Pseudo-solid-phase protecting groups and methods for synthesizing oligonucleotides and oligonucleotide conjugates - Patents.com

Abstract

Novel pseudo solid-phase protecting groups and methods for synthesizing oligonucleotides and oligonucleotide conjugates are provided. The pseudo solid-phase protecting groups are represented by formula II: * indicates the point of attachment to the oxygen atom of the protected nucleoside, nucleotide, oligonucleotide, or of a hydroxyl moiety of a nucleoside, nucleotide, or oligonucleotide conjugate; c is 0 or 1; a is an integer from 1 to 12; R3 is H; R5 is O-R6 or H, where R6 is a C8 to C40 aliphatic hydrocarbon group; each R4 is independently O-R6, where R6, in each occurrence, is independently a C8 to C40 aliphatic hydrocarbon group; b is 1 to 3; with the proviso that when b is 1, at least one of R3 and R5 is not H; and with the further proviso that the sum of all carbon atoms contained in the R3, R4, and R5 moieties present is greater than 23 and less than 200. [Formula 1] TIFF2024513470000122.tif4195

Description

[Technical field]

[0001] The present invention is a soluble support for producing oligonucleotides and a method for producing oligonucleotides using the soluble support. [Background technology]

[0002] Synthesis of nucleic acids usually relies on solid-phase synthesis, in which a growing oligonucleotide chain is built on a solid support by repeated coupling cycles. Various types of coupling chemistry have been developed, of which the phosphoramidite method is the most well-known. Solid-phase synthesis using the phosphoramidite method has been optimized and automated. Therefore, solid-phase synthesis has the advantage of speed and is the most widely used. However, the scale-up of solid-phase synthesis is difficult for several reasons, including excessive consumption of reagents and raw materials. A further disadvantage of solid-phase synthesis is that it is difficult to follow the progress of the reaction in real time and to analyze the structure of intermediates.

[0003] In recent years, liquid phase synthesis methods using soluble supports, also called pseudo-solid-phase protecting groups, have been developed. Specifically, synthesis methods using groups containing long-chain hydrocarbons as pseudo-solid-phase protecting groups have been reported (e.g., Patent Document 1, Patent Document 2, Patent Document 3). Groups containing long-chain hydrocarbons for use in organic synthesis and peptide synthesis have also been reported (e.g., Patent Document 4 and Patent Document 5).

[0004] Patent Document 1 discloses a method for synthesizing hydrophobic-group-linked nucleosides and hydrophobic-group-linked oligonucleotides. This patent discloses a method for synthesizing hydrophobic-group-linked nucleosides, in which a 5'-OH protected nucleoside, 5'-O-(1,1-bis(4-methoxyphenyl)-1-phenylmethyl)thymidine, is reacted with succinic anhydride to obtain 3'-O-succinyl-5'-O-(1,1-bis(4-methoxyphenyl)-1-phenylmethyl)thymidine, and the product is then reacted with 3,4,5-tris(octadecyloxy)benzoyl)piperazine to obtain 3'-O-(4-(3,4,5-tris(octadecyloxy)benzoyl)piperazin-1-ylsuccinyl)-5'-O-(4 and 4'-dimethoxytrityl)thymidine.

[0005] In hydrophobic group-linked nucleosides, the hydrophobic aromatic ring is linked to a linker via an amide bond, and the linker is linked to the 3'-OH position of the sugar via a succinyl group or the like. Another hydrophobic group-linked nucleoside is disclosed in Non-Patent Document 1. Matsuno discloses a hydrophobic group similar to that in Patent Document 1, which has a carbonyl (4-carbonylbenzyloxy) structure instead of a succinyl group to effect selective cleavage. Such hydrophobic groups can be selectively cleaved from oligonucleotides bearing 5'-DMTr, 2-CE and / or 2'-TBS groups via hydrogenation.

[0006] US Patent No. 5,999,633 discloses a pseudo solid-phase protecting group bound to a nucleoside and a method for producing an oligonucleotide using the pseudo solid-phase protecting group. The pseudo solid-phase protecting group is shown as "-LYZ". L is " ** CO-L1-L2-CO * ", exemplified by a succinyl group, where Y represents an oxygen atom or NR, and Z is a hydrophobic aromatic ring.

[0007] US Patent No. 5,399,633 discloses another pseudo-solid-phase protecting group that is easily and selectively removed in a short time and does not use toxic heavy metals or dangerous hydrogen gas in liquid-phase synthesis in particular. The linkage to the 3'-hydroxyl group of the nucleoside is established via an ester bond. [Prior art documents] [Patent documents]

[0008] [Patent Document 1] JP5548852 [Patent Document 2] WO2012 / 157723 [Patent Document 3] WO2020 / 017085 [Patent Document 4] WO2007 / 122847 [Patent Document 5] WO2007 / 034812 [Non-patent literature]

[0009] [Non-Patent Document 1] Matsuno et al. (Organic Letters, 2016, 18, pp. 800-803 Summary of the Invention [Problem to be solved by the invention]

[0010] There remains a need to obtain further pseudo-solid-phase protecting groups suitable for oligonucleotide synthesis.In particular, such protecting groups should be simple and cost-effective to generate.They should avoid premature cleavage from the growing oligonucleotide chain, but should be easy to complete cleavage under suitable conditions.Such suitable conditions may include treatment with base.The present invention provides such pseudo-solid-phase protecting groups. [Means for solving the problem]

[0011] The following terms and phrases are used throughout this application. Additional terms and phrases are explained in the detailed disclosure of the invention. Any and all of these definitions are applicable throughout this application unless otherwise indicated for a particular embodiment.

[0012] As used herein, the indefinite articles "a" and "an" may mean "at least one."

[0013] As used herein, the term "hydroxyl group" generally refers to a structure of formula -OH. The term "hydroxyl moiety" can refer to a hydroxyl group or a residue of a hydroxyl group resulting from the reaction of a hydroxyl group with another chemical group. Thus, a hydroxyl moiety may have a structure of the formula -O- or -OH. The expressions "amine group" (e.g., -NH-) and "amine moiety" (e.g., =N- or -NH-) are used analogously.

[0014] As used herein, "C 1~30 The expressions "group", "C1 to C30 group", "C1-C30 group" or "chemical group having 1 to 30 carbon atoms" are used interchangeably. They all refer to a chemical moiety containing 1 to 30 carbon atoms, but not more or less than that. Furthermore, the group may contain additional atoms, for example hydrogen atoms in the case of a C1 to C30 alkyl group. As a further example, a "C1 to C30 hydrocarbon group substituted with a C1 to C30 heteroalkyl group" may contain a total of 2 to 60 carbon atoms, but not more or less than that.

[0015] The term "protecting group" as used herein may be understood in the broadest sense as a group introduced into a molecule by chemical modification of a functional group to block said group from reaction in a subsequent process step, e.g. preventing side reactions at the backbone or at the bases of an oligonucleotide.

[0016] A molecule to which a pseudo solid-phase protecting group (PSPPG) is attached may be referred to herein as a "PSPPG-protected molecule", e.g., a PSPPG-protected nucleoside. As used herein, a pseudo solid-phase protecting group (PSPPG) is a protecting group that allows the PSPPG-protected molecule to be separated from other components of a liquid composition. This is often achieved by affecting the solubility of the PSPPG-protected molecule, thereby facilitating, for example, a precipitation process. For the synthesis of oligonucleotides, PSPPG may include hydrophobic moieties, particularly long-chain hydrocarbon moieties and aromatic moieties.

[0017] Nucleosides or nucleoside units can be glycosylamines that contain a sugar moiety, a typically (deoxy)ribose moiety, and a nucleobase. The term nucleoside unit can be used to refer to a nucleoside moiety in the context of a larger moiety, for example as part of an oligonucleotide. The terms nucleoside and nucleoside unit used herein include naturally occurring nucleosides, as well as non-natural compounds in which the ribose moiety and / or the nucleobase are modified or replaced by functional equivalents or abasic sites. Examples of natural nucleosides include, but are not limited to, adenosine, guanosine, cytidine, ribothymidine, uridine, deoxyuridine, deoxythymidine, inosine, deoxyadenosine, deoxyguanosine, deoxycytidine, and their methylated derivatives. Further examples are queuosine, alkaeosine, wybutosine, lysidine, and N6-threonylcarbamoyl adenosine. Unnatural nucleosides may contain modified ribose moieties, which may be, for example, "locked" (e.g., containing a methylene bridge connecting the 2'-oxygen and 4'-carbon), may exhibit an ethylene bridge, or may bear -F, -OMe (-O-CH3) and / or -methoxyethyloxy (-O-CH2-CH2-O-CH3, also known as MOE, -O-methoxyethyl) substituents. Furthermore, the ribose moiety may be replaced by functional equivalents, for example, another pentose such as arabinose, a hexose such as mannose, a morpholine derivative, or a glycerol moiety. The term nucleobase as used herein encompasses both unnatural nucleobases and naturally occurring nucleobases, such as, for example, adenine, guanine, uracil, cytosine, and thymine. Unnatural nucleobases are functional equivalents of natural nucleobases in that they are capable of specifically interacting with complementary nucleobases. The interaction between complementary nucleobases can be mediated by hydrogen bonds known as Watson-Crick base pairs. Such non-natural nucleobases can be derivatives of purines or pyrimidines, which are capable of specifically interacting with other nucleobases.

[0018] A nucleotide or nucleotide unit can include a nucleoside and a phosphate moiety. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0019] As used herein, the expression oligonucleotide(s) is used in the most general manner to refer to any oligomer comprising at least two nucleosides linked by a phosphodiester bond or by a structure similar to those shown, for example, in the following formulae A-1 and A-2, as well as any isomeric forms and stereoisomers thereof. In a preferred embodiment, an oligonucleotide, in the sense of the present invention, is of formula A-1 or formula A-2: [ka] (Wherein, in Formula A-1 and Formula A-2: * and ** each independently represents a point of attachment to a nucleoside moiety (i.e., the preceding and succeeding nucleoside moieties, respectively) or to a H (i.e., in the case of the termini of an oligonucleotide); X 1 is selected from S and O; X 2 can be any conceivable electron-withdrawing group, preferably selected from the group consisting of -OH, -OR, -NHR, -NR2, C3-C20-(hetero)aromatic moieties, -F, -Cl, -SH, -SeH, and -SR; R is any residue, preferably a residue having no more than 30 carbon atoms, preferably a residue selected from the group consisting of branched, unbranched or cyclic C1-C20 (hetero)alkyl residues, branched, unbranched or cyclic C2-C20 (hetero)alkenyl residues, branched, unbranched or cyclic C2-C20 (hetero)alkynyl residues, C6-C20 aryl residues, C3-C19 heteroaryl residues, the aforementioned residues being optionally substituted with deuterium, halogen, branched, unbranched or cyclic C1-C20 (hetero)alkyl residues, branched, unbranched or cyclic C2-C20 (hetero)alkenyl residues, branched, unbranched or cyclic C2-C20 (hetero)alkynyl residues, C1-C20 (hetero)cycloalkyl residues, C6-C20 aryl residues and / or C3-C19 heteroaryl residues, comprising two or more nucleoside moieties conjugated to each other via a linker comprising or consisting of a salt thereof.

[0020] The term "alkyl" or "heteroalkyl" used throughout the present invention can be understood in the broadest sense. It can be, for example, methyl or ethyl. Heteroalkyl can contain at least one heteroatom, such as, for example, N, O, S or P. It can also include alkoxy (e.g., methoxy (-O-CH3, -OMe)), and similar residues such as -NHCH3 or -N(CH3)2 or salts thereof. Alkyl may be a branched, unbranched or cyclic C1-C40, C1-C30 or C1-C20 (hetero)alkyl residue, a branched, unbranched or cyclic C2-C40, C2-C30 or C2-C20 (hetero)alkenyl residue, a branched, unbranched or cyclic C2-C40, C2-C30, C2-C20 (hetero)alkynyl residue, a C1-C20 (hetero)cycloalkyl residue, a C6-C20 aryl residue and / or a C3-C19 heteroaryl residue.

[0021] The terms "alkenyl" and "heteroalkenyl" used throughout this application may be understood in the broadest sense and may both have one, two or more C-C double bonds. Heteroalkenyl may contain at least one heteroatom, such as N, O, S or P. Preferably, alkenyl is branched, unbranched or cyclic C1-C20 (hetero)alkenyl, optionally substituted with deuterium, halogen, branched, unbranched or cyclic C1-C20 (hetero)alkyl residue, branched, unbranched or cyclic C2-C20 (hetero)alkenyl residue, branched, unbranched or cyclic C2-C20 (hetero)alkynyl residue, C1-C20 (hetero)cycloalkyl residue, C6-C20 aryl residue and / or C3-C19 heteroaryl residue.

[0022] The terms "alkynyl" and "heteroalkynyl" used throughout the present invention may be understood in the broadest sense and may both have one, two or more triple C-C bonds. Heteroalkynyl may contain at least one heteroatom, such as N, O, S or P. Preferably, alkynyl is branched, unbranched or cyclic C1-C20 (hetero)alkynyl, optionally substituted with deuterium, halogen, branched, unbranched or cyclic C1-C20 (hetero)alkyl residue, branched, unbranched or cyclic C2-C20 (hetero)alkenyl residue, branched, unbranched or cyclic C2-C20 (hetero)alkynyl residue, C1-C20 (hetero)cycloalkyl residue, C6-C20 aryl residue and / or C3-C19 heteroaryl residue.

[0023] The terms "aryl" and "heteroaryl" as used herein may be understood in the broadest sense. Heteroaryl may contain at least one heteroatom, such as, for example, N, O, S and / or P, preferably in a cyclic structure. Preferably, alkenyl is a branched, unbranched or cyclic C1-C20 (hetero)alkyl residue, a branched, unbranched or cyclic C2-C20 (hetero)alkenyl residue, a branched, unbranched or cyclic C2-C20 (hetero)alkynyl residue, a C1-C20 (hetero)cycloalkyl residue, a C6-C20 aryl residue and / or a C3-C19 heteroaryl residue.

[0024] In some embodiments, X 2 is selected from the group consisting of -OH, -SH, a C1-C6 alkoxy group, a C1-C6 alkyl sulfide group, a di(C1-C6-alkyl)amino group and a piperazino group, and the nitrogen atom at position 4 is protected by a protecting group and is optionally substituted.

[0025] The type of internucleoside linkage is not necessarily the same at all positions in an oligonucleotide, for example, the middle of an oligonucleotide strand may contain phosphodiester linkages and phosphorothioate linkages are present at the ends of the strand.

[0026] From the above formulas A-1 and A-2, it is clear that each nucleoside contains two moieties that can participate in internucleoside linkages. Thus, these hydroxyl or amine moieties are referred to throughout the text as "backbone moiety(s)" or "backbone group(s)". The backbone moiety of a nucleoside can be two hydroxyl moieties, or one hydroxyl moiety and one amine moiety.

[0027] The hydroxyl moiety participates in a phosphoester type linkage with the phosphorus (V) moiety located between adjacent nucleoside units. The amine moiety participates in a phosphoramidate type linkage with the phosphorus (V) moiety. One of the main chain hydroxyl moieties participates in an internucleoside linkage to the preceding nucleoside, and the other hydroxyl or amine moiety participates in an internucleoside linkage to the succeeding nucleoside. It should be noted that the expressions "preceding" and "following" nucleosides are used randomly in this context and do not reflect a specific orientation of the oligonucleotide strand. The expression "hydroxyl / amine moiety" as used herein includes hydroxyl / amine groups and moieties derived from hydroxyl / amine groups, which typically participate in an ester / amide bond that connects the residue of formula A-1 or A-2 to the nucleotide moiety.

[0028] The main chain hydroxyl and amine groups are typically located at corresponding positions of the consecutive nucleoside moieties arranged to form an oligonucleotide strand.As a result, each nucleoside may contain a first type of main chain hydroxyl moiety located at the first position of the nucleoside, and a second type of main chain hydroxyl / amine moiety located at the second position.The linkage between nucleosides typically involves a first type of main chain hydroxyl moiety derived from the preceding nucleoside, and a second type of main chain hydroxyl / amine moiety derived from the succeeding nucleoside.In RNA and DNA, for example, the main chain hydroxyl moiety is located at the 5' and 3' positions of the ribose moiety, i.e., each nucleoside contains a 5' main chain hydroxyl moiety and a 3' main chain hydroxyl moiety.

[0029] Each internucleoside phosphodiester linkage involves the 5' backbone hydroxyl moiety of the preceding nucleoside and the 3' backbone hydroxyl moiety of the succeeding nucleoside (in DNA and RNA). As a further example, in phosphorodiamidate morpholino oligomers (PMOs), the backbone moiety is the secondary amine moiety of the methylene morpholine ring (the "3' position") and the hydroxyl moiety attached to the methylene group of the methylene morpholine moiety (the "5' position"). Each internucleoside phosphodiester linkage involves the secondary amine moiety of the preceding nucleoside and the hydroxyl moiety of the succeeding nucleoside.

[0030] Obviously, the first and last nucleoside units are involved in only one internucleoside linkage and therefore contain free backbone hydroxyl groups (e.g., in the case of the phosphoribose backbone found in DNA and RNA, the so-called 5'-OH and 3'-OH groups) or a hydroxyl moiety that is linked to another moiety, e.g., a capping structure.

[0031] Oligonucleotides can be conjugated to additional moieties for various purposes. Conjugation can be via at least one of the terminal backbone hydroxyl moieties, or via other functional groups of the nucleoside moieties located at the end or inside of the oligonucleotide strand.For example, the 5'-OH group of the antisense strand of siRNA can be replaced by vinyl phosphonate to inhibit cellular degradation and improve efficacy.Another example is conjugation to N-acetylgalactosamine (GalNAc) for targeting oligonucleotide delivery to hepatocytes. GalNac structures, often branched to accommodate three or four GalNac moieties, can be conjugated, for example, to the 5'-OH group of an oligonucleotide (cf., e.g., WO2016055601), to the 3'-OH group (cf., e.g., WO2009073809), or a monovalent GalNac moiety can be conjugated via a linker to the 2' position of a subsequent ribose moiety within an oligonucleotide strand (cf., e.g., WO2019075419).

[0032] As used herein, the expression "nucleoside or (oligo)nucleotide conjugate" refers to any compound comprising a nucleoside or nucleotide covalently bound to another moiety, such as a peptide, protein, lipid, carbohydrate or hydrocarbon moiety.

[0033] The terms "oligonucleotide" and "polynucleotide" used herein can be understood interchangeably.From the above description, it follows that the non-limiting examples of oligonucleotide are deoxynucleic acid (DNA), ribonucleic acid (RNA), locked nucleic acid (LNA), restricted ethyl nucleic acid analog (cEt), bridged nucleic acid (BNA), tricyclo DNA, unlocked nucleic acid (UNA), small interfering RNA (siRNA), micro RNA, antisense oligonucleotide (ASO), gapmer, glycerol nucleic acid, phosphorodiamidate morpholino oligomer (PMO), phosphorothioate oligonucleotide, phosphorodithioate oligonucleotide, diastereomeric pure phosphorothioate oligonucleotide, and their derivatives and analogs.

[0034] The term "oligonucleotide" further includes conjugates of oligonucleotide moieties and other moieties such as peptides, carbohydrates, etc., unless otherwise indicated for a particular embodiment. A particular interesting example of an oligonucleotide is a phosphorothioate constructed from a nucleoside containing a ribose moiety modified with a 2' substituent selected from, for example, -F, -OMe (-O-CH3) or -methoxyethyloxy (-O-CH2-CH2-O-CH3, also known as MOE, -O-methoxyethyl substituent) and a nucleobase capable of Watson-Crick base pairing with a natural target sequence. Those skilled in the art will recognize that the above moieties have a number of stereoisomers, all of which are encompassed by the above definition. As used herein, an "oligonucleotide" can be a population of oligonucleotide molecules having essentially the same sequence, which is a mixture of a number of individual stereoisomers, or is enriched in one particular stereoisomer, or consists essentially of one particular stereoisomer.

[0035] It will be understood by those skilled in the art that the oligonucleotides used herein may optionally have any counterion known in the art, such as anions or cations, for example, chloride, acetate, carbonate, bicarbonate, sodium, potassium, magnesium, trifluoroacetate (TFA), bromide, perchlorate, ammonium, and / or cations or anions of the residues of protecting groups. Additionally, the oligonucleotides may optionally be covalently or non-covalently associated with trace amounts of one or more scavengers, such as, for example, triisopropylsilane (TIS), dithiothreitol (DTT), anisole, thioanisole, or 1,2-ethanedithiol.

[0036] In general terms, the present invention provides a compound of formula II: [ka] (In the formula: *indicates the point of attachment to an oxygen atom of a hydroxyl moiety of a protected nucleoside, nucleotide, oligonucleotide, or of a conjugate of a nucleoside, nucleotide, or oligonucleotide; c is 0 or 1; a is an integer from 1 to 12; R 3 is H or OR 7 and R 7 is a C1 to C6 alkyl group; R 5 OR 6 or H and R 6 contains a C1 to C40 aliphatic hydrocarbon group; R 4 Each of the following is independently OR 6 and R 6 contains a C1 to C40 aliphatic hydrocarbon group; Whereas b is 1 to 3; However, if b is 1, then R 3 and R 5 is not H; Also, however, there exists R 3 , R 4 and R 5 With the further proviso that the total sum of all carbon atoms contained in the moiety is greater than 23 and less than 200, the present invention relates to a pseudo solid-phase protecting group for protecting a nucleoside, nucleotide, oligonucleotide, or conjugate of a nucleoside, nucleotide, or oligonucleotide represented by the formula:

[0037] A further aspect of the invention relates to a pseudo solid-phase protecting group for protecting a nucleoside, a nucleotide, an oligonucleotide, or a conjugate of a nucleoside, a nucleotide or an oligonucleotide, the pseudo solid-phase protecting group being represented by formula II: [ka] (In the formula: *indicates the point of attachment to an oxygen atom of the nucleoside, nucleotide, oligonucleotide, or of a hydroxyl moiety of a conjugate of a nucleoside, nucleotide, or oligonucleotide to be protected; c is 0 or 1; a is an integer from 1 to 12; R 3 is H; R 5 OR 6 or H and R 6 is a C8 to C40 aliphatic hydrocarbon group; R 4 Each of the following is independently OR 6 and R 6 is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; Whereas b is 1 to 3; However, if b is 1, then R 3 and R 5 is not H; Also, however, there exists R 3 , R 4 and R 5 with the further proviso that the sum of all carbon atoms contained in the moiety is greater than 23 and less than 200.

[0038] The residue OR of the phenyl residue of the pseudo solid-phase protecting group of the present invention 6 OR of phenyl residues, if the sum of all is not greater than 4 (in other words, at least one residue of the phenyl residues is H) 6 It is understood that the sum of all carbon atoms in the alkyl group is preferably greater than 23 and less than 160 throughout the present invention.

[0039] R 3 In the formula where is H, throughout the present invention, the proviso is that when b is 1, R 5 is not H; 3 , R 4 and R 5It is also understood that the sum of all carbon atoms contained in the moiety is defined to be greater than 23 and less than 160.

[0040] Any residue OR 6 is connected via an oxygen atom to the core structure of formula II (i.e., -OR 6 It will be understood by those skilled in the art that the hyphen indicates that the bond to the core structure of formula II is attached to an oxygen atom. The same principle can be applied to one or more OR 6 The same applies to other formulas containing the moiety OR 6 , -OR 6 , OR 6 AND -OR 6 are used interchangeably. The hyphen does not represent an additional chemical bond not already depicted in the respective formula, e.g., Formula II.

[0041] The expression "aliphatic hydrocarbon group" as used herein, unless otherwise expressly specified, refers to a moiety that is non-aromatic and is constructed from carbon and hydrogen atoms. This means that, unless otherwise indicated for a particular embodiment, the "aliphatic hydrocarbon group" does not contain any heteroatoms (i.e. atoms of chemical elements other than carbon and hydrogen). When an aliphatic hydrocarbon group is said to "contain one or more heteroatoms", this means that one or more heteroatoms can be located at any position of the hydrocarbon group. Preferably, the heteroatoms are selected from O, S, N, Si or halogen. For example, the hydrocarbon group includes -O-, -S-, -COO-, -OCONH-, -CONH-, -Cl, -Br, -I or -NH2- moieties. This means that the hydrocarbon group takes the form of -alkyl-O-alkyl in such cases.

