" as used herein means a ribonucleotide or a deoxyribonucleotide or modified forms thereof and analogs thereof. Nucleotides include materials containing purines (e.g., adenine, hypoxanthine, guanine and derivatives and analogs thereof) and pyrimidines (e.g., cytosine, uracil, thymine and derivatives and analogs thereof).

Compound (I)

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

wherein

R is selected from- (CH)₂)_n-R^X1；

R^X1Selected from:

H*CH₃

^*represents said R^X1Radical and (CH)₂)_nAttachment point of linker

Wherein

R^X4Selected from the group including, but not limited to: branched or straight chain lower alkyl (C1-C20); halogen (F, Cl, Br, I); nitrile (CN); boric acid (BO)₂H₂) (ii) a Carboxylic acid (COOH); carboxylic acid ester (COOR)^X2) (ii) a Primary amide (CONH)₂) (ii) a Secondary amides (CONHR)^X2) (ii) a Tertiary amides (CONR)^X2R^X3) (ii) a Sulfonamides (SO)₂NH₂) (ii) a And N-alkylsulfonamides (SONHR)^X2)；

Wherein

R^X2、R^X3Independently selected from the group including, but not limited to: branched or straight chain lower alkyl (C1-C20); phenyl (C)₆H₅)；R^X4Substituted benzene ring (R)^X4C₆H₄) Wherein R is^X4As defined above; carboxylic acid (COOH); carboxylic acid ester (COOR)^X5) Wherein R is^X5Is branched or straight chain lower alkyl (C1-C20); and cycloalkyl, wherein R^X2＝R^X3＝(CH₂)_n；

Wherein n is 0 to 10;

wherein

X is selected from the group including, but not limited to: -H, -OH, -OMe, -O-allyl, -F, -OEt, -OPr, -OCH₂CH₂OCH₃And-an azido group;

wherein

R' is selected from the group including, but not limited to: -OH, -OAc, -OBz, -C (O) CH₂OCH₃and-OSiMe₂tBu；

Wherein

R' is selected from the group including, but not limited to: -H, 4' -Dimethoxytrityl (DMT) and triphosphate (-P (O) (OH)) - (OH) -O-P (O) (OH)₂) Or a salt thereof; and is

Wherein

Can be replaced by carbocyclic sugar analogs, α -isocephalitons, epimeric sugars such as arabinose, xylose or lyxose, pyranoses, furanoses, sedoheptulose, acyclic analogs and abasic nucleoside analogs such as methyl nucleoside.

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

wherein R, R' and X are as defined above. The compounds of this formula are useful for incorporating modified nucleosides into oligonucleotides by chemical synthesis.

In other embodiments, the present disclosure provides a compound of the formula:

wherein R, R' and X are as defined above. The compounds of this formula are useful for the incorporation of modified nucleosides into oligonucleotides by enzymatic synthesis.

The term "C-5 modified carboxyamide uridine" or "C-5 modified aminocarbonyl uridine" as used herein denotes uridine: having a carboxyamide (-C (O) NH-) modification at the C-5 position of said uridine, including, but not limited to, those moieties (R) described above. Examples of C-5 modified carboxyamide uridine include those described in U.S. Patents 5,719,273 and 5,945,527 and U.S. provisional application 61/422,957 entitled "nucleic acid Oligonucleotides" (the' 957 application), filed on 12, 2010, and 14. Representative C-5 modified pyrimidines include: 5- (N-benzylcarboxyamide) -2' -deoxyuridine (BndU), 5- (N-benzylcarboxyamide) -2' -O-methyluridine, 5- (N-benzylcarboxyamide) -2' -fluorouridine, 5- (N-isobutylcarboxyamide) -2' -deoxyuridine (iBudU), 5- (N-isobutylcarboxyamide) -2' -O-methyluridine, 5- (N-isobutylcarboxyamide) -2' -fluorouridine, 5- (N-tryptaminocarboxamide) -2' -deoxyuridine (TrpdU), 5- (N-tryptaminocarboxamide) -2' -O-methyluridine, 5- (N-tryptaminocarboxamide) -2' -fluorouridine, and mixtures thereof, 5- (N- [1- (3-trimethylammonium) propyl ] carboxamide) -2' -deoxyuridine chloride, 5- (N-naphthylmethylcarboxyamide) -2' -deoxyuridine (NapdU), 5- (N-naphthylmethylcarboxyamide) -2' -O-methyluridine, 5- (N-naphthylmethylcarboxyamide) -2' -fluorouridine or 5- (N- [1- (2, 3-dihydroxypropyl) ] carboxamide) -2' -deoxyuridine).

Specific examples of C-5 modified aminocarbonyluridines described herein for illustrative purposes only include the following compounds and the 5 '-triphosphates and 3' -phosphoramidites of said compounds and their salts, the syntheses of which are described in examples 1-5.

5- (4-fluorobenzylaminocarbonyl) -2' -deoxyuridine,

5- ((R) -2-furfurylmethylaminocarbonyl) -2' -deoxyuridine,

5- ((S) -2-furfurylmethylaminocarbonyl) -2' -deoxyuridine,

5- (2- (4-morpholino) ethylaminocarbonyl) -2' -deoxyuridine; and

5- (2- (1- (3-benzimidazolonyl)) ethylaminocarbonyl) -2' -deoxyuridine.

The chemical modifications of C-5 modified uridine described herein may also be combined with the following modifications, either alone or in any combination: sugar modifications at the 2' -position, modifications at exocyclic amines, and substitutions of 4-thiouridine, among others.

Salt (salt)

Corresponding salts, e.g., pharmaceutically acceptable salts, of the compounds may be conveniently or desirably prepared, purified and/or processed. In Berge et al, "pharmaceutical Acceptable Salts" (1977) J.pharm.Sci.66：1-19, examples of pharmaceutically acceptable salts are discussed.

For example, if the compound is anionic, or has a functional group that may be anionic (e.g., -COOH may be-COO^-) Salts may then be formed with suitable cations. 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 Ca²⁺And Mg²⁺And other cations such as Al⁺³. Examples of suitable organic cations include, but are not limited to: ammonium ion (i.e., NH)₄ ⁺) And substituted ammonium ions (e.g., NH)₃R^x+、NH₂R^x ₂ ⁺、NHR^x ₃ ⁺、NR^x ₄ ⁺). Some examples of suitable substituted ammonium ions are those derived from: ethylamine, diethylamine, dicyclohexylamine, triethylamine, butylamine, ethylenediamine, ethanolamine, diethanolamine, piperazine, benzylamine, phenylbenzylamine, choline, meglumine and tromethamineAlcohols, 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 (e.g., -NH)₂May be-NH₃ ⁺) Salts may then be formed with suitable anions. 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, edetic acid, ethanedisulfonic acid, ethanesulfonic acid, fumaric acid, glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxymaleic acid, hydroxynaphthalenecarboxylic acid, isethionic acid, lactic acid, lactobionic acid, lauric acid, maleic acid, malic acid, methanesulfonic acid, mucic acid, oleic acid, oxalic acid, palmitic acid, pamoic acid, pantothenic acid, phenylacetic acid, benzenesulfonic acid, propionic acid, pyruvic acid, salicylic acid, stearic acid, succinic acid, sulfanilic acid, tartaric acid, toluenesulfonic acid, and valeric acid. Examples of suitable polymeric organic anions include, but are not limited to, those derived from the following polymeric acids: tannic acid, carboxymethyl cellulose.

Unless otherwise indicated, reference to a particular compound also includes its salt form.

