Methods of chemical synthesis of oligonucleotides

Abstract

Methods for the synthesis of oligonucleotides and related compounds supported on a solid support. The method provides a process for the preparation of an oligonucleotide, comprising the steps of: a)

Description

Summary The invention relates to methods for the synthesis of oligonucleotides and oligonucleotide-like compounds, and to novel compounds obtainable thereby. Background of the Invention Solid-phase synthesis (SPS) is a common method for preparing oligonucleotides for both research and industrial manufacture. In SPS, oligonucleotides are assembled chemically on a solid support in a stepwise manner where repetitive cycles of coupling and deprotection reactions are carried out and are followed by washing steps. Although the stepwise approach has proven effective, its efficiency often deteriorates as the chain gets longer. This serious problem is due to the structurally related deletion, termination and modification sequences that are generated at each synthetic reaction step, which could eventually drive the whole process to failure. Despite its commercial success at a production scale for smaller oligonucleotides, it also produces significant levels of solvent and waste reagents. There is a high demand for synthetic nucleic acid oligomers in molecular biology and biomedical research and development. Synthetic nucleic acid oligomers (DNA, RNA or their analogues) are mainly prepared using column-based synthesizers. Particularly important and widespread applications for synthetic nucleic acid oligomers are primers for the polymerase chain reaction (PCR) {Critical Reviews in Biochemistry and Molecular Biology 26 (3 / 4), 301 -334, 1991) and the sequencing method according to Sanger {Proc. Nat. Acad. Sci. 74, 5463-5467, 1977). Another two fields of application are the production of microarrays or biochips from oligonucleotide probes and the preparation of interfering RNA (iRNA or RNAi) for the modulation of gene expression in target cells. The aforesaid fields of application of molecular biology provide valuable contributions in the development of active compounds, the production of active compounds, combinatorial biosynthesis (antibodies, effectors such as growth factors, neurotransmitters etc.), in biotechnology (e.g., enzyme design, pharming, biological production methods, bioreactors etc.), in molecular medicine in tissue engineering, in the development and application of new materials, in the development and use of diagnostic agents (microarrays, receptors and antibodies, enzyme design etc.) or in environmental engineering (specialized or tailor-made microorganisms, production methods, remediation, sensors etc.). The method according to the invention can thus be employed in all these areas. Prior art US9644225B2 relates to methods for the preparation of synthetic nucleic acids, such as double-stranded nucleic acids, which involves the preparation of a multiplicity of different nucleic acid fragments by solid-phase synthesis, and the joining together of at least two of the multiplicity of the nucleic acid fragments by binding to one another or by covalent linkage. In the methods at least some of the nucleic acid fragments have a high AT content and are used in an increased amount relative to other fragments for the joining step. Summary of the Invention According to a first aspect, the invention relates to a process for the preparation of an oligonucleotide, comprising the steps of: a) providing a solid support, the solid support having attached thereto: i. a first nucleotide fragment having a free hydroxyl group; and ii. a second nucleotide fragment having a phosphoramidite group; and b) coupling the free hydroxyl group of the first nucleotide fragment with the phosphoramidite group of the second nucleotide fragment to form a phosphite group connecting the first and second nucleotide fragments. According to a second aspect, the invention relates to a process for the preparation of an oligonucleotide, comprising the steps of: a) providing a solid support, the solid support having attached thereto: i. a first nucleotide fragment having a free hydroxyl group; and ii. a second nucleotide fragment having a free hydroxyl group; and b) coupling the free hydroxyl group of the first nucleotide fragment and the free hydroxyl group of the second nucleotide fragment with a divalent reagent. Detailed description of the Invention The inventors have improved standard solid phase oligonucleotide process in two ways. The first method is derived from an approach currently used to synthesise a random and diverse range of different oligonucleotides with potential therapeutic and other applications. A second aspect of the invention is the provision of an interchain assembly reaction which allows the synthesis of a wide spectrum of valuable oligonucleotides by chemically driving two or more precursor oligonucleotide chains synthesised on the support to react with each other to form the final oligonucleotide product (Figure 1). The combination of these two aspects allows the generation new oligonucleotide families with potential therapeutic (and other) applications, in an easier, faster, and cheaper reaction scheme. For example, in the methods of this invention, only a single purification step will be required at the end of the synthesis. In comparison with solution phase coupling, fewer steps are contemplated and higher yields are achieved. Most importantly, the inventive process inhibits a serious oligomerisation reaction by having one of the termini anchored to the support during the course of the reaction. In the broadest aspect, the invention relies on a coupling reaction between two oligonucleotide fragments both bound to a support, the first oligonucleotide fragment having a free hydroxyl group; and the second oligonucleotide fragment having a reactive phosphorus group. In contradistinction to known methodologies, both the first and second oligonucleotide fragments are chemically attached to the same support bead (or other particle). Solid supports (also called resins) as meant herein are the insoluble particles, typically 50-200 pm in diameter, to which the oligonucleotide is bound during synthesis. Any known type of solid support may be used in the methods of the invention, but controlled