[0042] In some embodiments, R 3 and R 5 At least one of R is H. In some embodiments, R 3 is H. In some embodiments, R 5 is H. In some embodiments, R3 is H and R 5 OR 6 In some embodiments, R 3 is H and R 5 is H. Further, in some embodiments, a is an integer from 1 to 3. Further, in some embodiments, b is an integer from 1 to 2. In some embodiments, a is an integer from 1 to 3 (i.e., a is 1, 2 or 3) and b is an integer from 1 to 2 (i.e., b is 1 or 2). In some embodiments, a is 1. In some embodiments, a is 2 or 3. In some embodiments, a is 1 and b is 1 to 3. In some embodiments, a is 1 and b is 1 or 2. As used herein, a variable is "1 to 3" if its value is selected from the group consisting of 1, 2 or 3. In some embodiments, a is 1 to 3 and b is 1 to 3 and R 6 is a C8 to C40 alkyl or heteroalkyl group. In some embodiments, a is 1, b is 1 to 3, and R 6 is a C8 to C40 alkyl or heteroalkyl group.

[0043] In some embodiments, a is an integer from 1 to 3. In some embodiments, when c is 0, a is an integer from 1 to 2 (i.e., a is 1 or 2). In some preferred embodiments, R 6 is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group, which is preferably a C8 to C40 alkyl or alkenyl group, particularly a C8 to C40 alkyl group. In some embodiments, R 6 is, in each occurrence, independently a C12 to C40 aliphatic hydrocarbon group, which is preferably a C12 to C40 alkyl or alkenyl group, particularly a C12 to C40 alkyl group. In some embodiments, R 6is, in each occurrence, independently a C12 to C30 aliphatic hydrocarbon group, which is preferably a C12 to C30 alkyl or alkenyl group, particularly a C12 to C30 alkyl group. In some embodiments, R 6 is, in each occurrence, independently a C15 to C25 aliphatic hydrocarbon group, which is preferably a C15 to C25 alkyl or alkenyl group, particularly a C15 to C25 alkyl group. In some embodiments, R 6 is, in each occurrence, independently a C18 to C22 aliphatic hydrocarbon group, which is preferably a C18 to C22 alkyl or alkenyl group, particularly a C18 to C22 alkyl group. In some embodiments, R 6 is, in each occurrence, independently a C18 to C22 aliphatic hydrocarbon group, which is preferably a straight-chain C18 to C22 alkyl or alkenyl group, in particular a straight-chain C18 to C22 alkyl group.

[0044] In some embodiments, * indicates the point of attachment to the oxygen atom of the hydroxyl moiety of the nucleoside or nucleotide to be protected.

[0045] In some embodiments, a is 1, b is 1 to 2, and R 6 is a C8 to C40 alkyl or heteroalkyl group. In some embodiments, a is 1, b is 1 to 2, and R 6 is a C8 to C40 alkyl or heteroalkyl group; R 3 is H and R 5 is H or OR 6 In some embodiments, a is 1, b is 1 to 2, and R 6 is a C8 to C40 alkyl group; R 3 is H and R 5 is H or OR 6 In the foregoing embodiments, the alkyl or heteroalkyl group can be, for example, a linear, i.e., unbranched, group.

[0046] In some embodiments, the R present in the pseudo solid-phase protecting group 3 , R 4 and R 5 The sum of all carbon atoms contained in the moiety is greater than 23 and less than 200, 185, 184, 180, 160, 140, 123, 120, 100, 80, 67, 60, 55 or 40.

[0047] From the above definition, R 6 It is clear that R may occur several times in a compound according to formula II. 6 The different instances of the groups are selected independently of each other, ie, they may be the same or different at each occurrence.

[0048] In some embodiments, R 6 is a C1-C30 hydrocarbon group optionally substituted with one or more moieties selected from the group consisting of a C1-C30 heteroalkyl group, a C1-C30 alkyl group, a C1-C30 heteroalkenyl group, and a C1-C30 alkenyl group. 6 is a C1-C40 hydrocarbon group optionally substituted with a moiety selected from the group consisting of, for example, alkyl, heteroalkyl, alkenyl, and heteroalkenyl groups, preferably C1-C30 alkyl, C1-C30 heteroalkyl, C1-C30 alkenyl, and C1-C30 heteroalkenyl groups. In some embodiments, R 6 is a C8 to C40 aliphatic hydrocarbon group containing one or more heteroatoms.

[0049] In some embodiments, R 6 is an aliphatic hydrocarbon group having 1 to 40, 1 to 30, 8 to 40, 8 to 30, 12 to 30, or 12 to 40 carbon atoms. 6The group has 1 to 40, 1 to 30, 8 to 40, 8 to 30, 12 to 30, or 12 to 40 carbon atoms and is selected from the group consisting of alkyl, heteroalkyl, alkenyl, or heteroalkenyl. These alkyl, heteroalkyl, alkenyl, or heteroalkenyl moieties can be optionally substituted. In some embodiments, R 6 has one of the structures depicted in FIG. 1, where the asterisk represents the point of attachment to the oxygen atom. In some embodiments, R 6 is a C8 to C40 alkyl or heteroalkyl group. In the foregoing embodiment, the alkyl or heteroalkyl group is a straight chain, i.e., unbranched, group.

[0050] In some embodiments of the pseudo solid phase protecting groups of the present invention, c is 1. In such embodiments, the pseudo solid phase protecting group according to formula II has the formula II-1: [ka] (In the formula, R 3 , R 4 , R 5 , R 6 , R 7 , a, b and * The same definitions as for formula II apply to pseudo solid-phase protecting groups according to formula II-1. This means that all embodiments and definitions given throughout the text may also apply to pseudo solid-phase protecting groups of formula II-1, with the proviso that c is always 1.

[0051] In some embodiments of the pseudo solid phase protecting groups of the present invention, c is 0. In such embodiments, the pseudo solid phase protecting group according to formula II has the formula II-2: [ka] (In the formula, R 3 , R 4 , R 5 , R 6 , R 7 , a, b and *The same definitions as for formula II apply to pseudo solid-phase protecting groups according to formula II-2. This means that all embodiments and definitions given throughout the text may also apply to pseudo solid-phase protecting groups according to formula II-2, with the proviso that c is always 0.

[0052] In some embodiments, the pseudo solid phase protecting group represented by formula II is Formula II-a: [ka] (In the formula: * indicates the point of attachment to an oxygen atom of the nucleoside, nucleotide, oligonucleotide, or of a hydroxyl moiety of a conjugate of a nucleoside, nucleotide, or oligonucleotide to be protected; c is 0 or 1; a is an integer from 1 to 3; R 5 OR 6 or H; R 8 , R 9 and R 10 Each of the following is independently OR 6 or H; R 6 is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; provided that the pseudo solid-phase protecting group according to formula II-a has exactly two or exactly three OR 6 and However, R 5 , R 8 , R 9 and R 10 with the further proviso that the total of all carbon atoms contained in the moiety is greater than 23 and less than 200.

[0053] The residue OR of the phenyl residue of the pseudo solid-phase protecting group of the present invention 6OR of a phenyl residue, if the sum of all is not greater than 3 (in other words, at least two residues of the phenyl residue are H). 6 It is understood that the sum of all carbon atoms in the alkyl group is preferably greater than 23 and less than 120 throughout the present invention. Thus, the pseudo solid-phase protecting group according to formula II-a has exactly three OR 6 If it contains a part, R 5 , R 8 , R 9 and R 10 The total number of all carbon atoms contained in the moiety may be greater than 23 and less than 120.

[0054] The residue OR of the phenyl residue of the pseudo solid-phase protecting group of the present invention 6 OR of a phenyl residue, if the sum of all is not greater than 2 (in other words, at least three residues of the phenyl residue are H). 6 It is understood that the sum of all carbon atoms in the alkyl group is preferably greater than 23 and less than 80 throughout the present invention. Thus, the pseudo solid-phase protecting group according to formula II-a has exactly two OR 6 If it contains a part, R 5 , R 8 , R 9 and R 10 The total number of all carbon atoms contained in the moiety may be greater than 23 and less than 80.

[0055] In some embodiments of the pseudo solid-phase protecting group represented by formula II-a, R 6 is, at each occurrence, independently a C8 to C40 aliphatic hydrocarbon group, which is preferably a C8 to C40 alkyl or alkenyl group, particularly a C8 to C40 alkyl group. In some embodiments of the pseudo solid-phase protecting group represented by formula II-a, R 6 is, at each occurrence, independently a C12 to C40 aliphatic hydrocarbon group, which is preferably a C12 to C40 alkyl or alkenyl group, particularly a C12 to C40 alkyl group. In some embodiments of the pseudo solid-phase protecting group represented by formula II-a, R6 is, at each occurrence, independently a C12 to C30 aliphatic hydrocarbon group, which is preferably a C12 to C30 alkyl or alkenyl group, particularly a C12 to C30 alkyl group. In some embodiments of the pseudo solid-phase protecting group represented by formula II-a, R 6 is, at each occurrence, independently a C15 to C25 aliphatic hydrocarbon group, which is preferably a C15 to C25 alkyl or alkenyl group, particularly a C15 to C25 alkyl group. In some embodiments of the pseudo solid-phase protecting group represented by formula II-a, R 6 is, at each occurrence, independently a C18-C22 aliphatic hydrocarbon group, which is preferably a C18-C22 alkyl or alkenyl group, particularly a C18-C22 alkyl group. In some embodiments of the pseudo solid-phase protecting group represented by formula II-a, R 6 is, in each occurrence, independently a C18 to C22 aliphatic hydrocarbon group, which is preferably a straight-chain C18 to C22 alkyl or alkenyl group, in particular a straight-chain C18 to C22 alkyl group.

[0056] In some embodiments of the pseudo solid-phase protecting group represented by formula II-a, a is 1. In some embodiments of the pseudo solid-phase protecting group represented by formula II-a, a is 2. In some embodiments of the pseudo solid-phase protecting group represented by formula II-a, a is 3. In some embodiments of the pseudo solid-phase protecting group represented by formula II-a, a is 2 or 3.

[0057] In some embodiments of the pseudo solid-phase protecting group represented by formula II-a, a is 1 and R 5 OR 6 If R 9 is H.

[0058] In some embodiments of the pseudo solid phase protecting group represented by formula II-a: - R 5 OR 6 and R 8 and R 10 Both are OR 6and R 9 is H; - R 5 OR 6 and R 8 and R 10 are both H and R 9 OR 6 is; or - R 5 is H and R 8 and R 10 Both are OR 6 and R 9 is H.

[0059] In some embodiments of the pseudo solid phase protecting group represented by formula II-a: - R 5 OR 6 and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3; - R 5 OR 6 and R 8 and R 10 are both H and R 9 OR 6 where a is an integer from 2 to 3; or - R 5 is H and R 8 and R 10 Both are OR 6 and R 9 is H and a is an integer from 1 to 3.

[0060] In some embodiments of the pseudo solid phase protecting group represented by formula II-a: - R 5 OR 6 and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3, and R 6 is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; - R 5 OR 6 and R 8 and R 10 are both H and R 9 OR 6 a is an integer from 2 to 3; R 6 is, in each occurrence, independently a C12 to C40 aliphatic hydrocarbon group; or - R 5 is H and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3, and R 6 is, in each occurrence, independently a C12 to C40 aliphatic hydrocarbon group.

[0061] In some embodiments of the pseudo solid phase protecting group represented by formula II-a: - R 5 OR 6 and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3, and R 6 is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group, and c is 1; - R 5 OR 6 and R 8 and R 10 are both H and R 9 OR 6 a is an integer from 2 to 3; R 6 is, in each occurrence, independently a C12 to C40 aliphatic hydrocarbon group, and c is 0 or 1; or - R 5 is H and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3, and R 6is independently, in each occurrence, a C12 to C40 aliphatic hydrocarbon group, and c is 0 or 1.

[0062] In some embodiments of the pseudo solid phase protecting group represented by formula II-a, when c is 0, a is an integer from 1 to 2 (ie, a is 1 or 2).

[0063] In some embodiments of the pseudo solid phase protecting group represented by formula II-a, c is 1. In such embodiments, the pseudo solid phase protecting group according to formula II-a has the formula II-a-1: [ka] (In the formula, R 5 , R 6 , R 8 , R 9 , R 10 , a and * The same definitions as for formula II-a apply to the pseudo solid-phase protecting group according to formula II-a-1. This means that any embodiment and definition relating to the pseudo solid-phase protecting group of formula II-a also applies to the pseudo solid-phase protecting group of formula II-a-1, with the proviso that c is always 1.

[0064] In some embodiments of the pseudo solid phase protecting group represented by formula II-a, c is 0. In such embodiments, the pseudo solid phase protecting group according to formula II-a has the formula II-a-2: [ka] (In the formula, R 5 , R 6 , R 8 , R 9 , R 10 , a and * The same definitions as for formula II-a apply to the pseudo solid-phase protecting group according to formula II-a-2. This means that any embodiment and definition relating to the pseudo solid-phase protecting group of formula II-a also applies to the pseudo solid-phase protecting group of formula II-a-2, with the proviso that c is always 0.

[0065] Further preferred embodiments of the pseudo solid-phase protecting groups according to formula II and formula II-a are shown in formulae III to VII below: [ka] In formula III, * indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 1 to 12; R 6 The above definition of applies. In some preferred embodiments, a is an integer from 1 to 3. In some embodiments, R 6 Each of R is independently a C12 to C40 aliphatic hydrocarbon group that optionally contains one or more heteroatoms. 6 Each of R is independently an aliphatic hydrocarbon or heteroalkyl group having 12 to 40 carbon atoms; preferably 12 to 30 carbon atoms. 6 Each of R is independently an aliphatic hydrocarbon group having from 12 to 30 carbon atoms. 6 is selected from the structures depicted in Figure 1. In some embodiments, R 6 Each of is independently a C12 to C40 alkyl group or a C12 to C40 heteroalkyl group. 6 Each of is independently a C12 to C40 alkyl group. In some embodiments, R 6 Each of is independently a linear C12 to C40 alkyl group.

[0066] [ka] In formula IV, * indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 1 to 12; R 6 The above definition of applies. In some preferred embodiments, a is an integer from 1 to 3. In some embodiments, R 6Each of R is independently a C8-C40 aliphatic hydrocarbon group that optionally contains one or more heteroatoms. 6 Each of R is independently an aliphatic hydrocarbon or heteroalkyl group having 8 to 40 carbon atoms; preferably 8 to 30 carbon atoms. 6 Each of R is independently an aliphatic hydrocarbon group having from 8 to 30 carbon atoms. 6 is selected from the structures depicted in Figure 1. In some embodiments, R 6 Each of is independently a C8 to C40 alkyl group or a C8 to C40 heteroalkyl group. 6 Each of is independently a C8 to C40 alkyl group. In some embodiments, R 6 Each of is independently a linear C8 to C40 alkyl group.

[0067] [ka]

[0068] In formula V, * indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 1 to 12, preferably from 2 to 12; R 6 The above definition of applies. In some preferred embodiments, a is an integer from 1 to 3, or an integer from 2 to 3. In some embodiments, R 6 Each of R is independently a C12 to C40 aliphatic hydrocarbon group that optionally contains one or more heteroatoms. 6 Each of R is independently an aliphatic hydrocarbon or heteroalkyl group having 12 to 40 carbon atoms; preferably 12 to 30 carbon atoms. 6 Each of R is independently an aliphatic hydrocarbon group having from 12 to 30 carbon atoms. 6is selected from the structures depicted in Figure 1. In some embodiments, R 6 Each of is independently a C12 to C40 alkyl group or a C12 to C40 heteroalkyl group. 6 Each of is independently a C12 to C40 alkyl group. In some embodiments, R 6 Each of is independently a linear C12 to C40 alkyl group.

[0069] [ka]

[0070] In formula VI: * indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 1 to 12, preferably from 2 to 12; R 6 The above definition of applies. In some preferred embodiments, a is an integer from 1 to 3, or an integer from 2 to 3. In some embodiments, R 6 Each of R is independently a C8-C40 aliphatic hydrocarbon group that optionally contains one or more heteroatoms. 6 Each of R is independently an aliphatic hydrocarbon or heteroalkyl group having 8 to 40 carbon atoms; preferably 8 to 30 carbon atoms. 6 Each of R is independently an aliphatic hydrocarbon group having from 8 to 30 carbon atoms. 6 is selected from the structures depicted in Figure 1. In some embodiments, R 6 Each of is independently a C8 to C40 alkyl group or a C8 to C40 heteroalkyl group. 6 Each of is independently a C8 to C40 alkyl group. In some embodiments, R 6 Each of is independently a linear C8 to C40 alkyl group.

[0071] [ka]

[0072] In formula VII: * indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 1 to 12; R 6 The above definition of applies. In some preferred embodiments, a is an integer from 1 to 3. In some embodiments, R 6 Each of R is independently a C12 to C40 aliphatic hydrocarbon group that optionally contains one or more heteroatoms. 6 Each of R is independently an aliphatic hydrocarbon or heteroalkyl group having 12 to 40 carbon atoms; preferably 12 to 30 carbon atoms. 6 Each of R is independently an aliphatic hydrocarbon group having from 12 to 30 carbon atoms. 6 is selected from the structures depicted in Figure 1. In some embodiments, R 6 Each of is independently a C12 to C40 alkyl group or a C12 to C40 heteroalkyl group. 6 Each of is independently a C12 to C40 alkyl group. In some embodiments, R 6 Each of is independently a linear C12 to C40 alkyl group.

[0073] For the avoidance of doubt, the expression "an aliphatic hydrocarbon group or a heteroalkyl group having from 12 to 40 carbon atoms" as used herein refers to a C12 to C40 aliphatic hydrocarbon group or a C12 to C40 heteroalkyl group as used herein. In the foregoing embodiment, the alkyl or heteroalkyl group is a straight chain, i.e., unbranched, group.

[0074] In some embodiments, the pseudo solid phase protecting group represented by any one of formulas II and II-a is represented by formulas III to VII: [ka] (In formula III, * indicates the point of attachment to the oxygen atom of the hydroxyl moiety to be protected; c is 0 or 1; a is an integer from 1 to 12, preferably 1 to 3; R 6 each independently is a C12 to C40 aliphatic hydrocarbon group; [ka] (In formula IV, * indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 1 to 12, preferably 1 to 3; R 6 each independently is a C8 to C40 aliphatic hydrocarbon group; [ka] (In formula V, * indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 2 to 12, preferably 2 to 3; R 6 each independently is a C12 to C40 aliphatic hydrocarbon group; [ka] (In formula VI, * indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 2 to 12, preferably 2 to 3; R 6 each independently represents a C8 to C40 aliphatic hydrocarbon group; and [ka] (In formula VII, * indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 1 to 12, preferably 1 to 3; R 6each of which is independently a C12 to C40 aliphatic hydrocarbon group).

[0075] In some embodiments, the pseudo solid-phase protecting group represented by any one of formulas II and II-a is a pseudo solid-phase protecting group represented by any one of formulas III to V above: In formula III, * indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 1 to 3; R 6 each independently represents a C12 to C40 aliphatic hydrocarbon group, preferably a C12 to C40 alkyl group, in particular a linear C12 to C40 alkyl group; In formula IV, * indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 1 to 3; R 6 each independently represents a C8 to C40 aliphatic hydrocarbon group, preferably a C8 to C40 alkyl group, particularly a linear C8 to C40 alkyl group; In formula V, * indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 2 to 3; R 6 Each of these is independently a C12 to C40 aliphatic hydrocarbon group, preferably a C12 to C40 alkyl group, in particular a linear C12 to C40 alkyl group.

[0076] In some embodiments of the pseudo solid phase protecting group represented by any one of formulas III to VII, when c is 0, a is an integer from 1 to 2 (i.e., a is 1 or 2). In some embodiments, in formula IV, c is always 1. In some embodiments, the pseudo solid-phase protecting group of the present invention is a pseudo solid-phase protecting group for protecting a nucleoside, nucleotide, or oligonucleotide. In such embodiments, in any one of formulas II, II-1, II-2, II-a, II-a-1, II-a-2, and III to VII: *indicates the point of attachment to the oxygen atom of the hydroxyl moiety of the nucleoside, nucleotide or oligonucleotide to be protected. In some embodiments, the pseudo-solid-phase protecting group of the present invention is a pseudo-solid-phase protecting group for protecting a nucleoside, nucleotide or oligonucleotide, and the term "oligonucleotide" does not include conjugates of oligonucleotides. In some embodiments, the pseudo-solid-phase protecting group of the present invention is a pseudo-solid-phase protecting group for protecting a nucleoside. In such an embodiment, in any one of formulas II, II-1, II-2, II-a, II-a-1, II-a-2 and III to VII: * indicates the point of attachment of the hydroxyl moiety of the protected nucleoside to an oxygen atom.

[0077] The present invention relates to a compound of formula I: [ka] (In the formula: R 1 is a protecting group; X is independently at each occurrence O or N; n is an integer equal to or greater than 0; Z is independently at each position an electron withdrawing group or H; Y is independently for each repeat unit, n, absent, O, or S; Each of the nucleosides x0 to xn may be the same or different; CA is a capping moiety or a single bond; c is 0 or 1; a is an integer from 1 to 12; R 3 is H or OR 7 and R 7 is a C1 to C6 alkyl group; R 5 OR 6 or H and R 6 contains a C1 to C40 aliphatic hydrocarbon group; R 4 Each of the following is independently OR6 and R 6 contains a C1 to C40 aliphatic hydrocarbon group; Whereas b is 1 to 3; However, if b is 1, then R 3 and R 5 is not H; Also, however, there exists R 3 , R 4 and R 5 with the further proviso that the sum of all carbon atoms contained in the moiety is greater than 23 and less than 200.

[0078] A further aspect of the present invention is a compound of formula I: [ka] (In the formula: R 1 is a protecting group; X is independently at each occurrence O or N; n is an integer equal to or greater than 0; Z is independently at each occurrence H or an electron withdrawing group; Y is independently for each repeat unit, n, absent, O, or S; Each of the nucleosides x0 to xn may be the same or different; CA is a capping moiety or a single bond; c is 0 or 1; a is an integer from 1 to 12; R 3 is H; R 5 OR 6 or H and R 6 is a C8 to C40 aliphatic hydrocarbon group; R 4 Each of the following is independently OR 6 and R 6 is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; Whereas b is 1 to 3; However, if b is 1, then R 3 and R 5 is not H; Also, however, there exists R 3 , R 4 and R 5 with the further proviso that the sum of all carbon atoms contained in the moiety is greater than 23 and less than 200.

[0079] With respect to the pseudo solid-phase protecting groups of the present invention, the integer a and the residues R 3 , R 5 , R 6 , R 4 , R 6 , R 7 , and the descriptions and explanations regarding the integer b are equally applicable to compounds according to formula I. For example, R 3 and R 5 may be H; a may be an integer from 1 to 3; b may be an integer from 1 to 2; R 6 may be a C1-C30 hydrocarbon group optionally substituted with one or more moieties selected from the group consisting of a C1-C30 heteroalkyl group, a C1-C30 alkyl group, a C1-C30 heteroalkenyl group, and a C1-C30 alkenyl group. In some embodiments of the compound according to Formula I, R 3 is H. In some embodiments of the compound according to formula I, a is an integer from 1 to 3 and b is an integer from 1 to 2. In some embodiments of the compound according to formula I, when c is 0, a is an integer from 1 to 2 (i.e., a is 1 or 2).

[0080] In some embodiments of the compound according to Formula I, CA is a single bond. In this case, the oxygen atom depicted adjacent to CA in Formula I is directly attached to the nucleoside x0 via a single bond (the single bond is CA).