Preparation of oligonucleotides

In one aspect, the disclosure provides methods of using the modified nucleosides described herein to prepare modified oligonucleotides, alone or in combination with other modified nucleosides and/or naturally occurring nucleosides. Automated synthesis of oligodeoxynucleosides is a common practice in many laboratories (see, e.g., Matteucci, m.d. and carothers, m.h., (1990) j.am.chem.soc.,103: 3185 and 3191, the contents of which are incorporated herein by reference). Synthesis of oligoribonucleosidesThe success is also well known (see, e.g., Scaringe, S.A. et al, Nucleic Acids Res.18: 5433-. As noted above, the phosphoramidites can be used to incorporate the modified nucleosides into oligonucleotides by chemical synthesis, and the triphosphates can be used to incorporate the modified nucleosides into oligonucleotides by enzymatic synthesis (see, e.g., Vaught, j.v. et al (2010) j.am.chem.soc.,1324141-4151; gait, M.J. "Oligonucleotide Synthesis a reactive approach" (1984) IRL Press (Oxford, UK); herdewijn, P. "Oligonucleotide Synthesis" (2005) (Humana Press, Totowa, NJ), each of which is incorporated herein by reference in its entirety).

The terms "modified," "modified," and any variants thereof, as used herein, when used in conjunction with an oligonucleotide, mean that at least one of the 4 constituent nucleotide bases (i.e., A, G, T/U and C) of the oligonucleotide is an analog or ester of a naturally occurring nucleotide. In some embodiments, the modified nucleotide confers nuclease resistance to the oligonucleotide. Additional modifications may include: backbone modifications, methylation, abnormal base pairing combinations such as the isobase isocytidine (isocytidine) and isoguanidine (isogluanidine), and the like. Modifications may also include 3 'and 5' modifications, such as capping. Other modifications may include: one or more of the naturally occurring nucleotides are substituted with an analog, internucleotide modifications such as no electrical linkage modifications (e.g., methyl phosphonates, phosphotriesters, phosphoramidates, carbamates, etc.) and charged linkage modifications (e.g., phosphorothioates, phosphorodithioates, etc.), intercalator modifications (e.g., acridine, psoralen, etc.), chelator-containing modifications (e.g., metals, radioactive metals, boron, and oxidative metals, etc.), alkylator-containing modifications, and linkage modifications with modifications (e.g., alpha anomeric nucleic acids, etc.). Furthermore, any hydroxyl groups typically present in the sugar of a nucleotide may be replaced by phosphonate groups or phosphate groups, protected by standard protecting groups, or activated to prepare additional linkages to additional nucleotides or solid supports. The 5 'and 3' terminal OH groups may be phosphorylated or substituted with amines, organic capping group moieties of about 1 to about 20 carbon atoms, polyethylene glycol (PEG) polymers (in one embodiment, about 10 to about 80kDa), PEG polymers (in another embodiment, about 20 to about 60kDa), or other hydrophilic or hydrophobic biological or synthetic polymers.

Polynucleotides may also contain similar forms of ribose or deoxyribose commonly known in the art, including 2' -O-methyl, 2' -O-allyl, 2' -O-ethyl, 2' -O-propyl, 2' -O-CH₂CH₂OCH₃As indicated above, one or more phosphodiester linkages may be replaced by alternative linkers, including embodiments in which the phosphate ester is replaced by P (O) S ("thiophosphate)"), P (S) S ("thiophosphate)"), (O) NR^x ₂("amide"), P (O) R^x、P(O)OR^x', CO or CH₂("methylal"), wherein each R is R^xOr R^x' is independently H or a substituted or unsubstituted alkyl (C1-C20), aryl, alkenyl, cycloalkyl, cycloalkenyl, or aralkyl (araldyl) group optionally containing an ether (-O-) linkage. All linkages in a polynucleotide need not be identical. Similar forms of substitution of sugars, purines and pyrimidines may be advantageous in designing the final product, as may alternative backbone structures (e.g., polyamide backbones).

Modification of the nucleotide structure, if present, may be effected before or after assembly of the polymer. The nucleotide sequence may be interrupted by non-nucleotide components. After polymerization, the polynucleotide may be further modified, such as by conjugation with a labeling component.

As used herein, "nucleic acid," "oligonucleotide," and "polynucleotide" are used interchangeably to refer to a polymer of nucleotides and include DNA, RNA, DNA/RNA hybrids, and modifications of these types of nucleic acids, oligonucleotides, and polynucleotides, including the attachment of various entities or moieties to a nucleotide unit at any position. The terms "polynucleotide", "oligonucleotide" and "nucleic acid" include double-or single-stranded molecules as well as triple-helical molecules. Nucleic acids, oligonucleotides and polynucleotides are broader terms than the term aptamers, and thus the terms nucleic acids, oligonucleotides and polynucleotides include polymers of nucleotides as aptamers, but the terms nucleic acids, oligonucleotides and polynucleotides are not limited to aptamers.

When referring to modification of a nucleic acid, the term "at least one nucleotide" as used herein refers to one, several or all nucleotides in the nucleic acid, indicating that any or all of A, C, T, G or U may be modified or unmodified at any one or all occurrences in the nucleic acid.

In other aspects, the disclosure provides methods of using the modified nucleosides described herein, alone or in combination with other modified nucleosides and/or naturally occurring nucleosides, to prepare aptamers and somamers (described below). In particular embodiments, the aptamers and somamers are prepared using the general SELEX or modified SELEX method described below.

As used herein, "nucleic acid ligand," "aptamer," "SOMAmer," and "clone" are used interchangeably to refer to a non-naturally occurring nucleic acid having a desired effect on a target molecule. Desirable effects include, but are not limited to: binding to the target, catalytically altering the target, reacting with the target in a manner that modifies or alters the target or the functional activity of the target, covalently binding to the target (as in the case of suicide inhibitors), and facilitating reactions between the target and other molecules. In one embodiment, the effect is specific binding affinity for a target molecule that is a three-dimensional chemical structure rather than a polynucleotide that binds to the nucleic acid ligand by a mechanism that does not rely on Watson/Crick base pairing or triple helix formation, wherein the aptamer is not a nucleic acid with a known physiological function bound by the target molecule. Aptamers to a given target include: a nucleic acid identified from a candidate mixture of nucleic acids by a method wherein the aptamer is a ligand of the target, the method comprising: (a) contacting the candidate mixture with the target, wherein nucleic acids in the candidate mixture having increased affinity for the target compared to other nucleic acids can be separated from the remainder of the candidate mixture; (b) isolating the affinity-increased nucleic acid from the remainder of the candidate mixture; and (c) amplifying the increased affinity nucleic acids to produce a ligand-enriched nucleic acid mixture, thereby identifying the aptamer to the target molecule. Affinity interactions are known to be a matter of degree; however, in this context, "specific binding affinity" of an aptamer to its target means that the degree of binding affinity of the aptamer to its target is typically much greater than its binding affinity to other non-target components in a mixture or sample. An "aptamer," "SOMAmer," or "nucleic acid ligand" is a collection of copies of a class or species of nucleic acid molecule having a particular nucleotide sequence. Aptamers can include any suitable number of nucleotides. By "aptamer" is meant more than one such collection of molecules. Different aptamers may have the same or different number of nucleotides. Aptamers may be DNA or RNA, and may be single-stranded, double-stranded, or contain double-stranded or triple-stranded regions.

"SOMAmer" or Slow Off-Rate modifying aptamer (Slow Off-Rate modifying aptamer) as used herein refers to aptamers with improved Off-Rate characteristics. SOMAmer can be prepared using the modified SELEX process described in U.S. patent publication 20090004667 entitled "Method for generating Aptamers with Improved Off-Rates".

As used herein, "protein" is used synonymously with "peptide", "polypeptide" or "peptide fragment". A "purified" polypeptide, protein, peptide, or peptide fragment is substantially free of cellular material or other contaminating proteins from a cell, tissue, or cell-free source from which the amino acid sequence is derived, or substantially free of chemical precursors or other chemical reagents when chemically synthesized.

SELEX method

The terms "SELEX" and "SELEX method" are used interchangeably herein and generally refer to a combination of: (1) selecting nucleic acids that interact with the target molecule in a desired manner (e.g., binding proteins with high affinity), (2) amplifying those selected nucleic acids. The SELEX method can be used to identify aptamers with high affinity for a particular target molecule or biomarker.