pore glass (CPG) and polystyrene are preferred. Controlled-pore glass is rigid and non-swelling with deep pores in which oligonucleotide synthesis takes place. Glass supports with 500 A (50 nm) pores are mechanically robust and are used routinely in the synthesis of short oligonucleotides. However, synthesis yields fall off dramatically when oligonucleotides more than 40 bases in length are prepared on resins of 500 A pore size. Although large-pore resins are more fragile, 1000 A CPG resin has proved to be satisfactory for the synthesis of oligonucleotides up to 100 bases in length, and 2000 A supports can be used for longer oligonucleotides. Suitable materials and methods for their preparation are described in Bioorganic Chemistry 140 (2023) 106806, which is included herein by reference. In another embodiment, the solid support is a polymer. Highly cross-linked polystyrene beads have the advantage of good moisture exclusion properties and they allow very efficient oligonucleotide synthesis, particularly on small scale (e.g. 40 nmol). Solid supports for oligonucleotide synthesis as used herein typically have a loading of 20-30 pmol of nucleoside per gram. Oligonucleotide synthesis at higher loadings becomes less efficient owing to steric hindrance between adjacent chains attached to the resin; however, polystyrene supports with loadings of up to 350 pmol / g are used in some applications, particularly for short oligonucleotides, and enable the synthesis of large quantities of oligonucleotides. In one embodiment, the first oligonucleotide fragment is synthesised in a conventional, stepwise fashion on the support. The 3’-terminal nucleotide in the form of an 5’-protected, if necessary base protected reactive derivative is covalently coupled either directly or by means of a suitable linker to the support, which is swollen in an organic solvent. The 5’-protective group is removed, and the subsequent protected nucleotides are added in a stepwise fashion. When the desired nucleotide fragment chain length has been obtained, the sidechain protective groups are removed, and the nucleotide is cleaved from the resin, which might be done in separate steps or at the same time. Any suitable nucleotide protection methodology may be employed. A preferred coupling strategy is based on the use of nucleoside building blocks having two different 5'-hydroxy function protecting groups (and optionally matching side-chain protective groups) which are mutually orthogonal. “Orthogonal” as used herein means that a first protecting group can be removed under conditions which do not remove a second protecting group. A preferred first 5'-hydroxy protecting group is selected from acid-labile protecting groups. “Acid-labile protecting group” as used herein refers to a protecting group that can be removed under acidic conditions, either protonic or Lewis-acid. Preferred first protecting groups are those based on a trityl (triphenylmethyl) group. Especially preferred first protecting groups are shown in Scheme 1 below. 1 R a: a R" aa H 2 3 R^R^R^CO^ 4 R^R^R^GQH^ Scheme 1 A particularly preferred first protecting group is dimethoxytrityl (DMTr), having structure 3 above. 5 A preferred second 5'-hydroxy protecting group is selected from acid-stable protecting groups. Such protecting groups are labile under non-acidic conditions, such as basic conditions, reducing or oxidizing conditions, or photolysis. A preferred class of second protecting group is acyl protecting groups. Simple alkyl acyl groups (such as acetyl or propionyl), and benzoyl groups may be employed in 10 this context. Carbonate protecting groups can also be employed in this context. Preferred carbamate protecting groups are shown in Scheme 2 below (B represents nucleobase). HO R - isobutyl R - p-nitrophenyl R = .ophenylazophenyl R = tluofen-9-meihy! R = p-chloraphenyl R = 2-trimethylsilytethyi R = S-phenylsulfonyiethyi R - o-nitrophenyl Scheme 2 15 In certain embodiments, a third protecting group may be used that is mutually orthogonal to both the first and second and protecting groups. Several protocols exist for the elaboration of oligonucleotides on solid supports. The phosphoramidite method developed by Caruthers in the 1980s is the preferred method for use with the present invention (see for example Caruthers MH etal. Methods Enzymol. 1987;154:287-313). The sequence is outlined in Scheme 3. Addition of further nucleotides Scheme 3 Referring to scheme 3 above, a first nucleotide is attached at the 3’-position to a solid support (0) via a succinate linker. This is protected in the 5’-position with a dimethoxytrityl (DMTr) group (5). The 5’-position is deprotected under acidic conditions to give (6) having a free 5’-hydroxyl group. (6) is coupled with phosphoramidite (7), again protected at the 5’-position with a DMTr group. Coupled dinucleotide phosphite (8) is the immediate product. This is oxidized to phosphate (9) under appropriate conditions. Thereafter, product (9) may be deprotected and coupled with further nucleotides to give the desired oligonucleotide fragment. As an alternative to oxidation, dinucleotide phosphite (8) may be treated with a sulfurization agent such as 3-(Dimethylaminomethylidene)amino-3H-1,2,4-dithiazole-3-thione (DDTT) or 3 / - / -1,2-benzodithiol-3-one 1,1-dioxide (Beaucage reagent) to give oligonucleotide phosphorothioates (OPS). At the conclusion of the synthetic sequence, the desired polynucleotide may be cleaved from the solid support, and the cyanoethyl protection removed from the phosphate groups using concentrated aqueous ammonia. As explained above, the first oligonucleotide fragment is synthesised using a conventional SPS sequence. Similarly, in some embodiments, the second oligonucleotide fragment is also synthesized using a conventional SPS sequence. In one embodiment, the first and second oligonucleotide fragments are synthesized 5 using the same protection groups. In this embodiment, the first and second oligonucleotide fragments are differentiated during the synthetic sequence by the use at one or more coupling steps of a mixture of activated 5’-protected nucleotides, such that a proportion of the growing first and second oligonucleotide fragments differ at one or more amino acid residues. Such an embodiment is illustrated in Scheme 4 10 below. HO-Nu-i Nu^OH +