[0081] The electron-withdrawing group Z attached to the phosphorus atom can be any group that reduces the electron density at the P atom. Z can be, for example, -OH, -OR, -NHR, -NR2, C3-C20-(hetero)aromatic moieties, -F, -Cl, -SH, -SeH, -BH3 - , -SR and -SeR, where R is independently a C1-C10 alkyl or a C1-C10 heteroalkyl group. In particular, R can be a C1-C10 alkyl substituted with an electron-withdrawing group, such as a beta-cyanoethyl group (i.e., a 2-cyanoethyl group). In some embodiments of compounds according to formula I, the electron-withdrawing group Z in each occurrence is -OR and R is a 2-cyanoethyl group. As used herein, unless otherwise indicated, a "2-cyanoethyl" protecting group for a hydroxyl moiety is attached to the oxygen atom being protected via its 1-position, i.e., a 2-cyanoeth-1-yl group.

[0082] In some embodiments of the compound according to formula I, Y is O or S, independently for each repeat unit n. In some embodiments of the compound according to formula I, when Z is H, Y is O (oxygen). In some embodiments of the compound according to formula I, Y is O or S, Z is, in each occurrence, -OR, and R is a 2-cyanoethyl group. In some embodiments of the compound according to formula I, Y is O or S, Z is, in each occurrence, -OR, R is a 2-cyanoethyl group, and CA is a single bond. As listed above, the hyphen in -OR is used simply to indicate that the OR group is attached to the core structure of the respective formula, e.g., formula I, via the oxygen atom from which the hyphen originates. The hyphen does not represent any additional chemical bond not already depicted in the respective formula, e.g., formula I.

[0083] R 1 is a hydroxyl or amine protecting group. 1 R may be a temporary protecting group used during repeated oligomer synthesis to avoid unintended oligomerization or multiple insertions of a component. 1is a triarylmethyl type protecting group, i.e. a protecting group that contains an optionally substituted triarylmethyl residue. The skilled artisan is well aware that the nature of such protecting groups varies depending on their substitution pattern. Substituents that stabilize the carbocation leaving group can facilitate cleavage of the protecting group. For example, a triarylmethyl protecting group can include at least one substituent selected from the group consisting of a methoxy group and a halogen atom. 1 Non-limiting examples include trityl (i.e. triphenylmethyl), 4-methyltrityl, 4,4'-dimethyltrityl, 4,4',4"-trimethyltrityl, (p-methoxyphenyl)diphenylmethyl (MMT), di(p-methoxyphenyl)phenylmethyl (DMT), tri(p-methoxyphenyl)methyl ether (TMT), 4,4'-dimethoxy-3"-[N-(imidazolylmethyl)]trityl (IDT), 4,4'-dimethoxy-3"-[N-(imidazolylethyl)carbamoyl]trityl ether (IET), bis(4-methoxyphenyl)-1'-pyrenylmethyl (Bmpm) and 4-(17-tetrabenzo[a,c,g,i]fluorenylmethyl)-4',4"-dimethoxytrityl (Tbf-DMT). The DMT group is particularly preferred. The protecting groups, including optionally substituted triarylmethyl residues, may be the same or different for the various components used in the process according to the invention.

[0084] In some embodiments of the compounds according to formula I, R 1 is a triarylmethyl-type protecting group as defined herein. In some embodiments of the compound according to formula I, R 1 is a triarylmethyl-type protecting group selected from the group consisting of a trityl group, a MMT group, and a DMT group. In some preferred embodiments of the compound according to formula I, R 1 is a DMT group. In some embodiments of the compound according to formula I, R 1 is a trityl group.

[0085] In some embodiments of the compound according to Formula I, n is an integer ranging from 0 to 200, 0 to 150, 0 to 120, 0 to 100, 0 to 90, 0 to 80, 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 30, 0 to 20, or 0 to 10. In some embodiments of the compound according to Formula I, the integer n is 0 or 1. In some embodiments of the compound according to Formula I, the integer n is 0. When the integer n is 0, the protecting group R 1 It will be understood by one of skill in the art that is still present in compounds of formula I and is attached to nucleoside x0.

[0086] In formula I, each nucleoside is symbolized by a circle that is connected to the oxygen atom of a first backbone hydroxyl moiety via a single bond and contains a second backbone hydroxyl or amine moiety. X represents a heteroatom of the second backbone hydroxyl or amine moiety, which is connected to the phosphorus atom of a subsequent internucleoside linking group, such as a phosphate group. X can be incorporated into the nucleoside molecule in any conceivable manner. For example, X can be the 5' position of a ribose moiety (i.e., X is an oxygen atom that is connected to the remainder of the nucleoside moiety via a single bond), or it can be part of a secondary amine group that forms part of a morpholine ring (i.e., X is a nitrogen atom that is connected to the remainder of the nucleoside moiety via two single bonds). X can be independently selected for each repeat unit n (i.e., for each nucleoside x0 and xn). In some embodiments, X is the same at every position in the compound according to formula I (ie, at each nucleoside x0 and xn).

[0087] In some embodiments of the compound according to formula I, c is 1. In some embodiments of the compound according to formula I, c is 0.

[0088] In some embodiments, the compound of formula I has formula Ib: [ka] (In the formula: R 1 is a protecting group; X is independently at each occurrence O or N; n is an integer equal to or greater than 0; Z is independently at each occurrence H or an electron withdrawing group; Y is independently for each repeat unit, n, absent, O, or S; Each of the nucleosides x0 to xn may be the same or different; CA is a capping moiety or a single bond; c is 0 or 1; a is an integer from 1 to 3; R 5 OR 6 or H; R 8 , R 9 and R 10 Each of the following is independently OR 6 or H; R 6 is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; provided that the compound according to formula Ib is exactly two or exactly three OR 6 and However, R 5 , R 8 , R 9 and R 10 provided that the total number of carbon atoms contained in the moiety is greater than 23 and less than 200.

[0089] When the compound of formula Ib is a compound of formula I, any of the embodiments and definitions relating to the compound of formula I may also apply to the compound of formula Ib. Thus, the integers n, X, Y, Z, CA, R 1 , x0 and xn, R 5 , R 6 , R 8 , R 9 and R 10 The specifications, explanations and embodiments relating to are equally applicable to compounds of formula Ib.

[0090] In some preferred embodiments of the compound according to formula Ib, x0, xn, R 1 , CA and Z, OR 6 Nothing contains a part.

[0091] In some preferred embodiments of the compounds of formula Ib, R 6 is, at each occurrence, independently a C8-C40 aliphatic hydrocarbon group, which is preferably a C8-C40 alkyl or alkenyl group, particularly a C8-C40 alkyl group. In some embodiments of the compound of formula Ib, R 6 is independently at each occurrence a C12 to C40 aliphatic hydrocarbon group, which is preferably a C12 to C40 alkyl or alkenyl group, particularly a C12 to C40 alkyl group. In some embodiments of the compound of formula Ib, R 6 is, at each occurrence, independently a C12-C30 aliphatic hydrocarbon group, which is preferably a C12-C30 alkyl or alkenyl group, particularly a C12-C30 alkyl group. In some embodiments of the compound of formula Ib, R 6 is, at each occurrence, independently a C15 to C25 aliphatic hydrocarbon group, which is preferably a C15 to C25 alkyl or alkenyl group, particularly a C15 to C25 alkyl group. In some embodiments of the compound of formula Ib, R 6 is, at each occurrence, independently a C18-C22 aliphatic hydrocarbon group, which is preferably a C18-C22 alkyl or alkenyl group, particularly a C18-C22 alkyl group. In some embodiments of the compound of formula Ib, R 6 is, in each occurrence, independently a C18 to C22 aliphatic hydrocarbon group, which is preferably a straight-chain C18 to C22 alkyl or alkenyl group, in particular a straight-chain C18 to C22 alkyl group.

[0092] In some embodiments of the compound of formula Ib, a is 1. In some embodiments of the compound of formula Ib, a is 2. In some embodiments of the compound of formula Ib, a is 3. In some embodiments of the compound of formula Ib, a is 2 or 3. In some embodiments of the compound of formula Ib, a is 1 and R5 is OR. 6 If R 9 is H. In some embodiments of compounds of formula Ib, when c is 0, a is an integer from 1 to 2 (ie, a is 1 or 2).

[0093] In some embodiments of the compound of formula Ib: - R 5 OR 6 and R 8 and R 10 Both are OR 6 and R 9 is H; - R 5 OR 6 and R 8 and R 10 are both H and R 9 OR 6 is; or - R 5 is H and R 8 and R 10 Both are OR 6 and R 9 is H.

[0094] In some embodiments of the compound of formula Ib: - R 5 OR 6 and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3; - R 5 OR 6 and R 8 and R 10 are both H and R 9 OR 6where a is an integer from 2 to 3; or - R 5 is H and R 8 and R 10 Both are OR 6 and R 9 is H and a is an integer from 1 to 3.

[0095] In some embodiments of the compound of formula Ib: - R 5 OR 6 and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3, and R 6 is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; - R 5 OR 6 and R 8 and R 10 are both H and R 9 OR 6 a is an integer from 2 to 3; R 6 is, in each occurrence, independently a C12 to C40 aliphatic hydrocarbon group; or - R 5 is H and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3, and R 6 is, in each occurrence, independently a C12 to C40 aliphatic hydrocarbon group.

[0096] In some embodiments of the compound of formula Ib, c is 1. In some embodiments of the compound of formula Ib, c is 0.

[0097] In some embodiments of compounds of formula Ib, Z is independently at each position -OR, R is a 2-cyanoethyl group, Y is independently O or S for each repeat unit n, and CA is a single bond: - R 5 OR 6 and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3, and R 6 is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; - R 5 OR 6 and R 8 and R 10 are both H and R 9 OR 6 a is an integer from 2 to 3; R 6 is, in each occurrence, independently a C12 to C40 aliphatic hydrocarbon group; or - R 5 is H and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3, and R 6 is, in each occurrence, independently a C12 to C40 aliphatic hydrocarbon group.

[0098] In some embodiments of compounds of formula Ib, Z is independently at each position -OR, R is a 2-cyanoethyl group, Y is independently O or S for each repeat unit n, and CA is a single bond: - R 5 OR 6 and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3, and R 6 is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group, and c is 1; - R 5 OR 6 and R 8 and R 10 are both H and R 9 OR 6a is an integer from 2 to 3; R 6 is, in each occurrence, independently a C12 to C40 aliphatic hydrocarbon group, and c is 1 or 0; or - R 5 is H and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3, and R 6 is independently, in each occurrence, a C12 to C40 aliphatic hydrocarbon group, and c is 1 or 0.

[0099] In particularly preferred embodiments of the compounds of the invention, X is O. In these embodiments, the compounds according to formula I may alternatively be reflected by formula Ia, where a, b, c, R 1 ,n,Z,Y,C,A,R 3 , R 4 , R 5 , R 6 and R 7 The definition of is as follows: [ka] As presented in relation to.

[0100] Similar formulas can be drawn for formulas VIII to XII (see below), where X is O and a, c, R 1 , n, Z, Y, C-A and R 6 The definitions of are as provided for each formula.

[0101] Along the same lines, when X is O, the compound according to formula Ib may alternatively be a compound according to formula Ic: [ka] Reflected by a, c, R 1 ,n,Z,Y,C,A,R 5 , R 6 , R 8 , R 9 and R 10are as provided for formula Ib.

[0102] In some embodiments of the compound according to formula Ia, c is 1. In some embodiments of the compound according to formula Ia, c is 0. In some embodiments of the compound according to formula Ic, c is 1. In some embodiments of the compound according to formula Ic, c is 0. In some embodiments of the compound according to formula Ia, when c is 0, a is an integer from 1 to 2 (i.e., a is 1 or 2). In some embodiments of the compound according to formula Ic, when c is 0, a is an integer from 1 to 2 (i.e., a is 1 or 2).

[0103] Exemplary structures according to formula I, particularly according to any one of formulae Ia, Ib, Ic and VIII to XII (see below), when linked, for example, via a hydroxyl moiety to a pseudo solid-phase protecting group according to formula II, particularly according to any one of formulae II-1, II-2, II-a, II-a-1, II-a-2 and III to VII, include the following moieties: DNA oligonucleotides, RNA oligonucleotides, LNA oligonucleotides, UNA oligonucleotides, phosphorothioate oligonucleotides, nucleosides linked by a phosphite triester moiety, and phosphorodiamidate morpholino oligomers (PMOs), as well as any of the conjugates described above. The following structures illustrate non-limiting examples of such compounds, which are represented by the following formulae: * ) to a pseudo solid phase protecting group according to any one of formulae II, II-1, II-2, II-a, II-a-1, II-a-2 and III to VII, resulting in a compound according to any one of formulae I, Ia, Ib, Ic and VIII to XII (see below). [ka]

[0104] Alternatively, to obtain a compound according to any one of formulae I, Ia, Ib, Ic and VIII to XII (see below), the above compounds can be similarly modified by cleaving the attachment point ( * ) to a non-nucleoside moiety comprising a hydroxyl moiety protected by a pseudo solid-phase protecting group according to any one of formulae II, II-1, II-2, II-a, II-a-1, II-a-2 and III to VII. Said non-nucleoside moiety is a capping moiety CA. Depending on the identity of Y, the phosphorus atom in any one of formulae I, Ia, Ib, Ic and VIII to XII (see below) can have the oxidation number III (Y is H) or V (Y is an electron-withdrawing group).

[0105] The expression "capping moiety" as used herein refers to any moiety that is conjugated to the terminal nucleoside of an oligonucleotide strand. The capping moiety is included in the final oligonucleotide product obtained after cleavage from PSPPG. In the case where the capping moiety is present between the first nucleoside and the pseudo solid-phase protecting group (PSPPG), it can be attached to the terminal backbone hydroxyl group. An example for such a strategy is the insertion of a 3'GalNac conjugate, for example as described in WO2009073809. Typical capping moieties are peptides, saccharides or lipids.

[0106] In the exemplary structure above, R" can be any possible substituent to the ribose 2' position. In particular, R" can be selected from the group consisting of: H, alkyl, heteroalkyl, amine, amide, ester, halogen, hydroxyl or thiol moiety. R" can further represent a linker moiety that connects to a tagging moiety or form a bridge to the 4' carbon atom of the ribose ring. Those skilled in the art will recognize a multitude of ribose-based nucleoside moieties that are substituted at the 2' position and can be assembled into oligomers, with phosphorus-based moieties in between. R' is a C1-C10 alkyl or a C1-C10 heteroalkyl. In particular, R' is a C1-C10 alkyl substituted with an electron-withdrawing group, for example, R' is a beta-cyanoethyl group (i.e., a 2-cyanoethyl group).

[0107] In some embodiments, the compound according to formula I or Ib is selected from the group consisting of formulas VIII to XII depicted below. [ka]

[0108] In formula VIII, c is 0 or 1; a is an integer from 1 to 12; R 1 is a protecting group; X is independently at each position O or N; n is an integer equal to or greater than 0; Z is independently at each position H or an electron withdrawing group; Y is independently absent, O or S for each repeat unit n; each of the nucleosides x0 through xn can be the same or different; CA is a capping moiety or a single bond; R 6 The above definition of applies. In some preferred embodiments, a is an integer from 1 to 3. In some embodiments, CA is a single bond. In some embodiments, R 1 is a triarylmethyl type protecting group, preferably a protecting group selected from the group consisting of DMT, trityl and MMT, more preferably a DMT group. In some embodiments, R 6Each of R is independently a C12 to C40 aliphatic hydrocarbon group that optionally contains one or more heteroatoms. 6 Each of R is independently an aliphatic hydrocarbon or heteroalkyl group having 12 to 40 carbon atoms; preferably 12 to 30 carbon atoms. 6 Each of R is independently an aliphatic hydrocarbon group having from 12 to 30 carbon atoms. 6 is selected from the structures depicted in Figure 1. In some embodiments, a is 1. In some embodiments, R 6 Each of R is independently a C12 to C40 alkyl group or a C12 to C40 heteroalkyl group. 6 Each of is independently a C12 to C40 alkyl group. In some embodiments, X is O and R 6 Each of is independently a C12 to C40 alkyl group or a C12 to C40 heteroalkyl group. In some embodiments, X is O and R 6 is independently a C12 to C40 alkyl group. In some embodiments, a is 1, X is O, and R 6 is independently a C12 to C40 alkyl group or a C12 to C40 heteroalkyl group. In some embodiments, a is 1, X is O, and R 6 is independently a C12 to C40 alkyl group. In some embodiments, a is an integer from 1 to 3, X is O, and R 6 is independently a C12 to C40 alkyl group. In some embodiments, a is an integer from 1 to 3, X is O, and R 6 Each of is independently a linear C12 to C40 alkyl group.

[0109] [ka] In formula IX, c is 0 or 1; a is an integer from 1 to 12; R1 is a protecting group; X is independently at each position O or N; n is an integer equal to or greater than 0; Z is independently at each position H or an electron withdrawing group; Y is independently absent, O or S for each repeat unit n; each of the nucleosides x0 through xn can be the same or different; CA is a capping moiety or a single bond; R 6 The above definition of applies. In some preferred embodiments, a is an integer from 1 to 3. In some embodiments, CA is a single bond. In some embodiments, R 1 is a triarylmethyl type protecting group, preferably a protecting group selected from the group consisting of DMT, trityl and MMT, more preferably a DMT group. In some embodiments, R 6 Each of R is independently a C12 to C40 aliphatic hydrocarbon group that optionally contains one or more heteroatoms. 6 Each of is independently a C8 to C40 aliphatic hydrocarbon group optionally containing one or more heteroatoms.

[0110] In some embodiments, R 6 Each of R is independently an aliphatic hydrocarbon or heteroalkyl group having 8 to 40 carbon atoms; preferably 8 to 30 carbon atoms. 6 Each of R is independently an aliphatic hydrocarbon group having from 8 to 30 carbon atoms. 6 is selected from the structures depicted in Figure 1. In some embodiments, a is 1. In some embodiments, R 6 Each of is independently a C8 to C40 alkyl group or a C8 to C40 heteroalkyl group. 6 Each of is independently a C8 to C40 alkyl group. In some embodiments, X is O and R 6Each of is independently a C8 to C40 alkyl group or a C8 to C40 heteroalkyl group. In some embodiments, X is O and R 6 is independently a C8 to C40 alkyl group. In some embodiments, a is 1, X is O, and R 6 is independently a C8 to C40 alkyl group or a C8 to C40 heteroalkyl group. In some embodiments, a is 1, X is O, and R 6 is independently a C8 to C40 alkyl group. In some embodiments, a is an integer from 1 to 3, X is O, and R 6 is independently a C8 to C40 alkyl group. In some embodiments, a is an integer from 1 to 3, X is O, and R 6 Each of is independently a linear C8 to C40 alkyl group.

[0111] [ka] In formula X, c is 0 or 1; a is an integer from 1 to 12, preferably from 2 to 12; R 1 is a protecting group; X is independently at each position O or N; n is an integer equal to or greater than 0; Z is independently at each position H or an electron withdrawing group; Y is independently absent, O or S for each repeat unit n; each of the nucleosides x0 through xn can be the same or different; CA is a capping moiety or a single bond; R 6 The above definition of applies. In some embodiments, a is an integer from 1 to 3. In some preferred embodiments, a is an integer from 2 to 3. In some embodiments, CA is a single bond. In some embodiments, R 1 is a triarylmethyl type protecting group, preferably a protecting group selected from the group consisting of DMT, trityl and MMT, more preferably a DMT group. In some embodiments, R 6Each of R is independently a C12 to C40 aliphatic hydrocarbon group that optionally contains one or more heteroatoms. 6 Each of R is independently an aliphatic hydrocarbon or heteroalkyl group having 12 to 40 carbon atoms; preferably 12 to 30 carbon atoms. 6 Each of R is independently an aliphatic hydrocarbon group having from 12 to 30 carbon atoms. 6 is selected from the structures depicted in Figure 1. In some embodiments, a is 1. In some embodiments, R 6 Each of is independently a C12 to C40 alkyl group or a C12 to C40 heteroalkyl group. 6 Each of is independently a C12 to C40 alkyl group. In some embodiments, X is O and R 6 Each of is independently a C12 to C40 alkyl group or a C12 to C40 heteroalkyl group. In some embodiments, X is O and R 6 is independently a C12 to C40 alkyl group. In some embodiments, a is 1, X is O, and R 6 is independently a C12 to C40 alkyl group or a C12 to C40 heteroalkyl group. In some embodiments, a is 1, X is O, and R 6 is independently a C12 to C40 alkyl group. In some embodiments, a is an integer from 1 to 3, X is O, and R 6 is independently a C12 to C40 alkyl group. In some embodiments, a is an integer from 1 to 3, X is O, and R 6 Each of is independently a linear C12 to C40 alkyl group.

[0112] [ka] In formula XI, c is 0 or 1; a is an integer from 1 to 12, preferably from 2 to 12; R1 is a protecting group; X is independently at each position O or N; n is an integer equal to or greater than 0; Z is independently at each position H or an electron withdrawing group; Y is independently absent, O or S for each repeat unit n; each of the nucleosides x0 through xn can be the same or different; CA is a capping moiety or a single bond; R 6 The above definition of applies. In some embodiments, a is an integer from 1 to 3. In some preferred embodiments, a is an integer from 2 to 3. In some embodiments, CA is a single bond. In some embodiments, R 1 is a triarylmethyl type protecting group, preferably a protecting group selected from the group consisting of DMT, trityl and MMT, more preferably a DMT group. In some embodiments, R 6 Each of R is independently a C12 to C40 aliphatic hydrocarbon group that optionally contains one or more heteroatoms. 6 Each of R is independently a C8-C40 aliphatic hydrocarbon group that optionally contains one or more heteroatoms. 6 Each of R is independently an aliphatic hydrocarbon or heteroalkyl group having 8 to 40 carbon atoms; preferably 8 to 30 carbon atoms. 6 Each of R is independently an aliphatic hydrocarbon group having from 8 to 30 carbon atoms. 6 is selected from the structures depicted in Figure 1. In some embodiments, a is 1. In some embodiments, R 6 Each of is independently a C8 to C40 alkyl group or a C8 to C40 heteroalkyl group. 6 Each of is independently a C8 to C40 alkyl group. In some embodiments, X is O and R 6 Each of is independently a C8 to C40 alkyl group or a C8 to C40 heteroalkyl group. In some embodiments, X is O and R6 is independently a C8 to C40 alkyl group. In some embodiments, a is 1, X is O, and R 6 is independently a C8 to C40 alkyl group or a C8 to C40 heteroalkyl group. In some embodiments, a is 1, X is O, and R 6 is independently a C8 to C40 alkyl group. In some embodiments, a is an integer from 1 to 3, X is O, and R 6 is independently a C8 to C40 alkyl group. In some embodiments, a is an integer from 1 to 3, X is O, and R 6 Each of is independently a linear C8 to C40 alkyl group.

[0113] [ka] In formula XII, a is an integer from 1 to 12; R 1 is a protecting group; X is independently at each position O or N; n is an integer equal to or greater than 0; Z is independently at each position H or an electron withdrawing group; Y is independently absent, O or S for each repeat unit n; each of the nucleosides x0 through xn can be the same or different; CA is a capping moiety or a single bond; R 6 The above definition of applies. In some preferred embodiments, a is an integer from 1 to 3. In some embodiments, CA is a single bond. In some embodiments, R 1 is a triarylmethyl type protecting group, preferably a protecting group selected from the group consisting of DMT, trityl and MMT, more preferably a DMT group. In some embodiments, R 6 Each of R is independently a C12 to C40 aliphatic hydrocarbon group that optionally contains one or more heteroatoms. 6Each of R is independently an aliphatic hydrocarbon or heteroalkyl group having from 12 to 40 carbon atoms; preferably from 12 to 30 carbon atoms. 6 Each of R is independently an aliphatic hydrocarbon group having from 12 to 30 carbon atoms. 6 is selected from the structures depicted in Figure 1. In some embodiments, a is 1. In some embodiments, R 6 Each of is independently a C12 to C40 alkyl group or a C12 to C40 heteroalkyl group. 6 Each of is independently a C12 to C40 alkyl group. In some embodiments, X is O and R 6 Each of is independently a C12 to C40 alkyl group or a C12 to C40 heteroalkyl group. In some embodiments, X is O and R 6 is independently a C12 to C40 alkyl group. In some embodiments, a is 1, X is O, and R 6 is independently a C12 to C40 alkyl group or a C12 to C40 heteroalkyl group. In some embodiments, a is 1, X is O, and R 6 is independently a C12 to C40 alkyl group. In some embodiments, a is an integer from 1 to 3, X is O, and R 6 is independently a C12 to C40 alkyl group. In some embodiments, a is an integer from 1 to 3, X is O, and R 6 Each of is independently a linear C12 to C40 alkyl group.