SELEX generally comprises: preparing a candidate mixture of nucleic acids, binding the candidate mixture to a desired target molecule to form an affinity complex, separating the affinity complex from unbound candidate nucleic acids, separating and isolating nucleic acids from the affinity complex, purifying the nucleic acids, and identifying specific aptamer sequences. The method may include multiple cycles to further improve the affinity of the selected aptamer. The method may comprise an amplification step at one or more points in the method. See, for example, U.S. Pat. No. 5,475,096 entitled "Nucleic Acid Ligands". The SELEX method can be used to prepare aptamers that bind covalently to its target as well as aptamers that bind non-covalently to its target. See, for example, U.S. Pat. No. 5,705,337 entitled "Systematic Evolution of Nucleic Acid Ligands by expression entity" Chemi-SELEX ".

The SELEX method can be used to identify high affinity aptamers containing modified nucleotides that impart improved characteristics to the aptamers, for example, increased in vivo stability or improved delivery characteristics. Examples of such modifications include: chemical substitution at ribose and/or phosphate and/or base positions. Aptamers Containing Modified Nucleotides identified by the SELEX method are described in U.S. patent 5,660,985 entitled "High affinity nucleic Acid Ligands binding Modified Nucleotides," which describes oligonucleotides Containing nucleotide derivatives that are chemically Modified at the 5 '-and 2' -positions of pyrimidines. U.S. Pat. No. 5,580,737 (see above) describes highly specific aptamers containing one or more substituted 2 '-amino (2' -NH) groups₂)2 '-fluoro (2' -F) and/or 2 '-O-methyl (2' -OMe) modified nucleotides. See also U.S. patent application entitled "SELEX and PHOTOSELEXPublication 20090098549, which describes nucleic acid libraries having expanded physical and chemical properties and their use in SELEX and photoSELEX.

SELEX can also be used to identify aptamers with desirable off-rate characteristics. See U.S. patent publication 20090004667 entitled "Method for generating Aptamers with Improved Off-Rates," which is incorporated by reference herein in its entirety, which describes an Improved SELEX Method for making Aptamers that can bind to a target molecule. Methods for producing aptamers and photoaptamers having slower rates of dissociation from their respective target molecules are described. The method comprises the following steps: contacting the candidate mixture with the target molecule, allowing formation of nucleic acid-target complexes, and performing a slow off-rate enrichment process, wherein nucleic acid-target complexes with fast dissociation rates dissociate and do not reform, while complexes with slow dissociation rates remain intact. In addition, the methods include the use of modified nucleotides in the preparation of candidate nucleic acid mixtures to produce aptamers with improved off-rate performance (see U.S. patent publication 20090098549, entitled "SELEX and PhotoSELEX"). (see also U.S. patent 7,855,054 and U.S. patent publication 20070166740). Each of these applications is incorporated by reference herein in its entirety.

"target", "target molecule" or "target" refers herein to any compound to which a nucleic acid can act in a desired manner. The target molecule can be, but is not limited to, a protein, peptide, nucleic acid, carbohydrate, lipid, polysaccharide, glycoprotein, hormone, receptor, antigen, antibody, virus, pathogen, toxic substance, substrate, metabolite, transition state analog, cofactor, inhibitor, drug, dye, nutrient, growth factor, cell, tissue, any portion or fragment of any of the foregoing, and the like. Essentially any chemical or biological effector can be a suitable target. Molecules of any size can serve as targets. The target may also be modified in some manner to enhance the likelihood or strength of interaction between the target and the nucleic acid. Targets may also include any minor changes to a particular compound or molecule, for example, in the case of proteins, such as minor changes in amino acid sequence, disulfide bond formation, glycosylation, lipidation, acetylation, phosphorylation, or any other manipulation and modification, such as conjugation to a labeling component, that does not substantially alter molecular identity. A "target molecule" or "target" is a set of copies of a class or a class of molecules or multi-molecular structures that are capable of binding an aptamer. "target molecule" or "target" means a collection of more than one such molecules. An embodiment of the SELEX process in which the target is a peptide is described in U.S. patent 6,376,190 entitled "Modified SELEXProcesses Without purifying Protein".

Chemical synthesis

Methods of chemical synthesis of the compounds provided in the present disclosure are described herein. These and/or other well known methods may be modified and/or altered in known ways to facilitate the synthesis of other compounds provided in the present disclosure.

Referring to scheme 1, in one approach, the C-5 modified aminocarbonylpyrimidines of the present disclosure are prepared as follows: reacting a pyrimidine modified at the 5-position with a trifluoroethoxycarbonyl group with an amine in the presence of a base; and isolating the C-5 modified aminocarbonylpyrimidine.

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

wherein

X is selected from the group including, but not limited to: -H, -OH, -OMe, -O-allyl, -F, -OEt, -OPr, -OCH₂CH₂OCH₃And-azido, and

wherein

May be replaced by carbocyclic sugar analogs, α -isocephalitons, epimeric sugars such as arabinose, xylose or lyxose, pyranoses, furanoses, sedoheptulose, acyclic analogs and abasic nucleoside analogs such as methyl nucleoside₂A compound of (1), wherein

R is selected from- (CH)₂)_n-R^X1；

R^X1Selected from:

H*-CH₃

^*represents said R^X1Radical and (CH)₂)_nAttachment point of linker

Wherein

R^X4Selected from: branched or straight chain lower alkyl (C1-C20); halogen (F, Cl, Br, I); nitrile (CN); boric acid (BO)₂H₂) (ii) a Carboxylic acid (COOH); carboxylic acid ester (COOR)^X2) (ii) a Primary amide (CONH)₂) (ii) a Secondary amides (CONHR)^X2) (ii) a Tertiary amides (CONR)^X2R^X3) (ii) a Sulfonamides (SO)₂NH₂) (ii) a N-alkyl Sulfonamides (SONHR)^X2)；

Wherein

R^X2And R^X3Independently selected from: branched or straight chain lower alkyl (C1-C20); phenyl (C)₆H₅)；R^X4Substituted benzene ring (R)^X4C₆H₄) Wherein R is^X4As defined above; carboxylic acid (COOH); carboxylic acid ester (COOR)^X5) Wherein R is^X5Is branched or straight chain lower alkyl (C1-C20); and cycloalkyl, wherein R^X2＝R^X3＝(CH₂)_n(ii) a And is

Wherein n is 0 to 10.

In particular embodiments, the amine is selected from:

in some embodiments, the base is a tertiary amine selected from triethylamine, diisopropylamine, and the like.

Referring to scheme 1, the present disclosure also provides a method for synthesizing a 3' -phosphoramidite of a C-5 modified aminocarbonyl pyrimidine, the method comprising: reacting the C-5 modified aminocarbonylpyrimidine with cyanoethyldiisopropylphosphoramidite in the presence of a base; and isolating the 3' -phosphoramidite. In some embodiments, the C-5 modified aminocarbonylpyrimidine has the structure:

wherein R and X are as defined above. In some embodiments, the base is a tertiary amine selected from triethylamine, diisopropylamine, and the like.

Referring again to scheme 1, the present disclosure also provides a method for synthesizing a 5' -triphosphate of a C-5 modified aminocarbonyl pyrimidine, the method comprising:

a) reacting a C-5 modified aminocarbonylpyrimidine having the formula:

wherein R and X are as defined above,

the 5 '-DMT group is subsequently cleaved with an acid to form a 3' -acetate having the structure:

b) subjecting the 3' -acetate of step a) to a Ludwig-Eckstein reaction followed by anion exchange chromatography; and

c) isolating a 5' -triphosphate of a C-5 modified aminocarbonyl pyrimidine having the structure:

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

In one alternative, the trifluoroethoxycarbonyl pyrimidine has the following structure:

referring to scheme 2, the compound of scheme 2 is formed by reacting the compound (7) with carbon monoxide and trifluoroethanol in the presence of a palladium catalyst and a base. The base is selected from the group including, but not limited to, tertiary amines selected from triethylamine and the like.

The present disclosure includes compounds prepared by each of the above-described methods.

Examples

The following examples are provided for illustrative purposes only and are not intended to limit the scope of the invention, which is defined by the appended claims. All embodiments described herein are implemented using standard techniques that are well known and conventional to those skilled in the art. The conventional Molecular biology techniques described in the examples below may be implemented as described in standard Laboratory manuals (such as Sambrook et al, Molecular Cloning: A Laboratory Manual, 3 rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (2001)).