[10] N(iPr)2 DMTrO-Nu?—O—PZ 2 \

[11] O(CH2)2CN 1. Coupling 2. Oxidize DMTrO-Nu2-Nu1 Nu-|-Nu2-ODMTr

[12] Acid DMTrO—Nu3 N(iPr)2 HO-Nu^Nu-, Nu1-Nu2-OH

[13]

[14] O(CH2)2CN DMTrO—Nu4 N(iPr)2 O—P O(CH2)2CN

[15] 1. Coupling 2. Oxidize Nun-Nu3-Nu2-Nu1 DMTrO-Nu3-Nu2-Nu1 Nu1 Nu2 Nu4-ODMTr

[16] HO-Nu3-Nu2-Nu-| Nu1-Nu2-Nu4-OH

[17] Nu1-Nu2-Nu4-Nun Acid First fragment

[18] Second fragment Scheme 4 Scheme 4 illustrates the principle of simultaneously elaborating two oligonucleotide fragments attached to the same solid support (e.g. bead). Solid support (black circle) is loaded with first nucleotide residue (Nui) which is deprotected to reveal 5’-hydroxyl group ready for a subsequent coupling reaction

[10] . Although two identical nucleotides are shown bound to the support, this is representative of the numerous such nucleotides in fact present. Resin bound nucleotide

[10] is coupled with DMTr-protected nucleotide phosphoramidite

[11] under suitable coupling conditions; many appropriate conditions are known. After this first coupling reaction, the resultant product is oxidized with e.g. with iodine in water / pyridine / tetrahydrofuran (THF). DMTr protected dinucleotide NuiNu2is bound to solid support

[12] , and is deprotected under usual conditions (3% trichloroacetic acid in dichloromethane) to give resin-bound dinucleotide

[13] having free 5’-hydroxyl groups. At this point, all dinucleotides attached to the resin are identical. A critical step occurs when resin-bound dinucleotide

[13] is further extended. Instead of being coupled with a single protected nucleotide as usual, resin-bound dinucleotide