[0114] In some embodiments, the compound according to formula I is a compound represented by any one of formulas VIII to XII, where c is 1. In some embodiments, the compound according to formula I is a compound represented by any one of formulas VIII to XII, where c is 0.

[0115] In some embodiments, the compound according to formula I is a compound according to a formula selected from the group consisting of formula VIII, formula IX, and formula XII. In some embodiments, the compound according to formula I is a compound according to a formula selected from the group consisting of formula VIII, formula IX, and formula XII, wherein R 1 is a triarylmethyl type protecting group, e.g., DMT, trityl, or MMT. In some embodiments, the compound according to formula I is a compound according to a formula selected from the group consisting of formula VIII, formula IX, and formula X. In some embodiments, the compound according to formula I is a compound according to a formula selected from the group consisting of formula VIII, formula IX, and formula X, and R 1 is a triarylmethyl type protecting group, such as DMT, trityl or MMT.

[0116] In some preferred embodiments, the compound represented by any one of formulas I and Ib is represented by formulas VIII to XII: [ka] (In formula VIII, c is 0 or 1; a is an integer from 1 to 12, preferably from 1 to 3; R 6 Each of R is independently a C12 to C40 aliphatic hydrocarbon group; 1 is a protecting group; X is independently at each position O or N; n is an integer equal to or greater than 0; Z is independently at each position H or an electron withdrawing group; Y is independently absent, O or S for each repeat unit n; each of the nucleosides x0 through xn can be the same or different; CA is a capping moiety or a single bond);

[0117] [ka] (In formula IX, c is 0 or 1; a is an integer from 1 to 12, preferably from 1 to 3; R 6each is independently a C8 to C40 aliphatic hydrocarbon group, preferably a C12 to C40 aliphatic hydrocarbon group; X is independently at each position O or N; n is an integer equal to or greater than 0; Z is independently at each position H or an electron withdrawing group; Y is independently absent or O or S for each repeat unit n; each of the nucleosides x0 to xn can be the same or different; CA is a capping moiety or a single bond);

[0118] [ka] (In formula X, c is 0 or 1; a is an integer from 2 to 12, preferably from 2 to 3; R 6 Each of R is independently a C12 to C40 aliphatic hydrocarbon group; 1 is a protecting group; X is independently at each position O or N; n is an integer equal to or greater than 0; Z is independently at each position H or an electron withdrawing group; Y is independently absent, O or S for each repeat unit n; each of the nucleosides x0 through xn can be the same or different; CA is a capping moiety or a single bond);

[0119] [ka] (In formula XI, c is 0 or 1; a is an integer from 2 to 12, preferably from 2 to 3; R 6 each independently represents a C8 to C40 aliphatic hydrocarbon group, preferably a C12 to C40 aliphatic hydrocarbon group; R 1 is a protecting group; X is independently at each position O or N; n is an integer equal to or greater than 0; Z is independently at each position H or an electron withdrawing group; Y is independently absent, O or S for each repeat unit n; each of the nucleosides x0 through xn can be the same or different; CA is a capping moiety or a single bond); and

[0120] [ka] (In formula XII, c is 0 or 1; a is an integer from 1 to 12, preferably from 1 to 3; R 6 each independently represents a C12 to C40 aliphatic hydrocarbon group optionally containing one or more heteroatoms; R 1 is a protecting group; X is independently at each position, O or N; n is an integer equal to or greater than 0; Z is independently at each position, H or an electron withdrawing group; Y is independently absent, O or S for each repeat unit n; each of the nucleosides x0 through xn can be the same or different; and CA is a capping moiety or a single bond.

[0121] In some embodiments of a compound according to any one of formulas VIII to XII, when c is 0, a is an integer from 1 to 2 (i.e., a is 1 or 2). In some embodiments, in formula IX, c is always 1. In some embodiments, in formula IX, c is always 0.

[0122] In some preferred embodiments, the compound represented by formula I is a compound according to any one of formulas VIII, IX, and X.

[0123] In some embodiments, the compound of formula I, particularly the compound of formula Ib, has the following formula Ib-1: [ka] (In formula Ib-1: Integers a and c, and R 5 , R 8 , R 9 , R 10 and R 6 is defined for formula Ib; R 1is a triarylmethyl type protecting group; n is an integer equal to or greater than 0; Y is independently O or S for each repeat unit, n; B N is a nucleobase which may be identical or different in each occurrence; R III are hydroxyl protecting groups, which may be the same or different in each occurrence; R II is independently selected at each occurrence from the group consisting of H, F, -O-(C1-C5 alkyl), -O-(C1-C5 alkyl)-O-(C1-C5 alkyl), -O-Si(C1-C5 alkyl)3 and -O-CH2-O-Si(C1-C5 alkyl)3; R I is independently, in each occurrence, H, or R II and R I together form a methylene or ethylene bridge of the chemical structure +-CH2-O-++, +-CH(CH3)-O-++ or +-CH2-CH2-O-++, where + is the 4'-carbon atom (i.e., R I is the point of attachment to the 2'-carbon atom (i.e., the carbon atom to which R II is the point of attachment to the carbon atom to which it is bonded.

[0124] "Integers a and c, and R 5 , R 8 , R 9 , R 10 and R 6 is defined for formula Ib" refers to the integers a and c, and / or R 5 , R 8 , R 9 , R 10 , R 6 Any of the definitions and embodiments relating to the integers a and c, and / or R 5 , R 8 , R 9 , R10 , R 6 It is understood to mean that this also applies to

[0125] In some embodiments of the compound of formula Ib-1, c is 1 or 0: - R 5 OR 6 and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3, and R 6 is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; - R 5 OR 6 and R 8 and R 10 are both H and R 9 OR 6 a is an integer from 2 to 3; R 6 is, in each occurrence, independently a C12 to C40 aliphatic hydrocarbon group; or - R 5 is H and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3, and R 6 is, in each occurrence, independently a C12 to C40 aliphatic hydrocarbon group.

[0126] In some embodiments of the compound of formula Ib-1, c is 1 or 0: - R 5 OR 6 and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3, and R 6 is, in each occurrence, independently a linear C18 to C22 alkyl group; - R 5 OR 6 and R 8and R 10 are both H and R 9 OR 6 a is an integer from 2 to 3; R 6 is, in each occurrence, independently a linear C18 to C22 alkyl group; or - R 5 is H and R 8 and R 10 Both are OR 6 and R 9 is H, a is an integer from 1 to 3, and R 6 is, in each occurrence, independently a straight chain C18 to C22 alkyl group.

[0127] In some embodiments of the compound of formula Ib-1, c is 1. In some embodiments of the compound of formula Ib-1, c is 0.

[0128] In some embodiments of the compound of formula Ib-1: n is an integer in the range of 0-200, 0-150, 0-120, 0-100, 0-90, 0-80, 0-70, 0-60, 0-50, 0-40, 0-30, 0-20, 0-10, or 0-1; Y is independently O or S for each repeat unit, n; B N is, at each occurrence, independently selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil; R III is a 2-cyanoethyl group for each repeat unit n; R IIis independently selected at each occurrence from the group consisting of H, F, -O-CH3 (i.e., methoxy), -O-CH2-CH2-O-CH3 (i.e., 2-methoxyethyl-1-oxy), -O-Si(CH3)3 (i.e., trimethylsilyloxy, TMS), -O-Si(CH3)2(C(CH3)3) (i.e., tert-butyl(dimethyl)silyloxy, TBDMS or TBS) and -O-CH2-O-Si(C(CH3)3)3 (i.e., ((triisopropylsilyl)oxy)-methyloxy, TOM); R I is H in each occurrence.

[0129] In some embodiments of the compound of formula Ib-1, c is 1. In some embodiments of the compound of formula Ib-1, c is 0.

[0130] B N When a nucleobase such as "is, at each occurrence, independently selected from the group consisting of adenine, guanine, cytosine, thymine and uracil" is stated, said nucleobase is optionally protected. In particular, the exocyclic amino groups of adenine, guanine and cytosine may be protected. Examples of suitable protecting groups are listed in Table 1 (see below).

[0131] In some embodiments of the compound of formula Ib-1: n is an integer of 0 or 1; Y is independently O or S for each repeat unit, n; B N is, at each occurrence, independently selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil; R III is a 2-cyanoethyl group for each repeat unit n; R II is independently selected at each occurrence from the group consisting of H, -O-CH3 (i.e., methoxy), and -O-Si(CH3)2(C(CH3)3) (i.e., tert-butyl(dimethyl)silyloxy, TBDMS, or TBS); R I is H in each occurrence.

[0132] In some embodiments of the compound of formula Ib-1: n is an integer of 0 or 1; Y is independently O for each repeat unit, n; B N is, at each occurrence, independently selected from the group consisting of adenine, whose exocyclic amino group is protected with benzoyl, guanine, whose exocyclic amino group is protected with isobutyryl, cytosine, thymine and uracil, whose exocyclic amino group is protected with benzoyl or acetyl; R III is a 2-cyanoethyl group for each repeat unit n; R II is independently selected at each occurrence from the group consisting of H, -O-CH3 (i.e., methoxy), and -O-Si(CH3)2(C(CH3)3) (i.e., tert-butyl(dimethyl)silyloxy, TBDMS, or TBS); R I is H in each occurrence.

[0133] In some embodiments of the compound according to formula Ib-1, when c is 0, a is an integer from 1 to 2 (ie, a is 1 or 2).

[0134] In some embodiments, the compounds of formula I, particularly the compounds of formula Ib-1, have the following formulae VIII-1, IX-1, and X-1: [ka] [ka] (In the formula, In formula VIII-1: Integers a and c, and R 6 is defined for formula VIII; In formula IX-1: Integers a and c, and R 6 is defined for formula IX; In formula X-1: Integers a and c, and R 6 is defined in terms of formula X; In formulas VIII-1, IX-1 and X-1, the integer n and R 1 , Y, B N , R I , R II and R III is a compound according to any one of the formulas Ib-1).

[0135] Thus, a, c and R for Formula VIII 6 Any of the definitions and embodiments of any one of the following may also be applied to the integers a and c for formula VIII-1, and to R 6 a, c and R for formula IX 6 Any of the definitions and embodiments of any one of the following may also be applied to the integers a and c for formula IX-1, and to R 6 a, c and R for formula X 6 Any of the definitions and embodiments of any one of the following may also be applied to the integers a and c of formula X-1, and to R 6 In the same manner, n, R 1 , Y, B N , R I , R II and R III Any of the definitions and embodiments of any one of the formulas VIII-1, IX-1 and X-1 may also be applied to the integer n, and R 1 , Y, B N , R I , R II and R III also applies.

[0136] In some embodiments of the compound of formula VIII-1, IX-1 or X-1, c is 1. In some embodiments of the compound of formula VIII-1, IX-1 or X-1, c is 0. In some embodiments of the compound of formula VIII-1, IX-1 or X-1, when c is 0, a is an integer from 1 to 2 (i.e., a is 1 or 2).

[0137] In another aspect, the present application provides a method for synthesizing an oligonucleotide, comprising steps a) to f): a) obtaining a nucleoside, nucleotide, oligonucleotide, or a conjugate of a nucleoside, nucleotide, or oligonucleotide bound to a pseudo solid-phase protecting group according to any one of formulas II, II-1, II-2, II-a, II-a-1, II-a-2, and III to VII, the nucleoside, nucleotide, oligonucleotide, or conjugate thereof being protected by a protecting group R 1 wherein the backbone hydroxyl or amine moiety is protected by b) Protecting group R 1 from the compound obtained in the previous step, thereby generating a free backbone hydroxyl or amine group; c) reacting the free backbone hydroxyl or amine group generated in step b) with a phosphorus moiety of a nucleoside or oligonucleotide component, the component being protected by a protecting group R 1 whereby a covalent linkage is created between the oxygen or nitrogen atom of the free backbone hydroxyl or amine group and the phosphorus atom of the moiety; d) optionally modifying the phosphorus moiety; e) optionally repeating the sequence of steps b) to c) or b) to d); and f) cleaving the oligonucleotide from the pseudo solid phase protecting group.

[0138] The above definitions and explanations regarding the pseudo solid phase protecting groups and compounds of the present invention are equally applicable to the methods of the present invention and vice versa.

[0139] As will be understood from what has been recited above, said "backbone hydroxyl or amine moiety" of the compound obtained in step a) is preferably a terminal hydroxyl or amine moiety, such as, for example, the DMT-protected 5'-OH group of a ribose-based nucleoside moiety (i.e. 5'-ODMT).

[0140] In some embodiments of the method comprising steps a) through f) above: said "main chain hydroxyl or amine moiety" of the compound obtained in step a) is a main chain hydroxyl moiety, said "main chain hydroxyl or amine groups" in steps b) and c) are main chain hydroxyl groups, and / or Said "backbone hydroxyl or amine moiety" of the nucleoside or oligonucleotide component of step c) is a backbone hydroxyl moiety.

[0141] In some embodiments of the method comprising steps a) through f) above: said "main chain hydroxyl or amine moiety" of the compound obtained in step a) is a terminal main chain hydroxyl moiety, said "main chain hydroxyl or amine groups" in steps b) and c) are terminal main chain hydroxyl groups; and / or Said "backbone hydroxyl or amine moiety" of the nucleoside or oligonucleotide component of step c) is a terminal backbone hydroxyl moiety.

[0142] In some embodiments of the method comprising steps a) to f) above, step a) comprises: obtaining a nucleoside, nucleotide or oligonucleotide bound to a pseudo solid phase protecting group according to any one of formulas II, II-1, II-2, II-a, II-a-1, II-a-2 and III to VII, wherein the nucleoside, nucleotide or oligonucleotide is protected by a protecting group R 1In these embodiments, the term "oligonucleotide" does not include conjugates thereof.

[0143] It should be understood that the methods of the present invention may be performed in a variety of ways, and that the sequence of steps performed does not necessarily correspond to the sequence in which the steps are set forth in the claims, unless otherwise indicated. Thus, any numbering of steps, e.g., a) through f) or i) through iii), is introduced merely for ease of reference and is not intended to reflect a required sequence of the steps, unless otherwise indicated.

[0144] As presented above, R 1 teeth, For example, it may be a triarylmethyl type protecting group such as trityl, methoxytrityl (i.e., (p-methoxyphenyl)diphenylmethyl, MMT), and dimethoxyltrityl (i.e., di(p-methoxyphenyl)phenylmethyl, DMT). 1 may be the same or different for different iterations of steps b) and c) of the method. In some embodiments of the method of the present invention, the protecting group R 1 is selected from the group consisting of a trityl group, a MMT group, and a DMT group, and may be the same or different for each repetition of steps b) and c). In some embodiments of the method of the present invention, the protecting group R 1 is a DMT group. In some embodiments of the methods of the invention, R 1 is R 1 is always a trityl group when it is the protecting group for the amine moiety, and R 1 is R 1is always a DMT group when is a protecting group for the hydroxyl moiety. Preferably, step a) involves obtaining the compound in an aprotic solvent or a mixture of aprotic solvents. Such solvents are not particularly limited, but halogenated hydrocarbon solvents such as dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, etc., aromatic hydrocarbon solvents such as benzene, toluene, xylene (o-, m- or p-xylene), mesitylene, etc., aliphatic hydrocarbon solvents such as pentane, hexane, cyclohexane, heptane, octane, etc., ether solvents such as diethyl ether, tetrahydrofuran, cyclopentyl methyl ether, etc., and ester solvents such as ethyl acetate, isopropyl acetate, etc. may be used, for example.

[0145] Step a) of the method described herein comprises obtaining a nucleoside, nucleotide or oligonucleotide, or a conjugate thereof, bound to a pseudo solid-phase protecting group as disclosed herein, which may be understood in the broadest possible sense.

[0146] For example, when the nucleoside, nucleotide, oligonucleotide, or conjugate of nucleoside, nucleotide or oligonucleotide obtained in step a) is bound to a pseudo solid-phase protecting group according to formula II, c is 1 and step a) comprises the following steps i) to iii): i) Protecting group R 1 , and possibly R 1 obtaining a nucleoside, nucleotide, oligonucleotide or conjugate thereof comprising a first free backbone hydroxyl moiety, a second backbone hydroxyl or an amine moiety protected by an additional protecting group orthogonal to said first free backbone hydroxyl moiety; ii) reacting the first free hydroxyl moiety with an activated carbonic acid derivative; And

[0147] iii) reacting the product of step ii) with a compound of formula XIII [ka] (In Formula XIII: a is an integer from 1 to 12; R 3 H and OR 7 Selected from R 7 is a C1 to C5 alkyl group; R 5 H and OR 6 R 6 contains a C1 to C40 aliphatic hydrocarbon group; R 4 Each of the following is independently OR 6 and R 6 contains a C1 to C40 aliphatic hydrocarbon group; Whereas b is 1 to 3; However, if b is 1, then R 3 and R 5 is not H; Also, however, there exists R 3 , R 4 and R 5 with the further proviso that the sum of all carbon atoms contained in the moiety is greater than 23 and less than 200.

[0148] Thus, a further aspect of the present invention is a process for preparing a compound according to formula I, wherein c is 1, comprising the following steps i) to iii): i) Protecting group R 1 , and possibly R 1 obtaining a nucleoside or oligonucleotide comprising a first free backbone hydroxyl moiety, a second backbone hydroxyl or an amine moiety protected by an additional protecting group orthogonal to said first free backbone hydroxyl moiety; ii) reacting the first free hydroxyl moiety with an activated carbonic acid derivative; and

[0149] iii) reacting the product of step ii) with a compound of formula XIII [ka] (In the formula: a is an integer from 1 to 12, preferably from 1 to 3; R 3 is H; R 5 OR 6 or H and R 6 is a C8 to C40 aliphatic hydrocarbon group; R 4 Each of the following is independently OR 6 and R 6 is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; Whereas b is 1 to 3; However, if b is 1, then R 3 and R 5 is not H; Also, however, there exists R 3 , R 4 and R 5 with the further proviso that the sum of all carbon atoms contained in the moiety is greater than 23 and less than 200.

[0150] As used herein, two protecting groups are considered "orthogonal" if they can be removed independently of one another, i.e., using different sets of reaction conditions. Nucleosides and oligonucleotides having acid-labile backbone protecting groups, such as DMT, and acid-resistant protecting groups for the phosphate and nucleobase moieties are readily available to those of skill in the art.

[0151] The activated carbonic acid derivative used in step ii) can be phosgene, or an activated diester of carbonic acid, such as, for example, disuccinimidyl carbonate, or an activated diamide of carbonic acid, such as, for example, 1,1'-carbonyldiimidazole. In the present context, an activated carbonic acid derivative is a compound in which the carbonyl carbon atom is bound to two leaving groups (e.g., two -Cl residues in phosgene, or two N-hydroxysuccinimide residues in disuccinimidyl carbonate, or two imidazole residues in 1,1'-carbonyldiimidazole), both of which can be displaced by nucleophilic attack of a free hydroxyl group. The skilled person will understand that the first such leaving group can be displaced by said first free hydroxyl moiety in step ii) and that the second such leaving group can be displaced by the hydroxyl group of the compound of formula XIII in step iii) to thereby realize the desired carbonic acid diester.

[0152] Preferably, in step i), the second backbone moiety is a hydroxyl moiety.Preferably, the reaction is carried out in an aprotic solvent.

[0153] The integers a and b for the pseudo solid-phase protecting group of formula II and the residue R 3 , R 4 , R 5 , R 6 and R 7 It will be understood by those skilled in the art that any of the definitions and embodiments for the protecting group R for compounds of formula I may also be applied to compounds of formula XIII. 1 Any of the definitions and embodiments relating to the protecting group R 1 This can also apply.

[0154] In some embodiments of the method comprising steps i) to iii) above, the method is for making a compound according to formula Ib, c is 1, and the compound of formula XIII is of formula XIII-b: [ka] (In formula XIII-b, the integer a and the residue R5 , R 8 , R 9 , R 10 and R 6 is a compound of formula Ib, which is represented by the integer a and the residue R 5 , R 8 , R 9 , R 10 and R 6 It is meant that any of the definitions and embodiments relating to may also apply to compounds of formula XIII-b.

[0155] In some embodiments of the method comprising steps i) to iii) above, the method is for making a compound according to any one of formulas VIII to XII, preferably VIII to X, wherein c is 1: when the compound of formula XIII is a compound of formula XIII-c and the compound of formula VIII is prepared: [ka] In formula XIII-c, the integer a and the residue R 6 is defined for formula VIII; when the compound of formula XIII is a compound of formula XIII-d and the compound of formula IX is prepared: [ka] In formula XIII-d, the integer a and the residue R 6 is defined for formula IX; - the compound of formula XIII is a compound of formula XIII-e, when a compound of formula X is prepared: [ka] In formula XIII-e, the integer a and the residue R 6 is defined in terms of formula X; when the compound of formula XIII is a compound of formula XIII-f and a compound of formula XI is prepared: [ka] In formula XIII-f, the integer a and the residue R 6 is defined with respect to formula XI; the compound of formula XIII is a compound of formula XIII-g, when the compound of formula XII is prepared: [ka] In formula XIII-g, the integer a and the residue R 6 is defined with respect to formula XII.

[0156] In some embodiments of the method comprising steps i) through iii) above, step i) comprises: 1 , and possibly R 1 The step of obtaining a nucleoside or oligonucleotide comprising a first free backbone hydroxyl moiety, a second backbone hydroxyl or an amine moiety protected by an additional protecting group orthogonal to the first free backbone hydroxyl moiety, a second free backbone hydroxyl or an amine moiety. In such embodiments, the term "oligonucleotide" preferably does not encompass conjugates thereof. In some embodiments of the method comprising steps i) to iii) above, step i) is: 1 , and possibly R 1 The process provides a nucleoside that includes a first free backbone hydroxyl moiety, a second backbone hydroxyl or an amine moiety protected by an additional protecting group orthogonal to the first free backbone hydroxyl moiety.

[0157] In some embodiments of the method including the above steps i) to iii), at least steps ii) and iii) are carried out in an aprotic non-nucleophilic solvent. Examples of suitable solvents for this purpose may be halogenated hydrocarbon solvents such as dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, etc., aromatic hydrocarbon solvents such as benzene, toluene, xylene (o-, m- or p-xylene), mesitylene, etc., aliphatic hydrocarbon solvents such as pentane, hexane, cyclohexane, heptane, octane, etc.

[0158] For example, when the nucleoside, nucleotide, oligonucleotide, or conjugate of a nucleoside, nucleotide or oligonucleotide obtained in step a) is bound to a pseudo solid-phase protecting group according to formula II, c is 0 and step a) can be carried out by the following steps I) and II): I) Protecting group R 1 , and possibly R 1 obtaining a nucleoside, nucleotide, oligonucleotide, or conjugate thereof, comprising a first free backbone hydroxyl moiety, a second backbone hydroxyl or amine moiety, the first free backbone hydroxyl or amine moiety being protected by an additional protecting group orthogonal to II) an oxygen atom of the first free main chain hydroxyl moiety of the compound obtained in step I) and R L is bonded to the carboxyl or carbonyl carbon atom of formula XIV: [ka] (In Formula XIV: R L is selected from the group consisting of -OH and a leaving group capable of being replaced by a hydroxyl moiety during ester bond formation; a is an integer from 1 to 12; R 3 H and OR 7 Selected from R 7 is a C1 to C5 alkyl group; R 5 H, and OR 6 R 6 contains a C1 to C40 aliphatic hydrocarbon group; R 4 Each of the following is independently OR 6 and R 6 contains a C1 to C40 aliphatic hydrocarbon group; Whereas b is 1 to 3; However, if b is 1, then R 3 and R 5 is not H; Also, however, there exists R 3 , R 4 and R 5 With the further proviso that the sum of all carbon atoms contained in the moiety is greater than 23 and less than 200.