The modified nucleosides described in examples 1-3 and 5 were prepared using the general methods described below. The nomenclature used herein is based on Matsuda et al Nucleic Acids Research 1997,25: 2784 the system described in 2791.

Route 1

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

5 '-O-Dimethoxytrityl-5- (4-fluorobenzylaminocarbonyl) -2' -deoxyuridine (3 a). By the method of 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 2003,312514-2523) to prepare the raw material 5' -O-dimethoxytriphenylMethyl-5-trifluoroethoxycarbonyl-2' -deoxyuridine (1). A solution of (1) (9.85g,15mmol), 4-fluorobenzylamine (2a) (2.25g,18mmol,1.3 equiv.), triethylamine (4.2mL,30mmol) and dry acetonitrile (30mL) was heated at 60-70 ℃ for 2-24 hours under an inert atmosphere. Quantitative conversion of (1) to amide (3a) was confirmed by thin layer chromatography (silica gel 60, 5% methanol/dichloromethane) or HPLC. The reaction mixture was concentrated in vacuo and purified by flash chromatography on silica gel using 0-3% methanol eluent in 1% triethylamine/99% ethyl acetate (Still, w.c.; Kahn, m.; Mitra, a.j.org.chem.1978,432923) purifying the residue. The fractions containing pure product were combined and evaporated. Traces of residual solvent were removed by co-evaporation with anhydrous acetonitrile followed by drying under high vacuum to give (3a) as a white solid (6.57g, 64% yield).¹H-NMR(300MHz,CD₃CN)2.20-2.40(2H,m),3.28(2H,d,J＝4.3Hz),3.76(6H,s),4.01(1H,dd,J＝3.8,4.2Hz),4.26-4.30(1H,m),4.48(2H,bd,J＝6.1Hz),6.11(1H,t,J＝6.5Hz),6.85-7.46(13H,m),7.03-7.36(4H,m),8.58(1H,s),9.01(1H,t,J＝6.1Hz)。MS(m/z)C₃₈H₃₆FN₃O₈681.25; measured value 680.4[ M-H]^-。

5 '-O-Dimethoxytrityl-5- ((R) -2-furfurylmethylaminocarbonyl) -2' -deoxyuridine (3 b). Compound (3b) was prepared as described for (3a) using (R) -2-furfurylmethylamine (2b) and isolated as a white solid (9.3g, 94% yield). The eluent for chromatography was 1% triethylamine/4% methanol/95% ethyl acetate.¹H-NMR(CD₃CN)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) C₃₆H₃₉N₃O₉657.27; measured value 656.5[ M-H]^-。

5 '-O-Dimethoxytrityl-5- ((S) -2-furfurylmethylaminocarbonyl) -2' -deoxyuridine (3 c). Compound (3c) was prepared as described for (3b) using (S) -2-furfurylmethylamine (2c) and isolated as a white solid (9.9g, 99% yield).¹H-NMR(CD₃CN)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) C₃₆H₃₉N₃O₉657.27; measured value 656.5[ M-H]^-。

5 '-O-Dimethoxytrityl-5- (2- (4-morpholino) ethylaminocarbonyl) -2' -deoxyuridine (3 d). Compound (3d) was prepared as described for (3a) using 2- (4-morpholino) -ethylamine (2d) and isolated as a white solid (8.2g, 80% yield). The eluent for chromatography was 5% methanol/2% triethylamine/93% dichloromethane.¹H-NMR(CD₃CN)2.21-2.39(2H,m),2.39-2.41(4H,m),2.48(2H,t,J＝6.2Hz),3.27-3.29(2H,m),3.41(2H,dt,J＝5.8,6.2Hz),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～6Hz)。MS(m/z)C₃₇H₄₂N₄O₉686.30; measured value 685.7[ M-H]^-。

5 '-O-Dimethoxytrityl-5- (2- (N-benzimidazolonyl) ethylaminocarbonyl) -2' -deoxyuridine (3 e). Compound (3e) was prepared as described for (3a) using N-benzimidazolonyl-2-ethylamine (2e) (CASRN 64928-88-7). The eluent for chromatography was 2% methanol/1% triethylamine/97% dichloromethane. The pure product was isolated as a brown solid (8.2g, 74.5% yield).¹H-NMR(CD₃CN)2.20-2.36(2H,m),3.27-3.29(2H,m),3.60(2H,q,J＝6.5Hz),3.758(3H,s),3.762(3H,s),3.97(2H,t,J＝6.5Hz),3.98-4.02(1H,m),4.27-4.30(1H,m),6.09(1H,t,J＝6.5Hz),6.86-7.48(13H,m),6.91-7.10(4H,m),8.52(1H,s),8.76(1H,t,J＝6.1Hz)。MS(m/z)C₄₀H₃₉N₅O₉733.27; measured value 732.0[ M-H]^-。

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

5 '-O-Dimethoxybenzyl-5- (4-fluorobenzylaminocarbonyl) -3' -O- [ (2-cyanoethoxy) (N, N-diisopropylamino) phosphinyl]-2' -deoxyuridine (4 a). A solution of DMT-protected nucleoside (3a) (4.00g,5.9mmol) in anhydrous dichloromethane (40mL) was cooled to about-10 ℃ under a dry argon atmosphere. Diisopropylethylamine (3.1mL,17.6mmol,3 equiv.) was added followed by dropwise addition of 2-cyanoethyldiisopropylphosphoramidite (1.7mL,7.7mmol,1.3 equiv.). The solution was stirred for 1 hour, and the end of the reaction was confirmed by thin layer chromatography (silica gel 60, ethyl acetate/hexane). The reaction mixture was partitioned between ice-cold 2% sodium bicarbonate solution (200mL) 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 chromatography on silica gel using a mobile phase of 1% triethylamine/99% ethyl acetate. The fractions containing the pure product are combined and evaporated in vacuo (<At 30 deg.C). Traces of residual chromatographic solvent were removed by co-evaporation with anhydrous acetonitrile and dried under high vacuum to give (4a) as a white stable foam (4.10g, 80% yield).¹H-NMR(CD₃CN,2 isomers) 1.02-1.16(12H, m),2.27-2.57(2H, m),2.51/2.62(2H,2t, J ═ 6.0/6.0Hz),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 ═ 3.5/3.7Hz),4.37-4.43(1H, m),4.44-4.47(2H, m),6.09/6.10(1H,2t, J ═ 6.4/7.1Hz),6.83-7.44(13H, m),7.01-7.30(4H, m),8.58/8.60(1H, 8.60 s), 8.5H, 1H, 5H, 5.5Hz), 8.5-7.5H, 5 bs (1H, m).³¹P-NMR(CD₃CN)148.01(s),148.06(s)。¹⁹F-NMR(CD₃CN)-117.65(m)。MS(m/z)C₄₇H₅₃FN₅O₉Calculated value of P881.36; measurement value 880.3[ M-H]^-。

5 '-O-Dimethoxytrityl-5- ((R) -2-furfurylmethylaminocarbonyl) -3' -O- [ (2-cyanoethoxy) (N, N-diisopropylamino) phosphinyl]-2' -deoxyuridine (4 b). Compound (4b) was prepared as described for (4 a). The 1: 1 mixture of diastereomeric phosphoramidites was isolated as a white solid foam (3.15g, 62% yield). The eluent for chromatography was 1% triethylamine/20% hexane/79% ethyl acetate.¹H-NMR(CD₃CN,2 kinds of heteroStructure) 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 ═ 6.0/5.7Hz),3.27-3.38(2H, m),3.44-3.97(9H, m overlap), 3.782(3H, s),3.786(3H, s),4.11-4.18(1H, m),4.39-4.48(1H, m),6.11/6.13(1H,2t, J ═ 5.6/6.1Hz),6.96-7.47(13H, m),8.58/8.60(1H,2s),8.75(1H, bt, J ═ 5.6.1 Hz), 9.36H, bs).³¹P-NMR(CD₃CN)148.09(s),148.13(s)。MS(m/z)C₄₅H₅₆N₅O₁₀Calculated value of P857.38; measured value 856.6[ M-H]^-。