[13] is reacted with a mixture of two different DMTr protected nucleotide phosphoramidites,

[14] and

[15] . The exact ratio of the two DMTr protected nucleotide phosphoramidites

[14] and

[15] will depend on the nature of the nucleotide acid residues Nus and Nu4. At the completion of this step, the resin will now have bound two different trinucleotide chains, NU1NU2NU3 and NU1NU2NU4, protected at the N terminus with DMTr groups

[16] . Although in Scheme 4 the coupling reaction of DMTr protected nucleotide phosphoramidites

[14] and

[15] occurs in a single step, it is also contemplated that these couplings could occur sequentially. For example, resin-bound dinucleotide

[13] in some embodiments is coupled with DMTr protected nucleotide phosphoramidites

[14] in a sub-stoichiometric amount, i.e. an amount insufficient to completely react with all free amine groups of resin-bound dinucleotide

[13] , and then subsequently with DMTr protected nucleotide phosphoramidite

[15] to give

[16] .

[17] is treated under usual DMTr deprotection conditions to give a solid support bearing two different trinucleotides

[17] , At this point, the solid support is bound to two different trinucleotides, namely the first and second nucleotide fragments. These may be elaborated further, i.e. coupled with one or more further nucleotides, to provide resin support bearing fully elaborated first nucleotide fragment and second nucleotide fragment

[18] . Although in Scheme 4 above, the first nucleotide fragment and second nucleotide fragment differ at a specific nucleotide residue, it will be apparent to the skilled person that the first nucleotide fragment and second nucleotide fragment could be differentiated at any point during oligonucleotide synthesis, i.e. differ at any residue in the nucleotide sequence. Similarly, although in Scheme 4 above, although the first nucleotide fragment and second nucleotide fragment differ at a nucleotide residue, it will be understood that the first nucleotide fragment and second nucleotide fragment may differ at more than one residue, such as two, three, four or more residues. Further, although Scheme 4 above shows two differentiated nucleotide fragments, it is within the scope of the invention that three or more nucleotide fragments are synthesized, attached to the same resin e.g. solid support. In an alternative embodiment, first and second nucleotide fragments may be synthesized sequentially using an orthogonal protecting group sequence as shown in Scheme 5 below. N( / Pr)2 f. |L CN DMTrO-Nu1-^-Nu2-OTBDMS—DMTrO-Nu1-^^-Nu2-OH + TBDMSO-Nu3-OX

[19]

[20]

[21] 1. Coupling 2. Oxidation DMTrO-Nu! Nu2-Nu3-OTBDMS

[22] DMTrO-Nu! -^-Nu2-Nu3-[...]-Nun-OTBDMS

[23] N( / Pr)2 H+ XPX ,CN 1. Coupling --► HO-Nu1-^B-Nu2-Nu3-[...]-Nu„-OTBDMS + DMTrO-Nu4-O O V 1 ° n * 2. Oxidation

[24]

[25] DMTrO- Nu4-Nu-j -^-Nu2-Nu3-[...]-Nun-OTBDMS

[26] DMTrO-Nun.-[...]-Nu4-Nu1J^|Nu2-Nu3-[...]-Nun-OTBDMS

[27] Nucleotide Fragment 1 Nucleotide Fragment 2 Scheme 5 Referring to Scheme 5 above, solid support (black circle) is initially loaded with a mixture of 5’-DMTr protected and 5’-tert butyl dimethyl silyl (TBDMS) protected nucleotides

[19] . Again, it will be apparent to the skilled person that each bead is bound to a large number of such nucleotides, rather than the two depicted. Treatment of

[19] with a source of fluoride ion selectively removes the TBDMS group and unmasks the free 5’ hydroxyl group of nucleotide Nu2

[20] . This is coupled with 5’ TBDMS protected nucleotide phosphoramidite AA3

[21] under suitable coupling conditions, followed by oxidation with iodine. At this stage, the solid support is coupled to TBDMS protected dinucleotide NU2NU3 and 5’-TBDMS protected nucleotide Nui

[22] , Subsequent elaboration of the 5’-TBDMS -protected nucleotide fragment via a conventional deprotection / coupling / oxidation sequence gives intermediate

[23] . Treatment with acid removes the 5’-DMTr protecting group from support-bound nucleotide Nui, which may be coupled in a conventional manner with 5’-DMTr protected nucleotide