[0159] The integers a and b for the pseudo solid-phase protecting group of formula II and the residue R 3 , R 4 , R 5 , R 6 and R 7 It will be understood by those skilled in the art that any of the definitions and embodiments for the protecting group R for compounds of formula I may also be applied to compounds of formula XIV. 1 Any of the definitions and embodiments relating to the protecting group R 1 This can also apply.

[0160] Thus, a further aspect of the present invention is a method for preparing a compound according to formula I, wherein c is 0, comprising the following steps I) and II): I) Protecting group R 1 , and possibly R 1 obtaining a nucleoside or oligonucleotide comprising a first free backbone hydroxyl moiety, a second backbone hydroxyl or an amine moiety, each of which is protected by an additional protecting group orthogonal to II) an oxygen atom of the first free main chain hydroxyl moiety of the compound obtained in step I) and R L is bonded to the carboxyl or carbonyl carbon atom of formula XIV: [ka] (In Formula XIV: R L is selected from the group consisting of -OH and a leaving group capable of being replaced by a hydroxyl moiety during ester bond formation; a is an integer from 1 to 12, preferably from 1 to 3; R 3 is H; R 5 OR 6 or H and R 6 is a C8 to C40 aliphatic hydrocarbon group; R 4 Each of the following is independently OR 6 and R 6 is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; Whereas b is 1 to 3; However, if b is 1, then R 3 and R 5 is not H; Also, however, there exists R 3 , R 4 and R 5 with the further proviso that the sum of all carbon atoms contained in the moiety is greater than 23 and less than 200.

[0161] Those skilled in the art will recognize suitable leaving groups R that are displaced by the hydroxyl moiety during ester bond formation. L and know which (reaction) conditions are suitable for this purpose. L Non-limiting examples of R include halogen atoms such as Cl, Br and F, with Cl being preferred. L When is a halogen atom, preferably Cl, the reaction to form the ester bond in step II) can be carried out in the presence of a non-nucleophilic base such as pyridine, collidine, trimethylamine (TEA) and diisopropylethylamine (DIPEA). R L is -OH (i.e., a hydroxyl group), the carboxyl group of the compound of formula XIV may be activated towards ester bond formation. In such a case, the reaction to form the ester bond in step II) may be carried out in the presence of one or more activating agents, also referred to as coupling agents, and optionally one or more additives. The coupling agent may be, for example, a carbodiimide coupling agent known to those skilled in the art. Examples of suitable carbodiimide coupling agents include N,N'-diisopropylcarbodiimide (DIC), N,N'-dicyclohexylcarbodiimide (DCC) and N-(3-dimethylaminopropyl)-N'-ethylcarbodiimide (EDC), or a mixture thereof. As known to those skilled in the art, such carbodiimide coupling agents can be used with coupling additives such as, for example, 4-(dimethylamino)pyridine (DMAP), 2-cyano-2-(hydroxyimino)acetate (Oxima), 1-hydroxybenzotriazole (HOBt), 1-hydroxy-7-azabenzotriazole (HOAt), 5-(hydroxyimino)-1,3-dimethylpyrimidine-2,4,6(1H,3H,5H)-trione (Oxima-B), ethyl 1-hydroxy-1,2,3-triazole-4-carboxylate (HOCt), N-hydroxysuccinimide (HOSu) and mixtures thereof. Mixtures of DCC and DMAP or DIC and DMAP, for example, are commonly used to achieve the formation of an ester bond between a carboxylic acid and an alcohol. As an alternative to carbodiimide coupling agents, so-called aminium / uronium salts are commonly used as coupling reagents. In this case, the addition of non-nucleophilic bases such as pyridine, collidine, TEA and / or DIPEA is common. Further additives such as DMAP can also be added as needed. Examples of aminium / uronium salts include (benzotriazolyl)tetramethyluronium tetrafluoroborate (TBTU), N-[(7-aza-1H-benzotriazol-1-yl)(dimethylamino)-methylene]-N-methylmethanaminium tetrafluoroborate N-oxide (TATU), 2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyluronium hexafluorophosphate (HBTU), N-[(7-aza-1H-benzotriazol-1-yl)(dimethylamino)-methylene]-N-methylmethanaminium hexafluorophosphate N-oxide (HATU), and 1-cyano-2-ethoxy-2-oxoethylideneaminooxy)dimethylamino-morpholino-carbenium-hexafluorophosphate (COMU). For example, a mixture of COMU, DIPEA and DMAP can be used. Instead of aminium / uranium salts, phosphonium salts such as benzotriazol-1-yl-oxy-tris-pyrrolidino-phosphonium-hexafluorophosphate (PyBOP) can be used.As an alternative activator, MSNT (1-(mesitylene-2-sulfonyl)-3-nitro-1,2,4-triazole) can be used, for example with N-methylimidazole as an additive.

[0162] In some embodiments of the method comprising steps I) and II) above, the method is for preparing a compound according to formula Ib, wherein c is 0, and the compound of formula XIV is represented by formula XIV-b: [ka] (In formula XIV-b, Integer a and residue R 5 , R 8 , R 9 , R 10and R 6 is defined for formula Ib; R L is a compound of formula XIV). Thus, the integer a and the residue R for formula Ib 5 , R 8 , R 9 , R 10 and R 6 Any of the definitions and embodiments relating to also apply to compounds of formula XIV-b.

[0163] In some embodiments of the method comprising steps I) and II) above, the method is a method for making a compound according to any one of formulas VIII to XII, preferably VIII to X, wherein c is 0: if a compound of formula VIII is to be prepared, the compound of formula XIV may be prepared according to formula XIV-c: [ka] (In formula XIV-c, the integer a and the residue R 6 is defined for formula VIII; R L is defined with respect to formula XIV); if a compound of formula IX is to be prepared, a compound of formula XIV may be prepared according to formula XIV-d: [ka] (In formula XIV-d, the integer a and the residue R 6 is defined for formula IX, and R L is defined with respect to formula XIV); if a compound of formula X is to be prepared, the compound of formula XIV may be prepared according to formula XIV-e: [ka] (In formula XIV-e, the integer a and the residue R 6 is defined in terms of the formula X, and R Lis defined with respect to formula XIV); if a compound of formula XI is to be prepared, the compound of formula XIV may be prepared according to formula XIV-f: [ka] (In formula XIV-f, the integer a and the residue R 6 is defined with respect to formula XI, and R L is defined with respect to formula XIV); If a compound of formula XII is to be prepared, the compound of formula XIV may be prepared according to formula XIV-g: [ka] (In formula XIV-g, the integer a and the residue R 6 is defined for formula XII, and R L is a compound of formula XIV).

[0164] In some embodiments of the method comprising steps I) and II) above, step I) comprises: reacting a protecting group R 1 , and possibly R 1 The step of obtaining a nucleoside or oligonucleotide comprising a first free backbone hydroxyl moiety, a second backbone hydroxyl or an amine moiety protected by an additional protecting group orthogonal to the first free backbone hydroxyl moiety, a second free backbone hydroxyl or an amine moiety. In such embodiments, the term "oligonucleotide" preferably does not encompass conjugates thereof. In some embodiments of the method comprising steps I) and II) above, step I) is: 1 , and possibly R 1 The process provides a nucleoside that includes a first free backbone hydroxyl moiety, a second backbone hydroxyl or an amine moiety protected by an additional protecting group orthogonal to the first free backbone hydroxyl moiety.

[0165] In some embodiments of the method comprising steps I) and II) above, R in any one of formulas XIV, XIV-b, XIV-c, XIV-d, XIV-e, XIV-f, and XIV-g is Lis -OH (i.e., a hydroxyl group).

[0166] In some embodiments of the method including the above steps I) and II), at least step II) is carried out in an aprotic non-nucleophilic solvent. Examples of suitable solvents for this purpose may be halogenated hydrocarbon solvents such as dichloromethane, 1,2-dichloroethane, chloroform, carbon tetrachloride, etc., aromatic hydrocarbon solvents such as benzene, toluene, xylene (o-, m- or p-xylene), mesitylene, etc., aliphatic hydrocarbon solvents such as pentane, hexane, cyclohexane, heptane, octane, etc.

[0167] Step b) of the method including the above steps a) to f) is to subject the compound obtained in step a) to acidic conditions to remove the protecting group R 1 Preferably, this is achieved by adding a proton donor to a solution of the compound obtained in step a). The skilled artisan will recognize that the proton donor can be added to a solution of the compound obtained in step a) by removing the protecting group R 1 The proton donor is selected depending on the nature of the acid. Examples of suitable proton donors include trifluoroacetic acid, cyanopyridinium trifluoroacetate and trifluoroethanol, triethylammonium trifluoroacetate, cyanoacetic acid, acetic acid, dichloroacetic acid, trichloroacetic acid, phosphoric acid, mesylic acid, tosylic acid and hydrochloric acid. For example, 2-10% (v / v) DCA in toluene or DCM can be used. Optionally, the cleavage is carried out in the presence of a carbocation scavenger.

[0168] The expression "carbocation scavenger" as used herein refers to a nucleophilic compound that can be used to bind to the formed carbocation or to formally donate a hydride ion to said carbocation, thereby preventing undesired side reactions of the carbocation. Examples of carbocation scavengers are alcohols and phenols [e.g., methanol, ethanol, phenol, cresol, water], phenol ethers [e.g., anisole, 1,3-dimethoxybenzene (dimethylanisole), 1,3,5-trimethoxybenzene]; thioethers [e.g., dimethylsulfide, ethylmethylsulfide, thioanisole-, silanes-, e.g., triisopropylsilane (TIS), triethylsilane (TES)], N-heterocycles [e.g., pyrrole, 3-methylpyrrole, 2,4-dimethylpyrrole indole, 2-methyl ... indole, succinimide, phthalimide], thiols and thiophenols [e.g., 1,2-ethanedithiol (EDT), 1,4-dithioerythritol (DTE), 1,4-dithiothreitol (DTT), 3,6-dioxa-1,8-octanedithiol (DODT), 1,4-benzenedimethanethiol (BDMT), 1,4-butanedithiol, 2-mercaptoethanol, cysteine, thiophenol, p-thiocresol], and polyalkylbenzenes [e.g., 1,3,5-trimethylbenzene, pentamethylbenzene].

[0169] Step b) further comprises, after completion of the cleavage reaction, adding a base to the solution, thus neutralizing the composition, before adding the subsequent protected component in step c). Preferred bases are N,N-diisopropylethylamine, pyridine, 4-cyanopyridine, trimethylamine, sodium carbonate and potassium carbonate. Those skilled in the art will carefully limit the exposure to base in this step to avoid premature cleavage from the pseudo solid-phase protecting group (PSPPG). Step b may involve separation of the PSPPG-protected compound from the deprotection reagent and from any by-products of the deprotection reaction. After the coupling cycle, i.e. in a repeat after step c), d) and b) or c) and b) could also be carried out successively, which could remove residual reagents, scavengers and by-products. In both cases, step b) may involve the addition of a polar solvent to precipitate the PSPPG-protected compound. At the same time, the solvent of the process can be removed, for example by evaporation. These procedures can be used to induce precipitation of the PSPPG protected compound. Polar solvents used for precipitation include, but are not limited to, water, acetonitrile, propionitrile, methanol, ethanol, 1-propanol, 2-propanol, preferably acetonitrile and methanol, and even more preferably acetonitrile.

[0170] Mixtures of these solvents may also be used. Precipitation may be followed by solid-liquid separation, such as filtration or centrifugation, and dissolution in a suitable process solvent or mixture of solvents. Thus, in some embodiments, step b) comprises removing the protecting group R 1 from the compound obtained in the previous step, thereby generating a free backbone hydroxyl or amine moiety; precipitating the compound thus obtained by adding a polar solvent; separating the precipitate from the surrounding liquid; and dissolving the precipitate by addition of a solvent.

[0171] Method steps c), i.e., coupling of the moiety to the growing oligonucleotide strand, and d), e.g., oxidation or sulfurization of the phosphorus(III) moiety, depend on the coupling chemistry selected and are routinely performed by those skilled in the art. For example, with pre-activated moieties used in PMO synthesis, step c) may involve simply incubating the pre-activated moiety with the product of step b) in an inactivating solvent (i.e., a solvent that does not interfere in the desired coupling reaction). As a further example, in the case of H-phosphonate oligonucleotide synthesis, coupling between the moiety and the free backbone moiety may be achieved in the presence of an activating agent such as pivaloyl chloride. As a further example, in the case of phosphoramidite oligonucleotide synthesis, a weak acid such as tetrazole is required to activate the reaction. Thus, step c) may optionally include the addition of an activating agent. Step c) may further include the removal of unreacted moieties by separation or by the addition of a quenching moiety that reacts with any remaining moieties.

[0172] The term "nucleoside or oligonucleotide component" as used herein refers to a mono- or oligomeric component that allows the assembly of an oligonucleotide strand, unless otherwise indicated. Those skilled in the art will readily understand that essentially any base-labile coupling chemistry can be compatible with the use of pseudo-solid-phase protecting groups that can be used in the methods of the present invention. As a result, the chemical nature of the phosphorus moiety can vary. The phosphorus moiety can be attached to the nucleoside or oligonucleotide via a backbone hydroxyl group. In some embodiments, the component can include an H-phosphonate moiety that is attached to the backbone hydroxyl group of the nucleoside or nucleotide component.

[0173] In other embodiments, the component can be a phosphorus V reagent, for example, as taught in WO2019200273. In other embodiments, the component can be a methylmorpholine-based nucleoside or oligonucleotide according to formula B, in which a backbone hydroxyl group is attached to an activated phosphorus moiety.

[0174] [ka] In formula B, m is an integer equal to or greater than 0; R 1 is a protecting group as previously described, preferably a trityl protecting group; L 1 is a leaving group, such as a halogen atom (preferably a chlorine atom, Cl), a methanesulfonyloxy group or a p-toluenesulfonyloxy group; X B is a di-(C1-C6-alkyl)amino group, preferably a dimethylamino group, or a 1-piperazinyl group, and the 4-position nitrogen atom is protected by a protecting group and optionally further substituted. Each of the bases 0 to m may be the same or different and may contain the same or different protecting groups. Preferably, each base (i.e., base 0 and any base m ) is a nucleobase selected from the group consisting of adenine, guanine, cytosine, thymine and uracil, where the exocyclic amino groups in adenine, guanine and cytosine are protected. Compounds according to formula B can be prepared by well known methods, for example as described in WO9109033.

[0175] In yet another embodiment of the method comprising steps a) through f) above, the component of step c) is a compound of formula C: [ka] (In the formula: R 1 is a protecting group as previously described; m is an integer equal to or greater than 0; Y is independently selected for each repeat unit m from the group consisting of S and O; Z C is independently selected for each position (i.e., at the terminal positions and for each repeat unit m) from the group consisting of O and S; R 2Care the same or different protecting groups for each position (i.e., the terminal positions and for each repeat unit m); R 3C and R 4C are protecting groups which may be the same or different; Each of the nucleosides 0 through m are phosphoramidite building blocks of the general structure shown in (which may be the same or different). Compounds according to formula C can be prepared according to well-known methods and are commercially available.

[0176] In some embodiments, R 3C and R 4C and both are C1-C5-alkyl groups, for example both are isopropyl groups. 2C In each occurrence, Z is a 2-cyanoethyl group. C is O in each occurrence. In some preferred embodiments, R 3C and R 4C are each independently a C1-C5 alkyl group, preferably R 3C and R 4C are both isopropyl groups, and Z C is O in each occurrence and R 2C is a 2-cyanoethyl group in each occurrence.

[0177] In some embodiments, different types of components using different coupling chemistries can be used in different coupling cycles, for example, phosphorothioate linked nucleosides can be introduced using P(V) chemistry and phosphodiester linked nucleosides can be introduced using P(III) chemistry.

[0178] In some embodiments, m is an integer from 0 to 2 (i.e., m is 0, 1, or 2), i.e., the moiety is a mononucleoside, dinucleotide, or trinucleotide moiety. Specific non-limiting examples of mononucleoside phosphoramidite moieties are shown in Figures 2 and 3. It should be noted that the nucleosides 0 to m and x0 to xn in Formulas I, Ib, Ia, Ic, VIII to XII, and C can differ from one another, for example, with respect to the identity and substitution pattern of the ribose or ribose analog moiety (also known as ribose surrogate), with respect to the nucleobase, and with respect to the presence and identity of any protecting groups.

[0179] Suitable protecting groups R 2C A typical example for is the 2-cyanoethyl group; R 3C and R 4C A typical example for both is the isopropyl group. Alternatively, R 2C and R 3C may form an ethylene bridge, and the phosphoramidite may take the form of a chiral group of formula D, ** represents the point of attachment to the backbone hydroxyl group. [ka]

[0180] The components used in the methods of the invention may exhibit further protecting groups, for example to block exocyclic amino groups of the nucleobases from side reactions.

[0181] In some embodiments, the nucleoside or oligonucleotide phosphoramidite moiety of formula C has the following formula C-1: [ka] (In formula C-1: m is an integer equal to or greater than 0, preferably 0, 1 or 2; R 1 is a triarylmethyl type protecting group, preferably a DMT group; B N is a nucleobase which may be identical or different at each occurrence, preferably a nucleobase which, at each occurrence, is independently selected from the group consisting of adenine, guanine, cytosine, thymine and uracil; Y is independently selected for each repeat unit m from the group consisting of S and O; Z C is independently selected for each position (i.e., for the terminal positions and for each repeat unit m) from the group consisting of O and S; R 2C are protecting groups which may be the same or different for each position (i.e., the terminal positions and for each repeat unit m); R 3C and R 4C are protecting groups which may be the same or different; R IV is independently selected at each occurrence from the group consisting of H, F, -O-(C1-C5 alkyl), -O-(C1-C5 alkyl)-O-(C1-C5 alkyl), -O-Si(C1-C5 alkyl)3 and -O-CH2-O-Si(C1-C5 alkyl)3; R V is independently H in each occurrence, or R V and R IV together form a methylene or ethylene bridge of the chemical structure +-CH2-O-++, +-CH(CH3)-O-++ or +-CH2-CH2-O-++, where + is the point of attachment to the 4'-carbon atom (i.e., R V is attached to the carbon atom, and ++ is the point of attachment to the 2'-carbon atom (i.e., R IV is a component of ).

[0182] In some embodiments, in a nucleoside or oligonucleotide phosphoramidite moiety of formula C-1: m is an integer equal to or greater than 0, preferably 0, 1 or 2; R 1is a triarylmethyl type protecting group, preferably a DMT group; B N is a nucleobase which may be identical or different at each occurrence, preferably a nucleobase which, at each occurrence, is independently selected from the group consisting of adenine, guanine, cytosine, thymine and uracil; Y is independently selected for each repeat unit m from the group consisting of S and O; Z C is O in each occurrence; R 2C is a 2-cyanoethyl group in each occurrence; R 3C and R 4C are each independently a C1-C5 alkyl group, preferably R 3C and R 4C are both isopropyl groups; R IV is independently selected at each occurrence from the group consisting of H, F, -O-CH3 (i.e., methoxy), -O-CH2-CH2-O-CH3 (i.e., 2-methoxyethyl-1-oxy), -O-Si(CH3)3 (i.e., trimethylsilyloxy, TMS), -O-Si(CH3)2(C(CH3)3) (i.e., tert-butyl(dimethyl)silyloxy, TBDMS or TBS), and -O-CH2-O-Si(C(CH3)3)3 (i.e., ((triisopropylsilyl)oxy)-methyloxy, TOM); R V is H in each occurrence.

[0183] In some embodiments, in a nucleoside or oligonucleotide phosphoramidite moiety of formula C-1: m is 0; R 1 is a DMT group; B Nis a nucleobase which may be identical or different at each occurrence, preferably a nucleobase which, at each occurrence, is independently selected from the group consisting of adenine, guanine, cytosine, thymine and uracil; Y is independently selected for each repeat unit m from the group consisting of S and O; Z C is O in each occurrence; R 2C is a 2-cyanoethyl group in each occurrence; R 3C and R 4C are both isopropyl groups; R IV is independently selected at each occurrence from the group consisting of H, -O-CH3 (i.e., methoxy), and -O-Si(CH3)2(C(CH3)3) (i.e., tert-butyl(dimethyl)silyloxy, TBDMS, or TBS); R V is H in each occurrence.

[0184] In some embodiments, in a nucleoside or oligonucleotide phosphoramidite moiety of formula C-1: m is 0; R 1 is a DMT group; B N is, at each occurrence, independently selected from the group consisting of adenine, guanine, cytosine, thymine, and uracil; Y is independently selected for each repeat unit m from the group consisting of S and O; Z C is O in each occurrence; R 2C is a 2-cyanoethyl group in each occurrence; R 3C and R 4C are both isopropyl groups; R IVis independently selected at each occurrence from the group consisting of H, -O-CH3 (i.e., methoxy), and -O-Si(CH3)2(C(CH3)3) (i.e., tert-butyl(dimethyl)silyloxy, TBDMS, or TBS); R V is H in each occurrence.

[0185] As listed above, it is understood that the nucleobases adenine, guanine, cytosine, thymine and uracil can optionally be protected (i.e., bear one or more protecting groups). In particular, the exocyclic amino groups in adenine, guanine and cytosine can be protected. Examples of suitable protecting groups are shown in Table 1 (see below).

[0186] In some embodiments, in a nucleoside or oligonucleotide phosphoramidite moiety of formula C-1: m is 0; R 1 is a DMT group; B N is independently selected at each occurrence from the group consisting of adenine, whose exocyclic amino group is protected with benzoyl, guanine, whose exocyclic amino group is protected with isobutyryl, cytosine, thymine and uracil, whose exocyclic amino group is protected with benzoyl or acetyl; Y is independently selected for each repeat unit m from the group consisting of S and O; Z C is O in each occurrence; R 2C is a 2-cyanoethyl group in each occurrence; R 3C and R 4C are both isopropyl groups; R IV is independently selected at each occurrence from the group consisting of H, -O-CH3 (i.e., methoxy), and -O-Si(CH3)2(C(CH3)3) (i.e., tert-butyl(dimethyl)silyloxy, TBDMS, or TBS); RV is H in each occurrence.

[0187] Suitable nucleoside derivatives are commercially available. Table 1, for example, provides a non-limiting overview of protecting groups commonly used for nucleoside building blocks.

[0188] [Table 1]

[0189] In some embodiments of the process of the present invention, the compound obtained in step a) is provided with a protecting group R 1 and the building block used in step c) comprises a backbone hydroxyl moiety protected by a protecting group R 1 Further, the building block used in step c) is a phosphoramidite building block.

[0190] Step d) of the method may involve the oxidation of a P(III) moiety to a P(V) moiety. As used herein, oxidation is a chemical reaction that increases the oxidation number of the moiety subjected to said reaction. Oxidation may therefore include the introduction of a =O moiety or of a =S moiety. Oxidation of an H-phosphonate moiety or of a phosphite triester moiety may thus lead to the formation of any internucleoside linkage according to formula A-1 or A-2 above, for example the formation of a phosphodiester, phosphorothioate, phosphoramidate, phosphotriester (also known as phosphate triester), or thiophosphate triester bond. Corresponding methods and reagents are well known and readily available to the skilled artisan. For example, a phosphotriester bond may be obtained by reacting the phosphite triester moiety with aqueous iodine in the presence of a weak base such as pyridine, collidine or lutidine. Sulfurization can be accomplished using reagents such as 3-(dimethylaminomethylidene)amino-3H-1,2,4-dithiazole-3-thione (DDTT), 3H-1,2-benzodithiol-3-one 1,1-dioxide (Beaucage reagent) or N,N,N'N'-tetraethylthiuram disulfide (TETD).

[0191] In some embodiments of the method of the present invention comprising the above steps a) to f), the phosphorus moiety of the nucleoside or oligonucleotide component used in step c) is a phosphorus (III) moiety; the method comprises step d of oxidizing or sulfurizing the phosphorus (III) atom to a phosphorus (V) atom, thereby creating a desired type of internucleoside linkage; and step e) optionally including repeating the sequence of steps b) to d) or repeating the sequence of steps b) and c) before carrying out step d). In this specification, the terms "internucleoside linkage" and "internucleoside linkage" may be used interchangeably.