5 '-O-Dimethoxytrityl-5- ((S) -2-furfurylmethylaminocarbonyl) -3' -O- [ (2-cyanoethoxy) (N, N-diisopropylamino) phosphinyl]-2' -deoxyuridine (4 c). Compound (4c) was prepared as described for (4 b). The 1: 1 mixture of diastereomeric phosphoramidites was isolated as a white solid foam (3.74g, 74% yield).¹H-NMR(CD₃CN,2 isomers) 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 ═ 6.0/6.0Hz),3.25-3.41(2H, m),3.44-4.14(9H, m overlap), 3.783(3H, s),3.786(3H, s),4.12-4.19(1H, m),4.40-4.49(1H, m),6.11/6.13(1H,2t, J ═ 6.3/6.3Hz),6.86-7.48(13H, m),8.58/8.60(1H,2s),8.75(1H, t, 5 bj), 9.36 bs, H, 1H, m).³¹P-NMR(CD₃CN)148.09(s),148.13(s)。MS(m/z)C₄₅H₅₆N₅O₁₀Calculated value of P857.38; measured value 856.5[ M-H]^-。

5 '-O-Dimethoxytrityl-5- (2- (4-morpholino) ethylaminocarbonyl) -3' -O- [ (2-cyanoethoxy) (N, N-diisopropylamino) phosphinyl group]-2' -deoxyuridine (4 d). Compound (4d) was prepared as described for (4a) except that the chromatographic eluate using 1% triethylamine/5% absolute ethanol/94% ethyl acetate was purified. The 1: 1 mixture of diastereomeric phosphoramidites was isolated as a white solid foam (3.9g, 75% yield).¹H-NMR(CD₃CN,2 isomers) 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 ═ 6.2/6.2Hz),3.27-3.76(8H, m overlap), 3.61-3.65(4H, m),3.781(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 (c), (2H, m),2.43-2.47, 2H, m overlap, 2H, J ═ 6.2, J ═ 6.613H,m),8.58/8.60(1H,2s),8.78(1H,bt,J～5.3Hz),9.78(1H,bs)。³¹P-NMR(CD₃CN)148.08(s),148.11(s)。MS(m/z)C₄₆H₅₉N₆O₁₀Calculated value of P886.4; measured value 885.7[ M-H]^-。

5 '-O-Dimethoxytrityl-5- (2- (N-benzimidazolonyl) ethylaminocarbonyl) -3' -O- [ (2-cyanoethoxy) (N, N-diisopropylamino) phosphinyl group]-2' -deoxyuridine (4 e). Compound (4e) was prepared as described for (4a) except that the chromatographic eluate using 1% triethylamine/10% anhydrous methanol/89% ethyl acetate was purified. The 1: 1 mixture of diastereomeric phosphoramidites was isolated as a white solid foam (1.6g, 31% yield).¹H-NMR(CD₃CN,2 isomers) 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 ═ 6.3/6.3Hz),6.85-7.48(13H, m),6.93-7.09(4H, m),8.57/8.60(1H,2s), 8.82/6.83, 3 bs, 1H, 3H, 8.83, 3H, b, 3H, m), 4.9-4.18 (1H, m).³¹P-NMR(CD₃CN)148.07(s),148.10(s)。

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

5- (4-Fluorobenzylaminocarbonyl) -3 '-O-acetyl-2' -deoxyuridine (5 a).

Nucleoside (3a) (3.00g,4.4mmol) was dissolved in a solution of anhydrous pyridine (30mL) and acetic anhydride (3 mL). The solution was stirred overnight and concentrated in vacuo to give 3' -O-acetyl-nucleoside. Residual solvent was removed by co-evaporation with anhydrous toluene (10 mL). The residue was dissolved in anhydrous dichloromethane (10mL) and treated with 3% trichloroacetic acid in dichloromethane (58 mL). The red solution was stirred overnight atDuring which the product crystallizes. The slurry was cooled to-20 ℃, filtered, and washed with diethyl ether. The residue was dried in vacuo to give (5a) as an off-white solid (1.10g, 59% yield).¹H-NMR(CD₃CN)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 ═ 3.1,5.1Hz),4.46(2H, d, J ═ 6.0Hz),5.19-5.26(2H, m overlap), 6.15(1H, t, J ═ 7.0Hz),7.15(2H, tt, J ═ 2.2,9.0Hz),7.31-7.38(2H, m),8.79(1H, s),9.14(1H, bt, J ═ 6.1Hz),11.95(1H, bs).¹⁹F-NMR(CD₃CN)-116.02(tt,J＝5.5,9.0Hz))。MS(m/z)C₁₉H₂₀FN₃O₇421.13; measured value 419.8[ M-H]^-。

5- ((R) -2-furfurylmethylaminocarbonyl) -3 '-O-acetyl-2' -deoxyuridine (5 b).

Compound (5b) was prepared from (4b) by the method described for (5a) and isolated by precipitation from a mixture of dichloromethane and ethyl acetate as a white solid (1.27g, 73% yield).¹H-NMR(CDCl₃)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＝6.5,7.7Hz),8.91(1H,t,J＝5.5Hz),9.17(1H,s),9.44(1H,bs)。MS(m/z)C₁₇H₂₃N₃O₈397.15; measured value 396.1[ M-H]^-。

5- ((S) -2-furfurylmethylaminocarbonyl) -3 '-O-acetyl-2' -deoxyuridine (5 c).

Compound (5c) is prepared from (4c) by the method described for (5a) and isolated by precipitation from a mixture of dichloromethane and diethyl ether,as a pale orange solid (1.35g, 77% yield).¹H-NMR(CDCl₃)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＝6.7,7.4Hz),8.90(1H,t,J＝5.5Hz),9.15(1H,s),9.37(1H,bs)。MS(m/z)C₁₇H₂₃N₃O₈397.15; measured value 395.9[ M-H]^-。

5- (2- (4-morpholino) ethylaminocarbonyl) -3 '-O-acetyl-2' -deoxyuridine (5 d).

Nucleoside (3d) (1.00g,1.37mmol) was dissolved in a solution of anhydrous pyridine (10mL) and acetic anhydride (1 mL). The solution was stirred overnight and concentrated in vacuo to give 3' -O-acetyl-nucleoside. 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 (20mL) (Leonard, N.J. tetrahedron Letters,1995, 36: 7833) and heated at about 50 ℃ for 3 hours. Complete cleavage of the DMT group was confirmed by thin layer chromatography (tlc). The reaction was stopped by pouring the red solution mixture into well stirred methanol (200 mL). The resulting yellow solution was concentrated in vacuo and the residue was dissolved in hot ethyl acetate (20 mL). After cooling, the product crystallized and the resulting slurry was aged at-20 ℃ and subsequently filtered and washed with ethyl acetate. The 3' -O-acetyl-nucleoside (5d) was isolated as a white solid (0.46g, 79% yield).¹H-NMR (DMSO-d6)2.07(3H, s),2.32-2.45(7H, m overlap), 2.49-2.52(1H, m),3.33-3.40(2H, m),3.57(4H, t, J ═ 4.5Hz),3.60-3.63(2H, m),4.09(1H, bdd, J ═ 3.2,5.2Hz),5.17-5.25(2H, m),6.14(1H, t, J ═ 7.0Hz),8.74(1H, s),8.89(1H, bt, J ═ 5.4Hz),11.90(1H, bs). MS (m/z) C₁₈H₂₆N₄O₈426.18; measured value 425.0[ M-H [)]^-。

5- (2- (3-acetyl-benzimidazolon-1-yl) ethylaminocarbonyl) -3 '-O-acetyl-2' -deoxyuridine (5 e).