[25] to give

[26] . Subsequent elaboration of the 5’-DMTr-protected nucleotide fragment via a conventional deprotection / coupling sequence gives intermediate support coupled to elaborated fragments 1 and 2 (first and second nucleotide fragments)

[27] , Coupling Reaction on Solid Support A further aspect of the invention relates to a method of coupling at least two nucleotide fragments which are tethered to the same solid support. Such nucleotide fragments may be (and in some embodiments are) prepared according to the methods hereinbefore described, they need not be, and the on-support coupling aspect is a separate embodiment which finds utility independent of the methods used to attach such nucleotide fragments to the resin. In this aspect, the invention relates to a process for the preparation of an oligonucleotide or oligonucleotide-like compound, comprising the steps of: a) providing a support, the support having attached thereto: i. a first nucleotide fragment having a first reactive group; and ii. a second nucleotide fragment having a second reactive group; b) coupling the first reactive group of the first nucleotide fragment with the second reactive group of the second nucleotide fragment to form a bond. As used herein, the term “first reactive group” and “second reactive group” mean that the first and second reactive groups are capable under suitable conditions of reacting to form a bond. Such a bond may be a direct chemical bond (e.g. a covalent bond), or may involve a moiety that is linked to the first and second reactive groups to form a linker. Non-limiting examples of such first and second reactive groups and the bonds formed are shown in Table 1 below. First reactive group Second reactive group Bond Amine (-NHR; R=e.g. H, Alkyl) Carboxyl group (-COX; X=e.g. halogen, OH, OAlkyl) Amide -NHRCO- Alcohol (-OH) Carboxyl group (-COX; X=e.g. halogen, OH, OAlkyl), phosphoramidite Ester -OCO- Phosphite Thiol (-SH) Thiol (-SH) Disulphide Alkene Alkene C-C bond Alcohol (-OH) Alcohol (-OH) Ester Table 1 In the examples provided in Table 1, suitable reaction conditions will be apparent to the skilled person. For example, reaction of an amine with a carboxylic acid can be effected with any one of the many known coupling reagents, e.g. carbodiimide reagents to form an amide bond. Similarly, many appropriate conditions are known for formation of an ester bond from an alcohol and a carboxylic acid (or active derivative thereof). Coupling of thiol groups to form disulphide groups can be achieved under oxidizing conditions, and alcohols may be coupled under e.g. dehydrating conditions. A C-C bond can be formed using a metathesis crosslinking approach currently used in stapling strategy. In addition to formation of bonds directly between the first and second reactive groups, it is also contemplated that the first and second reactive groups may be bound via a linker. “Linker” as used herein refers to a chemical moiety capable of reacting with both the first reactive group and the second reactive group, and will usually be a bivalent entity. Suitable linkers include dicarboxylic acids (including oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid and suberic acid), diols, diamines, and amino acids. In some embodiments, the first reactive group is a hydroxyl group, such as a primary, secondary or tertiary hydroxyl group. This hydroxyl group in some embodiments is the 5’ hydroxyl group of a nucleotide, such as the 5’ hydroxyl group of the terminal nucleotide of the In some embodiments, the first reactive group is an amine. Suitably, the amine group may be provided by the nucleobase of any nucleotide present on the first nucleotide fragment. Adenine, guanine and cytosine are preferred examples of nucleotides with an amine group; however, it is contemplated that any non-naturally-occurring nucleotide bearing an amine group on the side chain can fulfil this role. In some embodiments, the second reactive group is a phosphoramidite. Suitably, the phosphoramidite group may be attached to the 3’ position of the nucleotide present on the second nucleotide fragment. Coupling of the first and second nucleotide fragments is effected whilst both fragments are bound to the solid support. This confers unique advantages to the inventive methods in terms of regioselectivity and chemoselectivity; further, as both nucleotide fragments are bound to the same support, the local relative concentration is high, and the coupling reaction generally proceeds quickly and in high yield. In some embodiments, the first reactive group is a hydroxyl group which is 5’ hydroxyl group of the terminal nucleotide of the first nucleotide fragment, and the second reactive group is a phosphoramidite group which is connected to the 3’ position of the terminal nucleotide of the second nucleotide fragment. It will be understood that the terms “first,” “second” etc serve merely to distinguish the nucleotide fragments from one another and have no further significance; e.g. the first nucleotide fragment could equally comprise a phosphoramidite group, and the second nucleotide fragment a hydroxyl group. A generalised scheme illustrating the coupling of a first nucleotide fragment and a second nucleotide fragment is shown in Scheme 6.