[0192] In some embodiments of the method of the invention comprising steps a) through f) above, step c) involves reacting the free backbone hydroxyl group resulting from step b) with a phosphoramidite nucleoside or oligonucleotide component to form a phosphite triester bond, and step d) involves oxidizing or sulfurizing the phosphite triester bond. It is understood that in both cases (i.e., oxidation or sulfurization), the P(III) atom of said phosphite triester bond is converted to a P(V) atom, and that sulfurization involves the introduction of a sulfur atom into each internucleoside linking group, while oxidation involves the introduction of an oxygen atom into each internucleoside linking group.

[0193] In order to avoid the formation of deletion sequences that are difficult to remove, the method of the present invention comprises an additional step of blocking (also called "capping") unreacted free hydroxyl or amine groups after step c) or step d).Hydroxyl or amine groups are typically blocked (i.e., capped) by acetylation.Methods for acetylating hydroxyl or amine groups during oligonucleotide synthesis are known to those skilled in the art.For example, acetic anhydride can be used, optionally in the presence of a base such as TEA, DIPEA, pyridine, lutidine or collidine. It should be noted that the order of steps indicated in the claims and description of the method is not necessarily binding unless otherwise indicated. In some embodiments of the method including the above steps a) to f), these steps are performed in the order of a) to f). This means that in these embodiments, steps a), b), c), d), e) and f) are performed in this order. However, this does not exclude performing additional steps between any of steps a) to f). Furthermore, the method may include additional steps. For example, the assembled oligonucleotide moiety may be conjugated to another moiety before performing the cleavage step f) of the method. Furthermore, the skilled person may use intervening separation steps, in particular precipitation and washing steps, for example when the composition of the solvent changes between process steps or to remove trace amounts of reagents from previous method steps. Furthermore, the terminal (e.g., 5'-terminal) R introduced via the nucleoside or oligonucleotide component used in the final repetition of step c) may be used to remove the terminal (e.g., 5'-terminal) R 1 - To remove the protecting group, a single repetition of step b) can be carried out after step e) or after step f).

[0194] For example, the pseudo-solid-phase protecting groups and the growing oligonucleotide strands attached thereto can be precipitated after each repetition of step d) by increasing the polarity of the solvent. This can be accomplished by adding a polar solvent or a mixture of polar solvents. Examples of polar solvents include water, nitrile solvents such as acetonitrile and propionitrile, and alcohols such as methanol, ethanol, 1-propanol, and 2-propanol.

[0195] Step f) of cleaving the oligonucleotide from the pseudo-solid-phase protecting group may involve incubation with a base such as ammonia or a source of hydroxide ions. In some cases, sodium hydroxide in an aqueous alcoholic solvent or in a cyclic ether (e.g., tetrahydrofuran, 2-methyl-tetrahydrofuran) may be used. In some embodiments of the method comprising steps a) to f) above, step f) comprises incubation with a base, said base being preferably selected from the group consisting of ammonia, C1-C6-alkylamines (i.e., primary alkylamines containing 1 to 6 carbon atoms, e.g., methylamine or tert-butylamine), and a source of hydroxide ions. In this context, the term "incubation" means incubating the conjugate of the pseudo-solid-phase protecting group and the oligonucleotide with said base, preferably in a solution containing said base. Thus, in some embodiments of the method comprising steps a) to f) above, step f) comprises incubating the conjugate of the pseudo-solid-phase protecting group and the oligonucleotide in a solution comprising a base, the base being preferably selected from the group consisting of ammonia, C1-C6-alkylamine (particularly methylamine or tert-butylamine), and a source of hydroxide ions. In some embodiments of the method comprising steps a) to f) above, step f) comprises incubating the conjugate of the pseudo-solid-phase protecting group and the oligonucleotide in a solution, preferably an aqueous solution, comprising a source of hydroxide ions. In some embodiments of the method comprising steps a) to f) above, step f) comprises incubating the conjugate of the pseudo-solid-phase protecting group and the oligonucleotide in a solution, preferably an aqueous solution, comprising ammonia. As used herein, the term "source of hydroxide ions" refers to a salt comprising hydroxide ions, with sodium hydroxide, potassium hydroxide and ammonium hydroxide being preferred examples, and sodium hydroxide being particularly preferred. Those skilled in the art will appreciate that an aqueous solution of ammonia is also referred to as an ammonium hydroxide solution because it contains ammonium ions and hydroxide ions.Those skilled in the art will understand that a solution containing a source of hydroxide anions contains said source of hydroxide ions in a solvated form. The term "aqueous solution" refers to a solution containing water and optionally one or more water-miscible organic solvents such as acetonitrile, tetrahydrofuran, ethanol, and the like.

[0196] In some embodiments of the method comprising the above steps a) to f), wherein the pseudo-solid-phase protecting group attached to said nucleoside, nucleotide, oligonucleotide, or conjugate of nucleoside, nucleotide or oligonucleotide obtained in step a) is a pseudo-solid-phase protecting group according to any one of formulas II, II-1, II-2, II-a, II-a-1, II-a-2 and III to VII, and c is 1, step f) comprises incubating the conjugate of pseudo-solid-phase protecting group and oligonucleotide in a solution, preferably an aqueous solution comprising a source of hydroxide ions and / or a C1-C6-alkylamine (in particular methylamine or tert-butylamine), preferably a source of hydroxide ions. In some embodiments of the method comprising steps a) to f) above, wherein the pseudo-solid-phase protecting group attached to said nucleoside, nucleotide, oligonucleotide, or conjugate of nucleoside, nucleotide or oligonucleotide obtained in step a) is a pseudo-solid-phase protecting group according to any one of formulas II, II-1, II-2, II-a, II-a-1, II-a-2, and III to VII, and c is 1, step f) comprises incubating the conjugate of pseudo-solid-phase protecting group and oligonucleotide in an aqueous solution of sodium hydroxide, potassium hydroxide, or ammonium hydroxide, preferably sodium hydroxide. In some embodiments of the method comprising steps a) to f) above, where the pseudo-solid-phase protecting group attached to the nucleoside, nucleotide, oligonucleotide, or conjugate of nucleoside, nucleotide, or oligonucleotide obtained in step a) is a pseudo-solid-phase protecting group according to any one of formulas II, II-1, II-2, II-a, II-a-1, II-a-2, and III to VII, and c is 1, step f) comprises incubating the conjugate of the pseudo-solid-phase protecting group and the oligonucleotide in an aqueous solution of C1-C6-alkylamine, preferably tert-butylamine. For example, a 1:1 (v / v) mixture of water and tert-butylamine may be used.

[0197] In some embodiments of the method comprising steps a) to f) above, wherein the pseudo-solid-phase protecting group attached to said nucleoside, nucleotide, oligonucleotide, or conjugate of nucleoside, nucleotide or oligonucleotide obtained in step a) is a pseudo-solid-phase protecting group according to any one of formulas II, II-1, II-2, II-a, II-a-1, II-a-2 and III to VII, and c is 0, step f) comprises incubating the conjugate of pseudo-solid-phase protecting group and oligonucleotide in a solution, preferably an aqueous solution comprising ammonia and / or a C1-C6-alkylamine (e.g. methylamine or tert-butylamine), preferably ammonia. In some embodiments of the method comprising steps a) to f) above, wherein the pseudo-solid-phase protecting group attached to the nucleoside, nucleotide, oligonucleotide, or conjugate of nucleoside, nucleotide, or oligonucleotide obtained in step a) is a pseudo-solid-phase protecting group according to any one of formulas II, II-1, II-2, II-a, II-a-1, II-a-2, and III to VII, and c is 0, step f) comprises incubating the conjugate of pseudo-solid-phase protecting group and oligonucleotide in aqueous ammonia.Such aqueous ammonia may comprise, for example, 28% ammonia (w / w) in water, where w / w indicates that 28% refers to the weight % of NH3 in water.

[0198] The processes of the present invention are not particularly limited with respect to the temperature at which they are carried out and may typically be carried out at ambient temperature, for example at a temperature of from 15 to 35° C. However, it should be understood that the reaction may be carried out at any suitable temperature, for example at a temperature of from 0 to 90° C.

[0199] Some embodiments of the present invention provide a method for synthesizing an oligonucleotide, comprising steps a) to f):

[0200] a) Formula S-1 below: [ka] (In formula S-1: * represents the point of attachment of a pseudo solid phase protecting group according to any one of formulas II, II-1, II-2, II-a, II-a-1, II-a-2 and III to VII; p is an integer equal to or greater than 0; R 1 , B N , Y, Z C , R 2C , R IV and R V is defined with respect to formula C-1), obtaining a nucleoside or oligonucleotide of formula C-2; b) Protecting group R 1 from the compound obtained in the previous step, thereby generating a free backbone hydroxyl or amine group;

[0201] c) reacting the free backbone hydroxyl or amine groups produced in step b) with a compound of formula C-1: [ka] (In formula C-1: m, R 1 , B N , Y, Z C , R 2C , R 3C , R 4C , R IV and R V is defined as above) with a phosphorus moiety of a nucleoside or oligonucleotide component; d) optionally modifying the phosphorus moiety; e) optionally repeating the sequence of steps b) to c) or b) to d); and f) cleaving the oligonucleotide from the pseudo solid phase protecting group.

[0202] In some embodiments, the integer p in formula S-1 is an integer in the range of 0 to 200, 0 to 150, 0 to 120, 0 to 100, 0 to 90, 0 to 80, 0 to 70, 0 to 60, 0 to 50, 0 to 40, 0 to 30, 0 to 20, 0 to 10, or 0 to 1.

[0203] In some embodiments, the method comprising the above steps a) to f) further comprises the following step g): g) isolating the cleaved oligonucleotide from the support. Further includes:

[0204] In the present context, the term "oligonucleotide cleaved from the support" refers to the oligonucleotide cleaved from the pseudo solid phase protecting group, preferably obtained in step f).

[0205] The means of isolating oligonucleotides during chemical oligonucleotide synthesis form part of the common knowledge of the skilled artisan. Typically, such a step of isolating an oligonucleotide comprises one or more purification steps and one or more steps aimed at obtaining the oligonucleotide in solid form. For oligonucleotide purification, chromatographic methods, in particular ion exchange (especially anion exchange) chromatography and reversed phase (RP) HPLC, for example in the form of hydrophobic interaction HPLC, can typically be used. These techniques are known to the skilled artisan. Furthermore, the step of isolating an oligonucleotide can comprise an ultrafiltration and / or desalting step. For example, the solution obtained after cleaving the oligonucleotide from the pseudo solid phase protecting group (PSPPG) can be subjected to an ultrafiltration and / or desalting step, an ion exchange chromatography, and another ultrafiltration and / or desalting step. Alternatively, the oligonucleotide cleaved from the PSPPG can be subjected to a reversed phase (RP) HPLC, for example in the form of hydrophobic interaction HPLC. The latter method can be preferably carried out when the oligonucleotide still carries the 5'-terminal hydroxyl protecting group, for example a DMT group. The 5'-protecting group is further removed on a RP-HPLC column by passing an acidic solution through the column. If the 5'-terminal protecting group is removed before purification, for example before cleaving the oligonucleotide from PSPPG, ion exchange chromatography is preferred. Purification is typically followed by one or more steps aimed at obtaining the oligonucleotide in solid form. Freeze-drying and spray-drying steps can be used, for example. In some cases, it is desirable to obtain the oligonucleotide in the form of a salt with a counterion. In such cases, salt exchange can be performed, typically before the freeze-drying or spray-drying step.

[0206] The following figures, claims, and examples, including the experiments performed and results achieved, are presented for illustrative purposes only and should not be construed as limiting the scope of the claims. EXAMPLES

[0207] Analysis method The synthesized oligonucleotides were analyzed by high performance liquid chromatography (HPLC) coupled with MS-spectrometry using the following parameters: Method A: Solid phase: YMC-Triart C18, 12 nm, 1.9 μm, 2.1 × 100 mm; Mobile phase A: HO:HFIP:TEA (1000:1.5:0.2); Mobile phase B: THF:HFIP:TEA (1000:1.5:0.2), flow rate: 0.48 mL / min; Gradient (% of mobile phase B): 0–0.5 min; 65%, 0.5–1.0 min; 65–72%, 1.0–3.0 min; 72%, 3.0–5.0 min; 72–80%, 5.0–6.0 min; 80–85%, 6–6.5 min; 80–90% Column temperature: 40°C; detection wavelength: 254 nm. Method B: Solid phase: Column YMC-Triart C18, 12 nm, 1.9 μm, 2.1 × 100 mm; Mobile phase A: HO:TFA (1000:1); Mobile phase B: MeCN:TFA (1000:1); Flow rate 0.9 mL / min; Gradient (mobile phase B%): 0-1 min; 1%, 1-15 min; 50%, 15.01-18 min; 90%; Column temperature: 60 °C; Detection wavelength: 263 nm. Method C: Solid phase: YMC-Triart C18, 12nm, 1.9μm, 2.1×100mm; Mobile phase A: H2O:TFA (1000:1); Mobile phase B: MeCN:TFA (1000:1); Flow rate 0.3m L / min; gradient (mobile phase B%): 0~1min; 1%, 1~15min; 50%, 15.01~18min; 90%; column temperature: 60°C; detection wavelength: 263nm. MS method: Ion source: Dual ESI, Ionization mode: positive or negative, Vcap: 3500V, Gas temp: 325°C, Gas flow: 8L / min, Nebulizer: 50psig. EXAMPLES

[0208] Synthesis of DMTr-dA(Bz)-Kc(1). [ka] To a solution of DMTr-dA (0.30 mmol, 197 mg) and 1,1'-carbonyldiimidazole (0.60 mmol, 97.3 mg) in CH2Cl2 (10 mL) stirred at room temperature (rt, i.e., approximately 20 °C) was added 4-dimethylaminopyridine (0.60 mmol, 73.3 mg). The resulting reaction mixture was stirred at rt for 14 h, diluted with NH4Cl aq, and extracted with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give 205 mg (91%) DMTr-dA-1H-imidazole-1-carboxyl. To a solution of DMTr-dA-1H-imidazole-1-carboxyl (ca. 0.30 mmol) and DBU (0.23 mmol, 34.3 μL) in CH2Cl2 (15 mL) stirred at rt, 3,5-bis(docosyloxy)benzenemethanol, C 51 H 96 O3 (0.15 mmol, 114 mg) was added. The resulting reaction mixture was stirred at rt for 16 h and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give DMTr-dA-Kc in 99% yield (215 mg) as a white solid. Throughout the text, the terms "DMT" and "DMTr" are used interchangeably to refer to the di(p-methoxyphenyl)phenylmethyl group. 1H NMR (CDCl3, 500 MHz) δ 9.01 (1H, d), 8.71 (1H, s), 8.17 (1H, s), 8.02 (2H, d), 7.60 (1H, t), 7.52 (2H, t), 7.39 (2H, d), 7.28 (4H, d), 7.25 (2H, t), 7.20 (1H,t), 6.79 (4H, d), 6.51 (3H, dt), 6.44 (1H, s), 5.48 (1H, d), 5.11 (2H, s), 4.40 (1H, s), 3.93 (4H, t), 3.77 (6H, s), 3.46 (2H, m), 3.10 (1H, m), 2.75 (1H, dd), 1.77 (4H, quint), 1.44 (4H, m), 1.39-1.19 (72H, m), 0.88 (6H, t); HPLC (method A): Rt=4.30 min; MS (ESI - ): m / z calcd for [C 90 H 127 N5O 10 - H] - : 1439.0, found: 1439.0.

Example

[0209] Synthesis of DMTr-dT-Kc(2).

change

[0210] DMTr-dG( i Synthesis of Bu)-Kc(3). [ka] To a solution of DMTr-dG(iBu) (0.30 mmol, 192 mg) and 1,1'-carbonyldiimidazole (0.60 mmol, 97.3 mg) in CH2Cl2 (10 mL) stirred at rt was added 4-dimethylaminopyridine (0.60 mmol, 73.3 mg). The resulting reaction mixture was stirred at rt for 14 h, diluted with NH4Cl aq, and extracted with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give DMTr-dG(iBu)-1H-imidazole-1-carboxyl quantitatively (225 mg). To a solution of DMTr-dG(iBu)-1H-imidazole-1-carboxyl (ca. 0.30 mmol) and DBU (0.23 mmol, 34.3 μL) in CH2Cl2 (15 mL) stirred at rt, 3,5-bis(docosyloxy)benzenemethanol, C 51 H 96 O3 (0.15 mmol, 114 mg) was added. The resulting reaction mixture was stirred at rt for 16 h and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give DMTr-dG(iBu)-Kc as a white solid in quantitative yield (225 mg). 1H NMR (CDCl3, 500 MHz) δ 11.9 (1H, s), 7.76 (1H, s), 7.58 (1H, s), 7.49 (2H, d), 7.35 (4H, d), 7.25 (2H, t), 7.21 (1H, d), 6.79 (4H, dd), 6.48 (2H, d), 6.42 (1H, t), 6.14 (1H, dd), 5.51 (1H, d), 5.07 (2H, m), 4.26 (1H, q), 3.91 (4H, t), 3.77 (3H, s), 3.76 (3H, s), 3.47 (1H, dd), 3.26 (1H, m), 3.24 (1H, dd), 2.56 (1H, dd), 1.75 (5H, m), 1.42 (5H, m), 1.37 - 1.21 (71H, m), 0.97 (3H, d), 0.88 (6H, t), 0.81 (3H, d); HPLC (method A): Rt = 3.76 min; MS (ESI - ): m / z calcd for [C 87 H 131 N5O 11 - H] - : 1421.0, found: 1421.0.

Example

[0211] Synthesis of DMTr-dC(Bz)-Kc(4).

Chem.

[0212] Synthesis of DMTr-dT-Ka (5). [ka] To a solution of DMTr-dT (0.30 mmol, 163 mg) and 1,1'-carbonyldiimidazole (0.60 mmol, 97.3 mg) in CH2Cl2 (10 mL) stirred at rt was added 4-dimethylaminopyridine (0.60 mmol, 73.3 mg). The resulting reaction mixture was stirred at rt for 14 h, diluted with NH4Cl aq, and extracted with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give DMTr-dT-1H-imidazole-1-carboxyl quantitatively (197 mg). To a solution of DMTr-dT-1H-imidazole-1-carboxyl (ca. 0.30 mmol) and DBU (0.23 mmol, 34.3 μL) in CH2Cl2 (15 mL) stirred at rt, 3,4,5-tris(octadecyloxy)benzenemethanol, C 61 H 116 O4 (0.15 mmol, 137 mg) was added. The resulting reaction mixture was stirred at rt for 16 h and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give DMTr-dT-Ka as a white solid in quantitative yield (240 mg). 1H NMR (CDCl3, 500 MHz) δ 8.75 (1H, s), 7.61 (1H, s), 7.38 (2H, d), 7.27 (7H, m), 6.84 (4H, d), 6.56 (2H, s), 6.46 (1H, dd), 5.37 (1H, d), 5.05 (2H, s), 4.23 (1H, m), 3.96 (3H, t), 3.93 (2H, t), 3.79 (6H, s), 3.52 (1H, dd), 3.45 (1H, dd), 2.57 (1H, dd), 2.45 (1H, m), 1.79 (3H, quint), 1.73 (2H, quint), 1.46 (6H, m), 1.37 (3H, s), 1.35-1.17 (86H, m), 0.88 (9H, t); HPLC (method A): Rt=4.92 min; MS (ESI - ): m / z calcd for [C 93 H 146 N2O 12 - H] - : 1482.1, found: 1482.1.

Example

[0213] Synthesis of DMTr-rU(TBS)-Kc(6).

change

[0214] Synthesis of dT-Kc(7). To a solution of DMTr-dT-Kc 2 (0.15 mmol) in CH2Cl2 (20 mL) stirred at rt was added dichloroacetic acid (7.46 mmol, 616.2 μl). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (7.54 mmol, 1045.2 μl), and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give dT-Kc quantitatively (179 mg) as a white solid. 1H NMR (CDCl3, 500 MHz) δ 7.93 (1H, s), 7.41 (1H, d), 6.49 (2H, d), 6.42 (1H, t), 6.16 (1H, dd), 5.30 (1H, dt), 5.09 (2H, s), 4.19 (1H, q), 3.93 (4H, t), 3.93 (1H, m), 2.54 (1H, m), 2.44 (2H, m), 1.93 (3H, d), 1.76 (4H, quint), 1.44 (4H, quint), 1.38-1.21 (73H, m), 0.88 (6H, t); HPLC (Method A): Rt=3.49 min; MS (ESI - ): m / z calcd for [C 62 H 108 NO-H] - : 1023.8, found: 1023.8.