Compound (5e) was prepared as described for (5d) except that the product crystallized directly when the DMT cleavage reaction was poured into methanol. The diacetylnucleoside (5e) was isolated by filtration as a white solid (0.55g, 78% yield).¹H-NMR (DMSO-d6)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 ═ 6.4Hz),4.09(1H, bdd, J ═ 2.3,5.2Hz),5.15-5.25(2H, m),6.13(1H, dd, J ═ 6.3,7.6Hz),7.11(1H, ddd, J ═ 1.2,7.6,7.9Hz),7.22(1H, ddd, J ═ 1.2,7.6, 7.9), 7.33(1H, ddb, J ═ 0, 7.8, 7.9Hz), 7.8H, 8, H, 8, H, b, H, b, H. MS (m/z) C₂₃H₂₅N₅O₉515.17; measured value 513.9[ M-H]^-。

Example 4.3' -O-acetyl-nucleosides (5a-5d) alternative syntheses

3 '-O-acetyl-nucleosides (5a-d) were also synthesized by an alternative route (scheme 2) from the starting material 3' -O-acetyl-5 '-O-dimethoxytrityl-5-iodo-2' -deoxyuridine (7) (Vaught, J.D., Bock, C., Carter, J.s., Fitzwater, T.s., Otis, M.s., Schneider, D.s., Rolando, J.Waugh, S.s., Wilcox, S.K., Eaton, B.E.J.Am.Chem.Soc.2010,132, 4141-4151). Briefly, referring to scheme 2, palladium (II) -catalyzed trifluoroethoxycarbonylation of iodides yields activated ester intermediate (8). Condensation of (8) with an amine (2a-d) (1.3 equivalents, triethylamine (3 equivalents), acetonitrile, 60-70 ℃,2-24 hours) followed by cleavage of the 5' -O-DMT protecting group (3% trichloroacetic acid/dichloromethane or 1,1,1,3,3, 3-hexafluoro-2-propanol, room temperature) affords (5a-d), which is the same product as that produced via intermediate (3a-d) (scheme 1).

Route 2

3' -O-acetyl-5 ' -O-dimethoxytrityl-5- (2,2, 2-trifluoroethoxycarbonyl) -2' -deoxyuridine (8). A500 mL thick-walled glass pressure reactor was charged with argon and charged with 3' -O-acetyl-5 ' -O-dimethoxytrityl-5-iodo-2 ' -deoxyuridine (7) (15.9g,22.8mmol), anhydrous acetonitrile (200mL), triethylamine (7.6mL,54.7mmol) and 2,2, 2-trifluoroethanol (16.4mL,228 mmol). Vigorously stirring the resulting solution, degassing by evacuation to<100mmHg for 2 minutes. The flask was charged with argon and bis (benzonitrile) palladium (II) dichloride (175mg,0.46mmol) was added. The resulting yellow solution was degassed again and then charged with carbon monoxide (99.9%) from the gas manifold (note: poison!). The reaction mixture was stirred vigorously while maintaining a pressure of 1-10psi CO, and heated at 60-65 deg.C for 12 hours. The cooled reaction mixture was filtered (note: poison gas) to remove the black precipitate and concentrated in vacuo. The orange residue was partitioned between dichloromethane (120mL) and 10% sodium bicarbonate (80 mL). The organic layer was washed with water (40mL) and dried over sodium sulfate, filtered, and concentrated to leave an orange foam (17 g). The crude product was used as is or further purified by flash chromatography on silica gel (using 30% hexane/1% triethylamine/69% ethyl acetate eluent) to give (8) as a colorless solid foam (12.7g, 80% yield).¹H-NMR(CD₃CN))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＝6.1Hz),6.84-7.46(13H,m),8.53(1H,s)。¹⁹F-NMR(CD₃CN)-74.07(t,J＝8.8Hz)。MS(m/z)C₃₅H₃₃F₃N₂O₁₀698.21; measured value 697.4[ M-H]^-。

Example 5 coreSynthesis of glycoside 5' -O-triphosphate

5- (4-Fluorobenzylaminocarbonyl) -2 '-deoxyuridine-5' -O-triphosphate (tri-triethylammonium salt) (6 a). The composition is prepared by the methods of Ludwig and Eckstein (Ludwig, j. and Eckstein, f.j.org.chem.1989,54631), triphosphate (6a) was synthesized from 3' -O-acetyl-nucleoside (5a) on a 500. mu. mol scale (5X). The crude triphosphate product obtained after ammonolysis and evaporation was purified by anion exchange chromatography as described in the general procedure (below).

General procedure for anion exchange HPLC purification of nucleoside triphosphates. Nucleoside triphosphates were purified by anion exchange chromatography using an HPLC column equipped with Source Q resin (GE Healthcare) mounted on a preparative HPLC system with detection at 278 nm. The linear elution gradient used 2 buffers (buffer a: 10mM triethylammonium bicarbonate/10% acetonitrile, and buffer B: 1M triethylammonium bicarbonate/10% acetonitrile), and the gradient was run from low buffer B content to high buffer B at ambient temperature during elution. The desired product is usually the last material to elute from the column and shows a broad peak across a retention time of about 10-12 minutes (the earlier eluted product includes a variety of reaction by-products, most notably nucleoside diphosphate). During the product elution, several fractions were collected. Fractions were analyzed by reverse phase HPLC on a Waters 2795HPLC using a Waters Symmetry column (PN: WAT 054215). Fractions containing pure product (typically > 90%) were evaporated in a Genevac VC 3000D evaporator to give a colorless to pale brown resin. Fractions were reconstituted in deionized water and combined for final analysis. Product quantification was performed by analysis at 278nm using a Hewlett Packard 8452A diode array spectrophotometer. Product yield was calculated via equation a ═ CL, where: a is the ultraviolet absorbance, which is the estimated extinction coefficient, and L is the optical path length (1 cm).

The crude product (6a) was dissolved in about 5mL of buffer A (Table 1: preparative HPLC Condition 1). Each purification injection included: a filtered aliquot of about 1mL of this solution was injected into a Waters 625HPLC with a 486 detector equipped with a Resource Q6 mL column (product of GE Healthcare)Product code: 17-1179-01) at 12 mL/min, using a mobile phase gradient of 0% to 100% buffer B, for 50 minutes. For (6a), "_Estimating13700cm^-1M^-1]The isolated purified product was 130. mu. mol (26% yield).¹H-NMR(D₂O)1.15(27H, t, J ═ 7.3Hz),2.32-2.37(2H, m),3.07(18H, q, J ═ 7.3Hz),4.06-4.17(3H, m overlap), 4.42(2H, bd, J ═ 0.7Hz),4.49-4.53(1H, m),4.70 (c), (d>7H,bs,HOD),6.12(1H,t,J＝6.8Hz),6.96-7.26(4H,m),8.45(1H,s)。¹⁹F-NMR(D₂O)-116.18(m)。³¹P-NMR(D₂O)-10.58(d,J＝20Hz),-11.45(d,J＝20Hz),-23.29(t,J＝20Hz)。MS(m/z)C₁₇H₂₁FN₃O₁₅P₃619.02; measured value 618.0[ M-H]^-。

TABLE 1 preparative HPLC Condition 1

5- ((R) -2-furfurylmethylaminocarbonyl) -2 '-deoxyuridine-5' -O-triphosphate (tri-triethylammonium salt) (6 b). Triphosphate (6b) was synthesized from 3' -O-acetyl-nucleoside (5b) as described for (6 a). Crude product (6b) was purified in a single injection on a Waters 2767 preparative system with a Waters 2489 detector using a Waters AP-5 column (Waters PN: WAT023331,50 mm. times.100 mm) loaded with 196mL Source 15Q resin (GE Healthcare product code: 17-0947-05). The same buffer as above was used, but the elution gradient was modified to 25% to 80% buffer B, eluting at 50 mL/min for 90 min (Table 2: preparative HPLC Condition 2). A second purification was performed on a C18HPLC column to remove residual impurities (Table 4: preparative HPLC Condition 4). For (6b), "_Estimating10200cm^-1M^-1]The isolated purified product was 325. mu. mol (65% yield).¹H-NMR(D₂O)1.17(27H, t, J ═ 7.3Hz),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 ═ 7.3Hz),4.01-4.19(3H, m overlap), 4.52-4.56(1H, m),4.70 (3H, m), (1H, m)>7H,bs,HOD),6.15(1H,t,J＝6.8Hz),8.48(1H,s)。³¹P-NMR(D₂O)-10.50(d,J＝20Hz),-11.51(d,J＝20Hz),-23.25(t,J＝20Hz)。MS(m/z)C₁₅H₂₄FN₃O₁₆P₃595.04; measured value 594.1[ M-H]^-。

TABLE 3 preparative HPLC Condition 3

5- ((S) -2-furfurylmethylaminocarbonyl) -2 '-deoxyuridine-5' -O-triphosphate (tri-triethylammonium salt) (6 c). Triphosphate (6c) was synthesized from 3' -O-acetyl-nucleoside (5c) as described for (6 a). Crude product (6c) was purified in a single injection on a Waters 2767 preparative system with a Waters 2489 detector using a Waters AP-5 column (Waters PN: WAT023331,50 mm. times.100 mm) loaded with 196mL Source 15Q resin (GE Healthcare product code: 17-0947-05). The same buffer as above was used, but the elution gradient was modified to 25% to 80% buffer B, eluting at 50 mL/min for 90 min (Table 2: preparative HPLC Condition 2). A second purification was performed on a C18HPLC column to remove residual impurities (Table 4: preparative HPLC Condition 4). For (6c)_Estimating10200cm^-1M^-1]The isolated purified product was 255 μmol (51% yield).¹H-NMR(D₂O)1.17(27H, t, J ═ 7.3Hz),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 ═ 7.3Hz)4.01-4.19(3H, m overlap), 4.52-4.56(1H, m), 4.70: (>7H,bs,HOD),6.15(1H,t,J＝6.7Hz),8.48(1H,s)。³¹P-NMR(D₂O)-10.60(d,J＝20Hz),-11.42(d,J＝20Hz),-23.25(t,J＝20Hz)。MS(m/z)C₁₅H₂₄FN₃O₁₆P₃595.04; measured value 594.1[ M-H]^-。

5- (2- (4-morpholino) ethylaminocarbonyl) -2 '-deoxyuridine-5' -O-triphosphate (di-triethylammonium salt) (6 d). Triphosphate (6d) was synthesized from 3' -O-acetyl-nucleoside (5d) as described for (6 a). The crude product (6d) was purified using the same equipment and buffer as used for (6a), but the gradient was modified to increase the resolution of the product by buffer B from 15% to 60% within 50 min of elution (table 3: preparative HPLC condition 3). For (6d), "_Estimating10200cm^-1M^-1]The isolated purified product was 54. mu. mol (11% yield).¹H-NMR(D₂O)1.17(18H, t, J ═ 7.3Hz),2.37-2.41(2H, m),2.91-2.98(2H, m),3.09(12H, q, J ═ 7.3Hz),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 (c), (d>7H,bs,HOD),6.15(1H,bt,J＝6.3Hz),8.48(1H,s)。³¹P-NMR(D₂O)-9.99(d,J＝21Hz),-11.90(d,J＝20Hz),-23.19(t,J＝20Hz)。MS(m/z)C₁₆H₂₇N₄O₁₆P₃624.06; measured value 623.1[ M-H]^-。

TABLE 2 preparative HPLC Condition 2

TABLE 4 preparative HPLC Condition 4

5- (2- (N-benzimidazolonyl) ethylaminocarbonyl) -2 '-deoxyuridine-5' -O-triphosphate (di-triethylammonium salt) (6 e). Triphosphate (6e) was synthesized from 3' -O-acetyl-nucleoside (5e) as described for (6 a). The crude product (6e) was purified using the same equipment and buffer as used for (6a), but the gradient was modified to increase the resolution of the product by buffer B from 15% to 60% within 50 min of elution (table 3: preparative HPLC condition 3). For (6e)_Estimating13700cm^-1M^-1]The isolated purified product was 101. mu. mol (20% yield).¹H-NMR(D₂O)1.17(18H,t,J＝7.3Hz),2.17-2.36(2H,m),3.09(12H,q,J＝7.3Hz),3.60-3.73(2H,m),4.01(2H,t,J＝5.4Hz),4.03-4.15(3H,m),4.45-4.50(1H,m),4.70(>7H,bs,HOD),6.04(1H,t,J＝6.6Hz),6.95-7.12(4H,m),8.02(1H,s)。³¹P-NMR(D₂O)-10.35(d,J＝20Hz),-11.40(d,J＝20Hz),-23.23(t,J＝20Hz)。MS(m/z)C₁₉H₂₄N₅O₁₆P₃671.04; measurement 670.1[ M-H]^-。

The foregoing embodiments and examples are intended as examples only. No element from a particular embodiment, example, or from a particular embodiment or example is intended to be construed as a critical, required, or essential element or feature of any or all the claims. Further, elements described herein are not required for the practice of the appended claims unless explicitly described as "required" or "critical". Various changes, modifications, substitutions and other changes may be made to the disclosed embodiments without departing from the scope of the invention as defined by the appended claims. It is intended that the specification, including the examples, be considered as illustrative and not restrictive, and that all such modifications and alterations be included within the scope of the invention. The scope of the invention should, therefore, be determined by the appended claims and their legal equivalents (rather than by the examples given above). For example, the steps recited in any method claims may be executed in any order practicable and are not limited to the order presented in any embodiment, example, or claim.

Claims (22)

1. A C-5 modified aminocarbonyl pyrimidine having the structure:

wherein

R comprises- (CH)₂)_n-R^X1；

R^X1The method comprises the following steps:

represents R^x1Radical and (CH)₂)_nThe point of attachment of the linker;

R^X4the method comprises the following steps: F. cl, Br, I; CN; BO₂H₂；COOH；COOR^X2；CONH₂；CONHR^X2；CONR^X2R^X3；SO₂NH₂(ii) a And SONHR^X2；

R^X2、R^X3Independently selected from: a branched or straight chain C1-C20 alkyl group; c₆H₅；R^X4C₆H₄Wherein R is^X4As defined above; COOH; COOR^X5Wherein R is^X5Is a branched or straight chain C1-C20 alkyl group; and cycloalkyl, wherein R^X2＝R^X3＝(CH₂)_n(ii) a And is

Wherein n is 0 to 10; and is

X is: -H, -OH, -OMe, -O-allyl, -F, -OEt, -OPr, -OCH₂CH₂OCH₃And-an azido group;

r' is selected from: H. -Ac, -Bz, -C (O) CH₂OCH₃、-P-(N(iPr)₂)(OCH₂CH₂CN) and-SiMe₂tBu; and is

R' is selected from: H. 4,4' -dimethoxytrityl and-P (O) (OH) -O-P (O) (OH)₂。

2. The C-5 modified aminocarbonylpyrimidine of claim 1, wherein n is 1, 2 or 3.

3. The C-5 modified aminocarbonylpyrimidine of claim 1, wherein

R comprises- (CH)₂)_n-R^X1(ii) a And is

R^X1The method comprises the following steps:

and is

Wherein R is^X4Including F, Cl, Br, I or CN; and n is 0 to 10.

4. A 3' -phosphoramidite of a C-5 modified aminocarbonyl pyrimidine having the structure:

wherein

R comprises- (CH)₂)_n-R^X1；

R^X1The method comprises the following steps:

represents R^x1Radical and (CH)₂)_nThe point of attachment of the linker;

R^X4the method comprises the following steps: F. cl, Br, I; CN; BO₂H₂；COOH；COOR^X2；CONH₂；CONHR^X2；CONR^X2R^X3；SO₂NH₂(ii) a And SONHR^X2；

R^X2、R^X3Independently selected from: a branched or straight chain C1-C20 alkyl group; c₆H₅；R^X4C₆H₄Wherein R is^X4As defined above; COOH; COOR^X5Wherein R is^X5Is a branched or straight chain C1-C20 alkyl group; and cycloalkyl, wherein R^X2＝R^X3＝(CH₂)_n(ii) a And is

Wherein n is 0 to 10; and is

X is: -H, -OH, -OMe, -O-allyl, -F, -OEt, -OPr, -OCH₂CH₂OCH₃And-an azido group;

r' is selected from: H. 4,4' -dimethoxytrityl and-P (O) (OH) -O-P (O) (OH)₂。

5. The 3' -phosphoramidite of claim 4 wherein n-1, 2 or 3.

6. The 3' -phosphoramidite of claim 4 wherein

R comprises- (CH)₂)_n-R^X1(ii) a And is

R^X1The method comprises the following steps:

and is

Wherein R is^X4Including F, Cl, Br, I or CN; and n is 0 to 10.