[0215] [ka] EXAMPLES

[0216] Synthesis of DMTr-dA(Bz)-dT-Kc(8). To a solution of dT-Kc 7 (1.00 mmol) and DMTr-dA(Bz) phosphoramidite (2.00 mmol, 1.72 g) in CHCl (100 mL) stirred at rt, a 0.25 M solution of 5-benzylmercapto-1H-tetrazole in CHCN (2.00 mmol, 8 mL) was added. After checking the completion of the coupling by TLC (hexane: EtOAc = 1:1), a solution of AcO (1.31 mol, 124 μL), pyridine (1.54 mmol, 124 μL) and 1-methylimidazole (1.56 mmol, 124 μL) in CHCl (12 mL) was added. After stirring for 10 min at rt, a solution of 70% t-butyl hydroperoxide solution (0.96 mmol, 130.8 μL) in CHCl (13.5 mL) was added. The resulting reaction mixture was stirred at rt for 30 min and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give DMTr-dA(Bz)-dT-Kc as a creamy white solid in quantitative yield (2.19 g). HPLC (Method A): Rt = 4.81 min; MS (ESI - ): m / z calculated value [C 103 H 145 N8O 17 P-C2F3O2] - :1910.0, actual value:1910.0. EXAMPLES

[0217] Synthesis of dA(Bz)-dT-Kc(9). [ka] To a solution of DMTr-dA(Bz)-dT-Kc 8 (0.50 mmol) in CHCl (20 mL) stirred at rt was added dichloroacetic acid (7.46 mmol, 616.2 μl). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (7.54 mmol, 1045.2 μl) and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give dA(Bz)-dT-Kc as a white solid in 99% yield (737 mg). HPLC (Method A): Rt = 3.11 min; MS (ESI - ): m / z calculated value [C 86 H 129 N8O 15 P+C2F3O2] - : 1607.9, actual value: 1607.9. EXAMPLES

[0218] Synthesis of dA-dT(10). [ka] dA(Bz)-dT-Kc 9 (0.0669 mmol) was added to 1M aqueous NaOH / tetrahydrofuran (13.4 mL, 1:3 v / v). After stirring at 80° C. for 90 min, the resulting reaction mixture was cooled to room temperature. The aqueous layer was separated, neutralized with 6N HCl (1.1 mL), diluted with water, and lyophilized to give crude dA-dT (211 mg). HPLC (Method B): Rt=2.72min; MS (ESI + ): m / z calculated value [C 20 H 26 N7O 10 P+H] + :556.2, actual value:556.2. EXAMPLES

[0219] Synthesis of dT-Ka(11). [ka] To a solution of DMTr-dT-Ka 5 (0.15 mmol) in CH2Cl2 (20 mL) stirred at rt was added dichloroacetic acid (7.46 mmol, 616.2 μl). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (7.54 mmol, 1045.2 μl), and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give dT-Ka quantitatively (180 mg) as a white solid. 1H NMR (CDCl3, 500 MHz) δ 8.23 ​​(1H, s), 7.42 (1H, d), 6.57 (2H, s), 6.16 (1H, dd), 5.30 (1H, dt), 5.06 (2H, s), 4.19 (1H, q), 3.97 (4H, t), 3.94 (4H, t), 2.54 (2H, m), 2.44 (1H, ddd), 1.93 (3H, d), 1.80 (4H, quint), 1.73 (2H, quint), 1.61 (2H, s), 1.47 (6H, quint), 1.39-1.20 (82H, m), 0.88 (9H, t); HPLC (Method A): Rt=4.04 min; MS (ESI - ): m / z calcd for [C 72 H 128 N2O 10 -H] - : 1179.9, found: 1180.0. EXAMPLES

[0220] Synthesis of DMTr-dA(Bz)-dT-Ka (12). [ka] To a solution of dT-Ka 11 (1.00 mmol) and DMTr-dA(Bz) phosphoramidite (2.00 mmol, 1.72 g) in CHCl (100 mL) stirred at rt, a 0.25 M solution of 5-benzylmercapto-1H-tetrazole in CHCN (2.00 mmol, 8 mL) was added. After checking the completion of the coupling by TLC (hexane: EtOAc = 1:1), a solution of AcO (1.31 mol, 124 μL), pyridine (1.54 mmol, 124 μL) and 1-methylimidazole (1.56 mmol, 124 μL) in CHCl (12 mL) was added. After stirring for 10 min at rt, a solution of 70% t-butyl hydroperoxide solution (0.96 mmol, 130.8 μL) in CHCl (13.5 mL) was added. The resulting reaction mixture was stirred at rt for 30 min and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give DMTr-dA(Bz)-dT-Ka as a creamy white solid in quantitative yield (2.10 g). HPLC (Method A): Rt = 3.98 min; MS (ESI - ): m / z calculated value [C 113 H 166 N8O 18 PH] - : 1952.2, actual value: 1952.2. EXAMPLES

[0221] Synthesis of dA(Bz)-dT-Ka (13). [ka] To a solution of DMTr-dA(Bz)-dT-Ka 12 (0.5 mmol) in CH2Cl2 (20 mL) stirred at rt was added dichloroacetic acid (7.46 mmol, 616.2 μl). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (7.54 mmol, 1045.2 μl) and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give dA(Bz)-dT-Ka as a white solid in 86% yield (702 mg). HPLC (Method A): Rt = 3.35 min; MS (ESI - ): m / z calculated value [C92 H 148 N8O 16 PH] - : 1650.1, actual value: 1650.1. EXAMPLES

[0222] Synthesis of rU(TBS)-Kc(14). [ka] To a solution of DMTr-rU(TBS)-Kc 6 (0.15 mmol) in CH2Cl2 (20 mL) stirred at rt was added dichloroacetic acid (7.46 mmol, 616.2 μl). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (7.54 mmol, 1045.2 μl), and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give rU(TBS)-Kc quantitatively (179 mg) as a white solid. 1 H NMR (CDCl3, 500 MHz) δ 9.29 (1H, s), 7.58 (1H, d), 6.49 (2H, d), 6.42 (1H, t), 5.76 (1H, dd), 5.53 (1H, d), 5.15 (1H, dd), 5.11 (1H, d), 5.03 (1H, d), 4.77 (1H, t), 4.29 (1H, m), 3.92 (4H, t), 3.77 (1H, dd), 3.37 (1H, s), 1.76 (4H, quint), 1.44 (4H, quint), 1.38-1.19 (73H, m), 0.88 (6H, t), 0.84 (9H, s), 0.06 (3H, s), 0.02 (3H, s); HPLC (Method A): Rt=4.01 min; MS (ESI - ): m / z calcd for [C 67 H 120 N2O 10 Si-H] - : 1139.9, found: 1139.9. EXAMPLES

[0223] Synthesis of DMTr-rA(Bz,TBS)-rU(TBS)-Kc (15). [ka] To a solution of rU(TBS)-Kc 14 (0.5 mmol), DMTr-rA(Bz, TBS) phosphoramidite (1.00 mmol, 988.2 mg) in CHCl (100 mL) stirred at rt was added 0.25 M 5-benzylmercapto-1H-tetrazole solution (2.00 mmol, 8 mL) in CHCN. After checking the completion of the coupling by TLC (hexane: EtOAc = 1:1), a solution of AcO (1.31 mol, 124 μL), pyridine (1.54 mmol, 124 μL) and 1-methylimidazole (1.56 mmol, 124 μL) in CHCl (12 mL) was added. After stirring at rt for 10 min, a solution of 70% t-butyl hydroperoxide solution (0.96 mmol, 130.8 μL) in CH2Cl2 (13.5 mL) was added. The resulting reaction mixture was stirred at rt for 30 min and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give DMTr-rA(Bz,TBS)-rU(TBS)-Kc as a creamy white solid in 82% yield (841 mg). HPLC (Method A): Rt = 4.76 min; MS (ESI - ): m / z calculated value [C 114 H 171 N8O 19 PSi2-C2F3O2] - : 2156.2, actual value: 2156.2. EXAMPLES

[0224] Synthesis of rA(Bz, TBS)-rU(TBS)-Kc (16). [ka] To a solution of DMTr-rA(Bz,TBS)-rU(TBS)-Kc 15 (0.5 mmol) in CHCl (20 mL) stirred at rt was added dichloroacetic acid (7.46 mmol, 616.2 μl). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (7.54 mmol, 1045.2 μl) and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give rA(Bz,TBS)-rU(TBS)-Kc in 61% yield (531 mg). HPLC (Method A): Rt = 4.17 min; MS (ESI - ): m / z calculated value [C 93 H 153 N8O 17 PSi2-C2F3O2] - :1854.0, actual value:1854.0. EXAMPLES

[0225] Synthesis of rA-rU(17). [ka] To a solution of rA(Bz,TBS)-rU(TBS)-Kc 16 (5.7 μmol) in THF (1 mL) stirred at rt was added tetrabutylammonium fluoride monohydrate (36.5 mmol, 9.6 mg). The resulting reaction mixture was stirred at rt for 30 min and 1M NaOH (0.344 mL) was added. The resulting reaction mixture was stirred at 80° C. for 90 min and cooled to room temperature. The aqueous layer was separated and analyzed by HPLC (Method C): Rt=2.69 min; LC-MS to find rA-rU. MS (ESI + ): m / z calculated value [C 19 H 24 N7O 12 P+H] + :574.1, actual value:574.1. EXAMPLES

[0226] Exemplary synthesis of pseudo solid-phase protecting groups Methyl 2-(3,5-bis(docosyloxy)phenyl)acetate [ka] To a solution of methyl 2-(3,5-dihydroxyphenyl)acetate (2.5 mmol) and 1-bromodocosane (3 eq) in DMF / THF (1 / 1, 100 mL) stirred at rt was added K2CO3 (6 eq). The resulting reaction mixture was stirred at 80° C. for 18 h, diluted with MeCN (100 mL) at 60° C., and filtered. The collected solid was washed with MeCN (60° C.) and dried in vacuum to quantitatively obtain methyl 2-(3,5-bis(docosyloxy)phenyl)acetate as a white solid. 1 H NMR (CDCl3, 500 MHz) δ 6.40 (2H, d), 6.36 (1H, t), 3.91 (4H, t), 3.69 (3H, s), 3.54 (2H, s), 1.75 (4H, quint), 1.43 (4H, quint), 1.38-1.19 (72H, m), 0.88 (6H, t).

[0227] Methyl 3-(2,4-bis(docosyloxy)phenyl)propanoate [ka] To a solution of methyl 3-(2,4-dihydroxyphenyl)propionate (5 mmol) and 1-bromodocosane (3 eq) in DMF / THF (1 / 1, 100 mL) stirred at rt was added K2CO3 (6 eq). The resulting reaction mixture was stirred at 80° C. for 18 h, diluted with MeCN at 60° C., and filtered. The collected solid was washed with MeCN (60° C.) and concentrated in vacuo to quantitatively obtain methyl 3-(2,4-bis(docosyloxy)phenyl)propanoate as a white solid. 1 H NMR (CDCl3, 300 MHz) δ 7.01 (1H, d), 6.41 (1H, d), 6.36 (1H, dd), 3.91 (4H, dt), 3.65 (3H, s), 2.86 (2H, t), 2.58 (2H, t), 1.76 (4H, m), 1.44 (4H, m), 1.38-1.19 (72H, m), 0.88 (6H, t).

[0228] 2-(3,5-bis(docosyloxy)phenyl)acetic acid [ka] To a solution of methyl 2-(3,5-bis(docosyloxy)phenyl)acetate (2.5 mmol) in EtOH / THF (1 / 1, 50 mL) stirred at rt was added aq. KOH (1 M, 6 eq). The resulting reaction mixture was stirred at 90° C. for 5 h, neutralized with 1N aq. HCl at rt and filtered. The collected solid was washed with water / acetone and dried in vacuum to give 2-(3,5-bis(docosyloxy)phenyl)acetic acid in 47% (930 mg) as a white solid from 2-(3,5-dihydroxyphenyl)acetate (2.5 mmol) over two steps. 1 H NMR (CDCl3, 500 MHz) δ 6.32 (2H, s), 6.23 (1H, s), 3.80 (4H, m), 3.27 (2H, m), 1.65 (4H, m), 1.34 (4H, m), 1.31-1.19 (73H, m), 0.88 (6H, t).

[0229] 3-(2,4-bis(docosyloxy)phenyl)propanoic acid [ka] To a solution of methyl 3-(2,4-bis(docosyloxy)phenyl)propanoate (5.0 mmol) in EtOH / THF (1 / 1, 50 mL) stirred at rt was added aq. KOH (1 M, 6 eq). The resulting reaction mixture was stirred at 90° C. for 5 h, neutralized with 1N aq. HCl at rt, and filtered. The collected solid was washed with water / acetone and concentrated in vacuo to give 3-(2,4-bis(docosyloxy)phenyl)propanoic acid in 49% (1.96 g) as a white solid from methyl 3-(2,4-dihydroxyphenyl)propionate (5 mmol) over two steps. 1H NMR (CDCl3, 500 MHz) δ 7.03 (1H, d), 6.41 (1H, d), 6.37 (1H, dd), 3.92 (4H, q), 2.87 (2H, t), 2.62 (2H, t), 1.77 (4H, m), 1.44 (4H, m), 1.38-1.18 (73H, m), 0.88 (6H, t).

[0230] 2-(3,5-bis(docosyloxy)phenyl)ethane-1-ol [ka] To a solution of 3-(2,4-bis(docosyloxy)phenyl)propanoic acid (2.5 mmol) in THF (50 mL) stirred at 0° C. was added LiAlH4 (1.7 eq, solid form). The resulting reaction mixture was stirred at 0° C. to rt for 24 h, quenched with water and concentrated in vacuo. The collected solid was washed with MeOH and dried in vacuo to give 2-(3,5-bis(docosyloxy)phenyl)ethan-1-ol quantitatively (2.10 g) as a white solid. 1 H NMR (CDCl3, 300 MHz) δ 6.35 (2H, d), 6.34 (1H, d), 3.92 (4H, t), 3.84 (2H, q), 2.79 (2H, t), 1.76 (4H, m), 1.44 (4H, m), 1.38-1.20 (73H, m), 0.88 (6H, t).

[0231] 3-(2,4-bis(docosyloxy)phenyl)propan-1-ol [ka] To a solution of 3-(2,4-bis(docosyloxy)phenyl)propanoic acid (1 mmol) in THF (50 mL) stirred at 0° C. was added LiAlH4 (1.7 eq). The resulting reaction mixture was stirred at 0° C. to rt for 24 h, quenched with water, and concentrated in vacuo. The collected solid was washed with MeOH and dried in vacuo to give 3-(2,4-bis(docosyloxy)phenyl)propan-1-ol quantitatively (900 mg) as a white solid. 1 H NMR (CDCl3, 500 MHz) δ 7.01 (1H, d), 6.44 (1H, d), 6.41 (1H, dd), 3.93 (4H, m), 3.57 (2H, q), 2.66 (2H, t), 1.78 (6H, m), 1.44 (4H, m), 1.39-1.19 (73H, m), 0.88 (6H, t). EXAMPLES

[0232] Synthesis of DMTr-dT-Kc-C2 (18). [ka] To a solution of DMTr-dT (0.30 mmol, 163 mg) and 1,1'-carbonyldiimidazole (0.60 mmol, 97.3 mg) in CH2Cl2 (10 mL) stirred at rt was added 4-dimethylaminopyridine (0.60 mmol, 73.3 mg). The resulting reaction mixture was stirred at rt for 14 h, diluted with NH4Cl aq, and extracted with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give DMTr-dT-1H-imidazole-1-carboxyl quantitatively (197 mg). To a solution of DMTr-dT-1H-imidazole-1-carboxyl (ca. 0.30 mmol) and DBU (0.23 mmol, 34.3 μL) in CH2Cl2 (15 mL) stirred at rt was added 2-(3,5-bis(docosyloxy)phenyl)ethan-1-ol, C 52 H 98O3 (0.15 mmol, 116 mg) was added. The resulting reaction mixture was stirred at rt for 16 h and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give DMTr-dT-Kc-C2 in 89% (177 mg) as a white solid. 1 H NMR (CDCl3, 500 MHz) δ 7.99 (1H, s), 7.38 (2H, d), 7.30 (2H, q), 7.27 (2H, m), 7.25 (2H, m), 6.84 (4H, d), 6.44 (1H, dd), 6.34 (2H, m), 5.33 (1H, d), 4.32 (1H, t), 4.21 (1H, m), 3.90 (3H, t), 3.79 (6H, s), 3.79 (1H, m), 3.51 (1H, m), 3.44 (1H, m), 2.90 (1H, t), 2.55 (1H, m), 2.44 (1H, m), 1.75 (4H, MS (ESI + ): m / z calcd for [C 84 H 128 N2O 11 + C8H 19 N + H] + : 1471.1, found: 1471.1. EXAMPLES

[0233] Synthesis of DMTr-dT-Kb-C3 (19). [ka] To a solution of DMTr-dT (0.30 mmol, 163 mg) and 1,1'-carbonyldiimidazole (0.60 mmol, 97.3 mg) in CH2Cl2 (10 mL) stirred at rt was added 4-dimethylaminopyridine (0.60 mmol, 73.3 mg). The resulting reaction mixture was stirred at rt for 14 h, diluted with NH4Cl aq, and extracted with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered, and concentrated in vacuo to give DMTr-dT-1H-imidazole-1-carboxyl quantitatively (197 mg). To a solution of DMTr-dT-1H-imidazole-1-carboxyl (ca. 0.30 mmol) and DBU (0.23 mmol, 34.3 μL) in CH2Cl2 (15 mL) stirred at rt was added 3-(2,4-bis(docosyloxy)phenyl)propan-1-ol, C 53 H 100 O3 (0.15 mmol, 118 mg) was added. The resulting reaction mixture was stirred at rt for 16 h and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give DMTr-dT-Kb-C3 in 73% yield (147 mg) as a white solid. 1 H NMR (CDCl3, 500 MHz) δ 8.11 (1H, s), 7.62 (1H, d), 7.38 (2H, m), 7.29 (4H, m), 7.27 (2H, m), 7.25 (2H, m), 6.98 (1H, d), 6.84 (5H, m), 6.46 (1H, dd), 6.41 (1H, d), 6.37 (1H, dd), 5.34 (1H, d), 4.23 (1H, d), 4.13 (2H, t), 3.91 (3H, t), 3.79 (6H, s), 3.78 (1H, m), 3.52 (1H, dd), 3.45 (1H, dd), 2.62 (2H, t), 2.56 (1H, dd), 2.45 (1H, m), 1.94 (2H, quint), 1.76 (4H, quint), 1.47-1.14 (77H, m), 0.88 (6H, t); MS (ESI + ): m / z calcd for [C 85 H 130 N2O 11+ C8H 19 N + H] + : 1485.1, found: 1485.1. EXAMPLES

[0234] Synthesis of dT-Kc-C2 (20). [ka] To a solution of DMTr-dT-Kc-C2 18 (0.15 mmol) in CH2Cl2 (20 mL) stirred at rt was added dichloroacetic acid (7.46 mmol, 616.2 μl). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (7.54 mmol, 1045.2 μl), and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give dT-Kc-C2 in 68% yield (103 mg) as a white solid. 1 H NMR (CDCl3, 500 MHz) δ 8.44 (1H, s), 7.43 (1H, s), 6.35 (3H, m), 6.16 (1H, dd), 5.27 (1H, dt), 4.35 (2H, t), 4.16 (1H, m), 3.95 (1H, m), 3.91 (3H, t), 3.88 (1H, m), 2.91 (2H, t), 2.53 (1H, m), 2.43 (1H, ddd), 2.18 (3H, s), 1.93 (3H, s), 1.76 (4H, quint), 1.44 (4H, quint), 1.37-1.19 (71H, m), 0.88 (6H, t); MS (ESI + ): m / z calcd for [C 63 H 110 N2O9+ C8H 19 N + H] + : 1169.0, found: 1169.0. EXAMPLES

[0235] Synthesis of DMTr-dA(Bz)-dT-Kc-C2 (21). [ka] To a solution of dT-Kc-C2 20 (1.00 mmol), DMTr-dA(Bz) phosphoramidite (2.00 mmol, 1.72 g) in CHCl (100 mL) stirred at rt, a 0.25 M solution of 5-benzylmercapto-1H-tetrazole (2.00 mmol, 8 mL) in CHCN was added. After checking the completion of the coupling by TLC (hexane: EtOAc = 1:1), a solution of AcO (1.31 mmol, 124 μL), pyridine (1.54 mmol, 124 μL) and 1-methylimidazole (1.56 mmol, 124 μL) in CHCl (12 mL) was added. After stirring at rt for 10 min, a solution of 70% t-butyl hydroperoxide solution (0.96 mmol, 130.8 μL) in CH2Cl2 (13.5 mL) was added. The resulting reaction mixture was stirred at rt for 30 min and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give DMTr-dA(Bz)-dT-Kc-C2 in 68% yield (1.23 g) as a creamy white solid. MS (ESI + ): m / z calculated value [C 104 H 147 N8O 17 P+H] + : 1812.1, actual value: 1812.1. EXAMPLES

[0236] Synthesis of dA(Bz)-dT-Kc-C2 (22). [ka] To a solution of DMTr-dA(Bz)-dT-Kc-C2 21 (0.5 mmol) in CH2Cl2 (20 mL) stirred at rt was added dichloroacetic acid (7.46 mmol, 616.2 μl). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (7.54 mmol, 1045.2 μl), and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give dA(Bz)-dT-Kc-C2 in 84% yield (630 mg) as a creamy white solid. MS (ESI +): m / z calculated value [C 83 H 129 N8O 17 P+H] + : 1509.9, actual value: 1509.9. EXAMPLES

[0237] Synthesis of dT-Kb-C3 (23). [ka] To a solution of DMTr-dT-Kb-C3 19 (0.15 mmol) in CH2Cl2 (20 mL) stirred at rt was added dichloroacetic acid (7.46 mmol, 616.2 μl). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (7.54 mmol, 1045.2 μl), and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give dT-Kc-C3 as a white solid in 40% yield (46 mg). 1 H NMR (CDCl3, 500 MHz) δ 8.08 (1H, s), 7.71 (1H, m), 7.53 (1H, m), 6.99 (1H, d), 6.42 (1H, d), 6.38 (1H, dd), 6.18 (1H, m), 5.27 (1H, dt), 4.29 (1H, m), 4.15 (2H, m), 3.92 (5H, m), 3.66 (2H, m), 3.56 (1H, m), 3.38 (1H, s), 2.63 (2H, t), 2.53 (1H, m), 2.45 (1H, m), 1.94 (4H, m), 1.76 (4H, m), 1.44 (4H, m), 1.37-1.11 (70H, m), 0.88 (6H, t); MS (ESI + ): m / z calcd for [C 64 H 112 N2O9+ C8H 19 N + H] + : 1183.0, found: 1183.0. EXAMPLES

[0238] Synthesis of DMTr-dA(Bz)-dT-Kb-C3 (24). [ka] To a solution of dT-Kb-C3 23 (1.00 mmol), DMTr-dA(Bz) phosphoramidite (2.00 mmol, 1.72 g) in CHCl (100 mL) stirred at rt was added 0.25 M 5-benzylmercapto-1H-tetrazole solution (2.00 mmol, 8 mL) in CHCN. After checking the completion of coupling by TLC (hexane: EtOAc = 1:1), a solution of AcO (1.31 mmol, 124 μL), pyridine (1.54 mmol, 124 μL) and 1-methylimidazole (1.56 mmol, 124 μL) in CHCl (12 mL) was added. After stirring at rt for 10 min, a solution of 70% t-butyl hydroperoxide solution (0.96 mmol, 130.8 μL) in CH2Cl2 (13.5 mL) was added. The resulting reaction mixture was stirred at rt for 30 min and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give DMTr-dA(Bz)-dT-Kb-C3 in 93% yield (1.31 g) as a creamy white solid. MS (ESI + ): m / z calculated value [C 105 H 149 N8O 17 P+H] + : 1826.1, actual value: 1826.1. EXAMPLES

[0239] Synthesis of dA(Bz)-dT-Kb-C3 (25). [ka] To a solution of DMTr-dA(Bz)-dT-Kb-C3 24 (0.5 mmol) in CHCl (20 mL) stirred at rt was added dichloroacetic acid (7.46 mmol, 616.2 μl). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (7.54 mmol, 1045.2 μl), and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give dA(Bz)-dT-Kb-C3 in 87% yield (663 mg) as a creamy white solid. MS (ESI + ): m / z calculated value [C 84 H 131 N8O 15 P+H] + :1524.0, actual value:1524.0. EXAMPLES

[0240] Synthesis of DMTr-dT-Kb-C2 acid (26). [ka] To a solution of DMTr-dT (0.30 mmol, 163 mg), DIPEA (0.45 mmol, 78.6 μL), COMU (0.3 mmol, 128.5 mg) and DMAP (0.02 mmol, 2.4 mg) in CH2Cl2 (20 mL) stirred at rt, 3-(2,4-bis(docosyloxy)phenyl)propanoic acid, C 53 H 98 O4 (Kb-C2 acid tag from Example 18, 0.15 mmol, 120 mg) was added. The resulting reaction mixture was stirred at rt for 16 h and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give DMTr-dT-Kc-C2 acid in 57% (114 mg) as a white solid. 1H NMR (CDCl3, 500 MHz) δ 8.81 (1H, m), 7.61 (1H, s), 7.38 (2H, d), 7.27 (6H, m), 6.99 (1H, d), 6.84 (3H, d), 6.41 (1H, s), 6.37 (1H, dd), 5.44 (1H, s), 4.03 (1H, s), 3.90 (4H, dt), 3.79 (6H, s), 3.44 (1H, s), 3.41 (1H, m), 2.85 (2H, m), 2.61 (2H, t), 2.40 (1H, m), 1.76 (4H, dq), 1.44 (4H, m), 1.38-1.13 (79H, m), 0.88 (6H, t); MS (ESI + ): m / z calcd for [C 83 H 126 N2O 10 + C8H 19 N + H] + : 1455.1, found: 1455.1. EXAMPLES

[0241] Synthesis of DMTr-dT-Kc-C1 acid (27). [ka] To a solution of DMTr-dT (0.30 mmol, 163 mg), DIPEA (0.45 mmol, 78.6 μL), COMU (0.3 mmol, 128.5 mg) and DMAP (0.02 mmol, 2.4 mg) in CH2Cl2 (20 mL) stirred at rt was added 2-(3,5-bis(docosyloxy)phenyl)acetic acid, C 52 H 96 O4 (0.15 mmol, 118 mg) was added. The resulting reaction mixture was stirred at rt for 16 h and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give DMTr-dT-Kc-C1 acid in 80% (158 mg) as a white solid. 1H NMR (CDCl3, 300 MHz) δ 9.10 (1H, s), 7.63 (1H, s), 7.37 (2H, m), 7.26 (5H, m), 6.83 (5H, d), 6.46 (2H, m), 6.37 (4H, m), 5.47 (1H, m), 4.11 (1H, m), 3.90 (6H, t), 3.78 (6H, s), 3.55 (2H, s), 3.46 (2H, s), 3.21 (1H, d), 2.97 (1H, d), 2.47 (2H, q), 1.74 (4H, quint), 1.42 (4H, m), 1.35-1.17 (70H, m), 0.88 (6H, t); MS m / z: [M + C8H 19 N + H] + calcd for C 91 H 146 N3O 10 + , 1441.1; found 1441.1. EXAMPLES

[0242] Synthesis of DMTr-2'O-methyl-rU-Kb-C2 acid (28). [ka] To a solution of DMTr-2'O-methyl-rU (0.30 mmol, 163 mg), DIPEA (0.45 mmol, 78.6 μL), COMU (0.3 mmol, 128.5 mg) and DMAP (0.02 mmol, 2.4 mg) in CH2Cl2 (20 mL) stirred at rt, 3-(2,4-bis(docosyloxy)phenyl)propanoic acid, C 53 H 98 O4 (0.15 mmol, 120 mg) was added. The resulting reaction mixture was stirred at rt for 16 h and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give DMTr-2'O-methyl-rU-Kb-C2 acid as a white solid in quantitative yield (252 mg). 1H NMR (CDCl3, 500 MHz) δ 9.07 (1H, s), 7.92 (1H, d), 7.36 (1H, m), 7.29 (2H, m), 7.25 (2H, m), 7.02 (1H, m), 6.84 (3H, m), 6.41 (2H, m), 6.37 (1H, dt), 6.34 (1H, dd), 6.01 (1H, d), 5.96 (1H, m), 5.31 (1H, d), 5.26 (1H, m), 3.92 (2H, m), 3.88 (2H, m), 3.79 (3H, s), 3.75 (6H, s), 3.69 (1H, t), 3.43 (1H, MS (ESI + ): m / z calcd for [C 83 H 126 N2O 10 + C8H 19 N + H] + : 1441.1, found: 1441.1. EXAMPLES

[0243] Synthesis of dT-Kb-C2 acid (29). [ka] To a solution of DMTr-dT-Kb-C2 acid 26 (0.15 mmol) in CH2Cl2 (20 mL) stirred at rt was added dichloroacetic acid (7.46 mmol, 616.2 μl). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (7.54 mmol, 1045.2 μl), and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give dT-Kc-C2 as a white solid in 46% yield (70 mg). 1H NMR (CDCl3, 500 MHz) δ 8.96 (1H, s), 7.48 (1H, d), 7.00 (1H, d), 6.43 (1H, d), 6.37 (1H, dd), 6.16 (1H, dd), 5.31 (1H, dt), 3.99 (1H, q), 3.92 (4H, dt), 3.87 (2H, m), 2.87 (2H, t), 2.75 (1H, m), 2.63 (2H, t), 2.41 (1H, m), 2.30 (1H, ddd), 1.92 (3H, s), 1.84 (1H, m), 1.77 (4H, dq), 1.46 (4H, m), 1.38-1.20 (71H, m), 0.88 (6H, t); MS (ESI + ): m / z calcd for [C 63 H 110 N2O8+ C8H 19 N + H] + : 1153.0, found: 1153.0.