7. A 5' -triphosphate of a C-5 modified aminocarbonyl pyrimidine having the structure:

wherein

R comprises- (CH)₂)_n-R^X1；

R^X1The method comprises the following steps:

represents R^x1Radical and (CH)₂)_nThe point of attachment of the linker;

R^X4the method comprises the following steps: F. cl, Br, I; CN; BO₂H₂；COOH；COOR^X2；CONH₂；CONHR^X2；CONR^X2R^X3；SO₂NH₂(ii) a And SONHR^X2；

R^X2、R^X3Independently selected from: a branched or straight chain C1-C20 alkyl group; c₆H₅；R^X4C₆H₄Wherein R is^X4As defined above; COOH; COOR^X5Wherein R is^X5Is a branched or straight chain C1-C20 alkyl group; and cycloalkyl, wherein R^X2＝R^X3＝(CH₂)_n(ii) a And is

Wherein n is 0 to 10; and is

X is: -H, -OH, -OMe, -O-allyl, -F, -OEt, -OPr, -OCH₂CH₂OCH₃And-an azido group;

r' is selected from: H. -Ac, -Bz, -C (O) CH₂OCH₃、-P-(N(iPr)₂)(OCH₂CH₂CN) and-SiMe₂tBu。

8. The 5' -triphosphate of claim 7, wherein n-1, 2 or 3.

9. The 5' -triphosphate of claim 7, wherein

R comprises- (CH)₂)_n-R^X1(ii) a And is

R^X1The method comprises the following steps:

and is

Wherein R is^X4Including F, Cl, Br, I or CN; and n is 0 to 10.

10. An oligonucleotide comprising at least one modified nucleotide selected from the group consisting of a C-5 modified aminocarbonylpyrimidine of any one of claims 1 to 3, at least one 3 '-phosphoramidite of any one of claims 4 to 6, or at least one 5' -triphosphate of any one of claims 7 to 9.

11. The oligonucleotide of claim 10, wherein the oligonucleotide further comprises at least one chemical modification comprising a chemical substitution at one or more positions independently selected from a ribose position, a deoxyribose position, a phosphate position, and a base position.

12. The oligonucleotide of claim 11, wherein the chemical modification is independently selected from the group consisting of: 2' -sugar-modified, 2' -amino (2' -NH)₂)2 '-fluoro (2'-F), 2' -O-methyl (2' -OMe), 2' -O-ethyl (2' -OEt), 2' -O-propyl (2' -OPr), 2' -O-CH₂CH₂OCH₃5-pyrimidine modifications, backbone modifications, methylation, 3 'capping, and 5' capping.

13. The oligonucleotide of any one of claims 10-12, wherein the oligonucleotide is an aptamer.

14. A process for preparing a C-5 modified aminocarbonylpyrimidine, the process comprising:

reacting a pyrimidine modified at the 5-position with a trifluoroethoxycarbonyl group with an amine in the presence of a tertiary amine base; and

isolating the C-5 modified aminocarbonyl pyrimidine;

wherein the trifluoroethoxycarbonylpyrimidine has the following structure:

wherein

X is selected from: -H, -OH, -OMe, -O-allyl, -F, -OEt, -OPr, -OCH₂CH₂OCH₃And-an azido group; and is

Wherein the amine is RNH₂；

Wherein

R is selected from- (CH)₂)_n-R^X1；

R^X1The method comprises the following steps:

and is

Represents R^x1Radical and (CH)₂)_nThe point of attachment of the linker;

R^X4the method comprises the following steps: F. cl, Br, I; CN; BO₂H₂；COOH；COOR^X2；CONH₂；CONHR^X2；CONR^X2R^X3；SO₂NH₂(ii) a And SONHR^X2；

R^X2、R^X3Independently selected from: a branched or straight chain C1-C20 alkyl group; c₆H₅；R^X4C₆H₄Wherein R is^X4As defined above; COOH; COOR^X5Wherein R is^X5Is a branched or straight chain C1-C20 alkyl group; and cycloalkyl, wherein R^X2＝R^X3＝(CH₂)_n(ii) a And is

Wherein n is 0 to 10.

15. The method of claim 14, wherein n-1, 2, or 3.

16. The method of claim 14, wherein

R comprises- (CH)₂)_n-R^X1(ii) a And is

R^X1The method comprises the following steps:

and is

Wherein R is^X4Including F, Cl, Br, I or CN; and n is 0 to 10.

17. A process for preparing a 3' -phosphoramidite of a C-5 modified aminocarbonyl pyrimidine or salt thereof, the process comprising:

reacting the C-5 modified aminocarbonylpyrimidine with cyanoethyldiisopropylphosphoramidite in the presence of a tertiary amine; and

isolating the 3' -phosphoramidite;

wherein the C-5 modified aminocarbonyl pyrimidine has the following structure:

wherein

R comprises- (CH)₂)_n-R^X1；

R^X1The method comprises the following steps:

represents R^x1Radical and (CH)₂)_nThe point of attachment of the linker;

R^X4the method comprises the following steps: F. cl, Br, I; CN; BO₂H₂；COOH；COOR^X2；CONH₂；CONHR^X2；CONR^X2R^X3；SO₂NH₂(ii) a And SONHR^X2；

R^X2And R^X3Independently selected from: a branched or straight chain C1-C20 alkyl group; c₆H₅；R^X4C₆H₄Wherein R is^X4As defined above; COOH; COOR^X5Wherein R is^X5Is a branched or straight chain C1-C20 alkyl group; and cycloalkyl, wherein R^X2＝R^X3＝(CH₂)_n(ii) a And is

Wherein n is 0 to 10; and is

X is: -H, -OH, -OMe, -O-allyl, -F, -OEt, -OPr, -OCH₂CH₂OCH₃And-an azido group.

18. The method of claim 17, wherein n-1, 2, or 3.

19. The method of claim 17, wherein

R comprises- (CH)₂)_n-R^X1(ii) a And is

R^X1The method comprises the following steps:

and is

Wherein R is^X4Including F, Cl, Br, I or CN; and n is 0 to 10.

20. A process for preparing a 5' -triphosphate of a C-5 modified aminocarbonyl pyrimidine, said process comprising:

a) reacting a C-5 modified aminocarbonylpyrimidine having the formula:

the 5 '-DMT group is subsequently cleaved with an acid to form a 3' -acetate having the structure:

b) subjecting the 3' -acetate of step a) to a Ludwig-Eckstein reaction followed by anion exchange chromatography; and

c) isolating a 5' -triphosphate of a C-5 modified aminocarbonyl pyrimidine having the structure:

wherein

R comprises- (CH)₂)_n-R^X1；

R^X1The method comprises the following steps:

represents R^x1Radical and (CH)₂)_nThe point of attachment of the linker;

R^X4the method comprises the following steps: F. cl, Br, I; CN; BO₂H₂；COOH；COOR^X2；CONH₂；CONHR^X2；CONR^X2R^X3；SO₂NH₂(ii) a And SONHR^X2；

R^X2And R^X3Independently selected from: a branched or straight chain C1-C20 alkyl group; c₆H₅；R^X4C₆H₄Wherein R is^X4As defined above; COOH; COOR^X5Wherein R is^X5Is a branched chainOr a linear C1-C20 alkyl group; and cycloalkyl, wherein R^X2＝R^X3＝(CH₂)_n(ii) a And is

Wherein n is 0 to 10; and is

X is: -H, -OH, -OMe, -O-allyl, -F, -OEt, -OPr, -OCH₂CH₂OCH₃And-an azido group.

21. The method of claim 20, wherein n-1, 2, or 3.

22. The method of claim 20, wherein

R comprises- (CH)₂)_n-R^X1(ii) a And is

R^X1The method comprises the following steps:

and is

Wherein R is^X4Including F, Cl, Br, I or CN; and n is 0 to 10.