Example

[0244] Synthesis of DMTr-dA(Bz)-dT-Kb-C2 acid (30).

change

[0245] Synthesis of dA(Bz)-dT-Kb-C2 acid (31). [ka] To a solution of DMTr-dA(Bz)-dT-Kb-C2 acid 30 (0.5 mmol) in CHCl (40 mL) stirred at rt was added dichloroacetic acid (14.9 mmol, 1.23 mL). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (14.9 mmol, 2.09 mL), and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give dA(Bz)-dT-Kb-C2 acid in 87% yield (601 mg) as a creamy white solid. MS (ESI + ): m / z calculated value [C 83 H 129 N8O14 P+H] + : 1493.9, actual value: 1493.9. EXAMPLES

[0246] Synthesis of dT-Kc-C1 acid (32). [ka] To a solution of DMTr-dT-Kc-C1 acid 27 (0.15 mmol) in CH2Cl2 (20 mL) stirred at rt was added dichloroacetic acid (7.46 mmol, 616.2 μl). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (7.54 mmol, 1045.2 μl), and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give dT-Kc-C1 acid in 75% yield (114 mg) as a white solid. 1 H NMR (CDCl3, 500 MHz) δ 8.66 (1H, s), 7.49 (1H, d), 6.39 (2H, d), 6.35 (2H, m), 6.22 (1H, dd), 5.35 (1H, dt), 4.07 (1H, dt), 3.91 (6H, m), 3.56 (1H, s), 3.55 (1H, m), 2.97 (1H, d), 2.55 (1H, m), 2.44 (1H, m), 2.37 (1H, dd), 1.92 (3H, d), 1.75 (4H, m), 1.42 (4H, m), 1.38-1.19 (70H, m), 0.88 (6H, t); MS (ESI + ): m / z calcd for [C 62 H 108 N2O8+ C8H 19 N + H] + : 1139.0, found: 1139.0. EXAMPLES

[0247] Synthesis of DMTr-dA(Bz)-dT-Kc-C1 acid (33). [ka] To a solution of dT-Kc-C1 acid 32 (1.00 mmol), DMTr-dA(Bz) phosphoramidite (2.00 mmol, 1.72 g) in CHCl (100 mL) stirred at rt was added 0.25 M 5-benzylmercapto-1H-tetrazole solution (2.00 mmol, 8 mL) in CHCN. After checking the completion of coupling by TLC (hexane:EtOAc = 1:1), a solution of AcO (1.31 mmol, 124 μL), pyridine (1.54 mmol, 124 μL) and 1-methylimidazole (1.56 mmol, 124 μL) in CHCl (12 mL) was added. After stirring at rt for 10 min, a solution of 70% t-butyl hydroperoxide solution (0.96 mmol, 130.8 μL) in CH2Cl2 (13.5 mL) was added. The resulting reaction mixture was stirred at rt for 30 min and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give DMTr-dA(Bz)-dT-Kc-C1 acid in 80% yield (1.42 g) as a creamy white solid. MS (ESI + ): m / z calculated value [C 103 H 145 N8O 16 P+H] + : 1782.1, actual value: 1782.0. EXAMPLES

[0248] Synthesis of dA(Bz)-dT-Kc-C1 acid (34). [ka] To a solution of DMTr-dA(Bz)-dT-Kc-C1 acid 33 (0.5 mmol) in CH2Cl2 (40 mL) stirred at rt was added dichloroacetic acid (14.9 mmol, 1.23 mL). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (14.9 mmol, 2.09 mL), and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give dA(Bz)-dT-Kc-C1 acid in 90% yield (665 mg) as a creamy white solid. MS (ESI + ): m / z calculated value [C82 H 127 N8O 14 P+H] + : 1479.9, actual value: 1480.0. EXAMPLES

[0249] Synthesis of 2'-O-methyl-rU(Me)-Kb-C2 acid (35). [ka] To a solution of DMTr-2'O-methyl-rU-Kb-C2 acid 28 (0.15 mmol) in CHCl (20 mL) stirred at rt was added dichloroacetic acid (7.46 mmol, 616.2 μl). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (7.54 mmol, 1045.2 μl) and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give 2'O-methyl-rU-Kb-C2 acid quantitatively (169 mg) as a white solid. 1 H NMR (CDCl3, 500 MHz) δ 7.69 (1H, d), 7.01 (1H, d), 6.20 (1H, d), 6.36 (1H, dd), 5.74 (1H, d), 5.68 (1H, d), 5.24 (1H, t), 4.29 (1H, m), 4.19 (1H, t), 4.12 (1H, dt), 3.92 (4H, m), 3.70 (1H, d), 3.42 (3H, s), 2.94 (1H, d), 2.88 (3H, m), 2.70 (2H, t), 1.77 (4H, m), 1.44 (4H, m), 1.38-1.19 (72H, m), 0.88 (6H, t); MS (ESI + ): m / z calcd for [C 63 H 110 N2O9+ C8H 19 N + H] + : 1169.0, found: 1169.0. EXAMPLES

[0250] Synthesis of DMTr-2'O-methyl-rA(Bz)-2'O-methyl-rU-Kb-C2 acid (36). [ka] To a solution of 2'O-methyl-rU-Kb-C2 acid 35 (1.00 mmol), DMTr-2'O-methyl-rA(Bz) phosphoramidite (2.00 mmol, 1.72 g) in CHCl (100 mL) stirred at rt was added 5-(benzylthio)-1H-tetrazole (3.00 mmol, 577 mg). After checking the completion of the coupling by TLC (hexane: EtOAc = 1:1), a solution of AcO (1.31 mmol, 124 μL), pyridine (1.54 mmol, 124 μL) and 1-methylimidazole (1.56 mmol, 124 μL) in CHCl (12 mL) was added. After stirring at rt for 10 min, a solution of 70% t-butyl hydroperoxide solution (0.96 mmol, 130.8 μL) in CHCl (13.5 mL) was added. The resulting reaction mixture was stirred at rt for 30 min and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give DMTr-2′O-methyl-rA(Bz)-2′O-methyl-rU-Kb-C acid in 79% yield (1.45 g) as a creamy white solid. MS (ESI + ): m / z calculated value [C 105 H 149 N8O 18 P+H] + :1842.1.0, actual value:1842.1. EXAMPLES

[0251] Synthesis of 2'O-methyl-rA(Bz)-2'O-methyl-rU-Kb-C2 acid (37). [ka] To a solution of DMTr-2'O-methyl-rA(Bz)-2'O-methyl-rU-Kb-C2 acid 36 (0.5 mmol) in CHCl (20 mL) stirred at rt was added dichloroacetic acid (7.46 mmol, 616.2 μl). The resulting reaction mixture was stirred at rt for 1 h, diluted with MeOH, neutralized with triethylamine (7.54 mmol, 1045.2 μl) and concentrated in vacuo. The precipitate was filtered and washed with MeOH to give 2'O-methyl-rA(Bz)-2'O-methyl-rU-Kb-C2 acid in 46% yield (358 mg) as a creamy white solid. MS (ESI + ): m / z calculated value [C 84 H 131 N8O 16 P+H] + : 1539.9, actual value: 1540.0. EXAMPLES

[0252] Synthesis of 2'O-methyl-rA-2'O-methyl-rU (38). [ka] 2'O-methyl-rA(Bz,Me)-2'O-methyl-rU-Kb-C2 acid (0.0660 mmol) was added to a 1M aqueous NaOH / tetrahydrofuran solution (13.4 mL, 1:3 v / v). After stirring at 80°C for 90 min, the resulting reaction mixture was cooled to room temperature. The aqueous layer was separated, neutralized with 6N HCl, diluted with water, and lyophilized to give crude 2'O-methyl-rA-2'O-methyl-rU (176 mg). MS (ESI + ): m / z calculated value [C 21 H 28 N7O 12 P+H] + : 602.2, actual value: 602.2. EXAMPLES

[0253] Synthesis of dA-dT (39). [ka] dA(Bz)-dT-Kb-C3 25 (0.0676 mmol) was added to a 1M aqueous NaOH / tetrahydrofuran solution (13.4 mL, 1:3 v / v). After stirring at 80° C. for 90 min, the resulting reaction mixture was cooled to room temperature. The aqueous layer was separated, neutralized with 6N HCl, diluted with water, and lyophilized to give crude dA-dT (231 mg). MS (ESI + ): m / z calculated value [C 20 H 26 N7O 10 P+H] + :556.2, actual value:556.2. [Brief description of the drawings]

[0254] [Figure 1] Non-limiting examples of R6 moieties. * represents the point of attachment of the R4, R5, R8, R9 or R10 moiety of a pseudo solid-phase protecting group or compound according to any one of formulas I, Ia, Ib, Ic, Ib-1, II, II-1, II-2, II-a, II-a-1, II-a-2, XIII, XIII-b to XIII-g, XIV and XIV-b to XIV-g to an oxygen atom or to an oxygen atom represented by any one of formulas III to VII, VIII to XII, VIII-1 to X-1. [Diagram 2] Non-limiting examples of building blocks suitable for the synthesis of oligonucleotides and phosphoribose backbones: (a) building blocks for synthesizing DNA by the phosphoramidite approach; (b) building blocks for synthesizing RNA by the phosphoramidite approach; (c) phosphoramidite building blocks and 2'-O-methoxyribose; (d) phosphoramidite building blocks and 2'-O-(2-methoxyethoxy)ribose; (e) phosphoramidite building blocks and 2'-fluororibose. [Diagram 3] FIG. 1 shows non-limiting examples of phosphoramidite building blocks for synthesizing oligonucleotide analogs containing ribose surrogates: (a) protected building blocks for obtaining arabinose nucleic acid (ANA), (b) building blocks for obtaining 2′-fluoroarabinose nucleic acid (FANA), (c) building blocks for obtaining threose nucleic acid (TNA).

[0255] The following types of building blocks are obtained as stereoisomers and the structures shown represent one of these compounds: (d) building blocks to obtain 1',3'-anhydrothreitol nucleic acids (HNA), (e) building blocks to obtain locked nucleic acids (LNA), (f) building blocks to obtain ethylene-bridged nucleic acids (ENA), (g) building blocks to obtain tricyclic nucleic acids (tc-DNA), (h) building blocks to obtain cyclohexene nucleic acids (CeNA), (i) building blocks in which the ribose is replaced by glycerol (GNA). The protected building block (j) to obtain unlocked nucleic acids (UNA) is obtained by oxidative ring cleavage of the ribose.

Claims

1. A pseudo solid-phase protecting group for protecting a nucleoside, a nucleotide, an oligonucleotide, or a conjugate of a nucleoside, a nucleotide or an oligonucleotide, the protecting group having the formula II: 【Chemistry 1】 (In the formula: ＊ indicates the point of attachment to an oxygen atom of a protected nucleoside, nucleotide, oligonucleotide, or of a hydroxyl moiety of a conjugate of a nucleoside, nucleotide, or oligonucleotide; c is 0 or 1; a is an integer from 1 to 12; R ３ is H; R ５ is O-R ６ or H, R ６ is a C8 to C40 aliphatic hydrocarbon group; R ４ Each of the is independently O-R ６ and R ６ is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; b is 1 to 3; However, when b is 1, R ３ and R ５ is not H; In addition, however, the existing R ３ , R ４ and R ５ with the further proviso that the total of all carbon atoms contained in the moiety is greater than 23 and less than 200.

2. 2. The pseudo solid-phase protecting group according to claim 1, wherein a is an integer of 1 to 3 and b is an integer of 1 to 2.

3. Formula II-a: 【Chemistry 2】 (In the formula: ＊ indicates the point of attachment to an oxygen atom of a protected nucleoside, nucleotide, oligonucleotide, or of a hydroxyl moiety of a conjugate of a nucleoside, nucleotide, or oligonucleotide; c is 0 or 1; a is an integer from 1 to 3; R ５ is O-R ６ or H; R ８ , R ９ and R １０ Each of the is independently O-R ６ or H; R ６ is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; With the proviso that the pseudo solid-phase protecting group according to formula II-a has exactly two or exactly three O-R ６ and However, R ５ , R ８ , R ９ and R １０ 2. The pseudo solid phase protecting group of claim 1, represented by the formula: with the further proviso that the total of all carbon atoms contained in the moiety is greater than 23 and less than 200.

4. a is 1, R ５ is O-R ６ If R ９ The pseudo solid-phase protecting group according to claim 3, wherein

5. Formulas III to VII: 【Chemistry 3】 (In formula III, ＊ indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 1 to 12; R ６ each independently is a C12 to C40 aliphatic hydrocarbon group; 【Chemistry 4】 (In formula IV, ＊ indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 1 to 12; R ６ each of which is independently a C8 to C40 aliphatic hydrocarbon group; 【Chemistry 5】 (In formula V, ＊ indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 2 to 12; R ６ each independently is a C12 to C40 aliphatic hydrocarbon group; 【Chemistry 6】 (In formula VI, ＊ indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 2 to 12; R ６ each of which is independently a C8 to C40 aliphatic hydrocarbon group; 【Chemistry 7】 (In formula VII, ＊ indicates the point of attachment of the protected hydroxyl moiety to the oxygen atom; c is 0 or 1; a is an integer from 1 to 12; R ６ Each of the following is independently a C12 to C40 aliphatic hydrocarbon group:

6. The pseudo solid-phase protecting group of claim 1, wherein c is 1.

7. The pseudo solid-phase protecting group according to claim 1 , wherein c is 0.

8. Formula I: 【Chemistry 8】 (In the formula: R １ is a protecting group; X is independently at each occurrence O or N; n is an integer equal to or greater than 0; Z is independently at each occurrence an electron withdrawing group or H; Y is independently for each repeat unit n, absent, O or S; Each of the nucleosides x0 to xn may be the same or different; CA is a capping moiety or a single bond; c is 0 or 1; a is an integer from 1 to 12; R ３ is H; R ５ is O-R ６ or H, R ６ is a C8 to C40 aliphatic hydrocarbon group; R ４ Each of the is independently O-R ６ and R ６ is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; b is 1 to 3; However, when b is 1, R ３ and R ５ is not H; In addition, however, the existing R ３ , R ４ and R ５ with the further proviso that the sum of all carbon atoms contained in the moiety is greater than 23 and less than 200.

9. 9. The compound of claim 8, wherein a is an integer from 1 to 3 and b is an integer from 1 to 2.

10. Formula Ib: 【Chemistry 9】 (In the formula: R １ is a protecting group; X is independently at each occurrence O or N; n is an integer equal to or greater than 0; Z is independently at each occurrence H or an electron withdrawing group; Y is independently for each repeat unit n, absent, O or S; Each of the nucleosides x0 to xn may be the same or different; CA is a capping moiety or a single bond; c is 0 or 1; a is an integer from 1 to 3; R ５ is O-R ６ or H; R ８ , R ９ and R １０ Each of the is independently O-R ６ or H; R ６ is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; With the proviso that the compound according to formula Ib has exactly two or exactly three O-R ６ and However, R ５ , R ８ , R ９ and R １０ 9. The compound of claim 8, wherein the total of all carbon atoms contained in the moiety is greater than 23 and less than 200.

11. a is 1, R ５ is O-R ６ If R ９ The compound of claim 10, wherein

12. Formulas VIII to XII: 【Chemistry 10】 (In Formula VIII, c is 0 or 1; a is an integer from 1 to 12; R ６ are each independently a C12 to C40 aliphatic hydrocarbon group; R １ is a protecting group; X is independently at each position O or N; n is an integer equal to or greater than 0; Z is independently at each position H or an electron withdrawing group; Y is independently absent or is O or S for each repeat unit n; each of the nucleosides x0 through xn can be the same or different; CA is a capping moiety or a single bond); 【Chemistry 11】 In Formula IX, c is 0 or 1; a is an integer from 1 to 12; R ６ are each independently a C8 to C40 aliphatic hydrocarbon group; R １ is a protecting group; X is independently at each position O or N; n is an integer equal to or greater than 0; Z is independently at each position H or an electron withdrawing group; Y is independently absent or O or S for each repeat unit n; Each of the amino acids x0 to xn is the same or different; CA is a capping moiety or a single bond; 【Chemistry 12】 (In formula X, c is 0 or 1; a is an integer from 2 to 12; R ６ are each independently a C12 to C40 aliphatic hydrocarbon group; R １ is a protecting group; X is independently at each position O or N; n is an integer equal to or greater than 0; Z is independently at each position H or an electron withdrawing group; Y is independently absent or is O or S for each repeat unit n; each of the nucleosides x0 through xn can be the same or different; CA is a capping moiety or a single bond); 【Chemistry 13】 In formula XI, c is 0 or 1; a is an integer from 2 to 12; R ６ are each independently a C8 to C40 aliphatic hydrocarbon group; R １ is a protecting group; X is independently at each position O or N; n is equal to 0; and is a larger integer; Z is independently at each position H or an electron withdrawing group; Y is independently absent or is O or S for each repeat unit n; each of the nucleosides x0 through xn can be the same or different; CA is a capping moiety or a single bond; and 【Chemistry 14】 (In Formula XII, c is 0 or 1; a is an integer from 1 to 12; R ６ each independently represents a C12 to C40 aliphatic hydrocarbon group optionally containing one or more heteroatoms; R １ is a protecting group; X is independently at each position, O or N; n is an integer equal to or greater than 0; Z is independently at each position, H or an electron withdrawing group; Y is independently absent or is O or S for each repeat unit n; each of the nucleosides x0 through xn can be the same or different; and CA is a capping moiety or a single bond.

13. The compound of claim 8 , wherein c is 1.

14. The compound of claim 8 , wherein c is 0.

15. A method for synthesizing an oligonucleotide comprising steps a) to f): a) obtaining a nucleoside, a nucleotide, an oligonucleotide, or a conjugate of a nucleoside, a nucleotide, or an oligonucleotide, which is bound to a pseudo solid-phase protecting group according to claim 1, the nucleoside, the nucleotide, the oligonucleotide, or the conjugate thereof being bound to a protecting group R １ including a backbone hydroxyl or amine moiety protected by b) Protecting group R １ from the compound obtained in the previous step, thereby generating a free backbone hydroxyl or amine group; c) reacting the free backbone hydroxyl or amine group generated in step b) with a phosphorus moiety of a nucleoside or oligonucleotide component, the component being protected by a protecting group R １ whereby a covalent linkage is created between the oxygen or nitrogen atom of the free backbone hydroxyl or amine group and the phosphorus atom of the moiety; d) optionally modifying the phosphorus moiety; e) optionally repeating the sequence of steps b) to c) or b) to d); and f) cleaving the oligonucleotide from the pseudo solid phase protecting group.

16. the phosphorus moiety of the nucleoside or oligonucleotide building block used in step c) is a phosphorus(III) moiety; the method includes a step d) of oxidizing or sulfurizing the phosphorus(III) atom to a phosphorus(V) atom, thereby creating an internucleoside linkage of the desired type; and The method according to claim 15, wherein step e) comprises optionally repeating the sequence of steps b) to d) or repeating the sequence of steps b) and c) before carrying out step d).

17. 16. The method of claim 15, wherein step c) comprises reacting the free backbone hydroxyl group resulting from step b) with a phosphoramidite nucleoside or oligonucleotide component, thereby forming a phosphite triester bond, and step d) involves oxidizing or sulfurizing the phosphite triester bond.

18. 16. The method of claim 15, wherein step f) comprises incubating the conjugate of the pseudo solid phase protecting group and the oligonucleotide in a solution containing a base, said base being selected from the group consisting of ammonia, C1-C6-alkylamines, and a source of hydroxide ions.

19. The following step g):

16. The method of claim 15, further comprising the step of: g) isolating the cleaved oligonucleotide from the support.

20. 9. A method for preparing a compound according to claim 8, wherein c is 1, comprising the steps of: i) to iii) i) a first free backbone hydroxyl moiety, as well as a protecting group R １ and optionally R １ obtaining a nucleoside or oligonucleotide containing a second backbone hydroxyl or amine moiety protected by an additional protecting group orthogonal to the ii) reacting the first free hydroxyl moiety with an activated carbonic acid derivative; and iii) reacting the product of step ii) with a compound of formula XIII 【Chemistry 15】 (In the formula: a is an integer from 1 to 12; R ３ is H; R ５ is O-R ６ or H, R ６ is a C8 to C40 aliphatic hydrocarbon group; R ４ Each of the is independently O-R ６ and R ６ is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; b is 1 to 3; However, when b is 1, R ３ and R ５ Provided that at least one of is not H. ； In addition, however, the existing R ３ , R ４ and R ５ with the further proviso that the sum of all carbon atoms contained in the moiety is greater than 23 and less than 200.

21. 9. A method for producing a compound according to claim 8, wherein c is 0, comprising the following steps I) and II): I) a first free backbone hydroxyl moiety, as well as a protecting group R １ and optionally R １ obtaining a nucleoside, nucleotide, oligonucleotide, or conjugate thereof, that includes a second backbone hydroxyl or amine moiety protected by an additional protecting group orthogonal to II) an oxygen atom of the first free main chain hydroxyl moiety of the compound obtained in step I) and R Ｌ is bonded to the carboxyl or carbonyl carbon atom of formula XIV: 【Chemistry 16】 (In Formula XIV: R Ｌ is selected from the group consisting of -OH and a leaving group capable of being replaced by a hydroxyl moiety during ester bond formation; a is an integer from 1 to 12; R ３ is H; R ５ is O-R ６ or H, R ６ is a C8 to C40 aliphatic hydrocarbon group; R ４ Each of the is independently O-R ６ and R ６ is, in each occurrence, independently a C8 to C40 aliphatic hydrocarbon group; b is 1 to 3; However, when b is 1, R ３ and R ５ is not H; In addition, however, the existing R ３ , R ４ and R ５ with the further proviso that the sum of all carbon atoms contained in the moiety is greater than 23 and less than 200.

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