[28]

[29] Scheme 6 Referring to Scheme 6, “Naa” represents an unspecified nucleotide, n, m, p and q are natural numbers (including zero). Structure

[28] shows solid support (black circle) 5 bound to first (upper) and second (lower) nucleotide fragment. The first nucleotide fragment contains a phosphoramidite group at the 3’ position of the terminal nucleotide. The second nucleotide fragment contains a free 5’ hydroxyl group on the terminal nucleotide. Coupling of the phosphoramidite group and the free 5’ hydroxyl group is achieved under standard conditions (tetrazole in acetonitrile) to give

[29] . 10 The coupled nucleotide may be oxidized and cleaved from the support under standard conditions (which will depend on the nature of the support) to yield oligonucleotide

[30] . [Naa]n

[30] During synthetic sequences according to this aspect of the invention, it may be necessary or convenient to protect nitrogenous base groups not participating in the coupling reaction described above. Methods of protecting such groups will be known to those skilled in the art, and are described for example in Sonveaux, E. (1994). 5 Protecting Groups in Oligonucleotide Synthesis. In: Protocols for Oligonucleotide Conjugates. Methods in Molecular Biology, vol 26. Humana Press. Although the structures made available for the first time by means of the inventive methodology are referred to as “oligonucleotides,” they may designated “modified oligonucleotides” as they may include non-natural nucleotide residues and / or linking 10 groups. In one aspect, the invention relates to a process wherein a first oligonucleotide fragment is attached to the solid support at the 3’ terminus, optionally via a linker, according to a conventional methodology for solid phase oligonucleotide synthesis. The second oligonucleotide fragment meanwhile is attached to the solid support at a 15 position other than the 3’ terminus; the 3’ terminus is present as a protected hydroxyl group which can be activated as a phosphoramidite in order to couple with the 5’ terminus of the first oligonucleotide fragment. Such a scheme is shown in Scheme 7 below.

[35]

[36] Scheme 7 Xi, X2 = protecting groups; base = nucleobase (independently selected); Naa = independently selected nucleotide reside; n, m = integers; DMTr = dimethoxytrityl. 5 Referring to Scheme 7 above,

[31] represents a solid support (black disc) having attached first (lower) and second (upper) nucleoside residues. First nucleoside residue is attached at the 3’ position via a linker (not shown); the 5’ hydroxyl group is protected with a DMTr group. Second nucleoside residue is attached via a linker (not shown) at a position other than the 3’ position, e.g. via an amino group present on the nucleobase. 3’ and 5’ hydroxyl groups of the second nucleoside are protected with protecting groups Xi and X2 which are orthogonal with respect to one another, and also with respect to the DMTr group present on the first nucleoside residue. In a first sequence, the first nucleoside residue is deprotected at the 5’ position and elaborated using an orthodox phosphoramidite coupling / oxidation sequence illustrated in Scheme 3 above. Once the desired nucleotide sequence has been assembled

[32] , the second nucleotide sequence is constructed. Protecting group X2 is removed, and using phosphoramidate chemistry, the second nucleotide sequence is constructed. This will differ from the sequence used to assemble the first nucleotide sequence, because the conditions for removal of the protecting group X2 will differ from those used to remove the DMTr group. The coupling and oxidation steps will be identical. Once the desired nucleotide sequence has been assembled the solid support is attached to two fully elaborated nucleotide sequences

[33] . First and second nucleotide sequences are coupled whilst still attached to the solid support as described hereinafter. Protecting group Xi is removed from the 3’ hydroxyl and reacted with 2-cyanoethyl N,N,N',N'-tetraisopropylphosphorodiamidite. The terminal 5’ hydroxyl group of the first nucleotide chain is deprotected under mild acidic conditions to give

[34] , the precursor to the intramolecular coupling reaction that is unique to this invention. Under standard phosphoramidite coupling conditions (acetonitrile / azole catalyst), first and second nucleotide sequences are coupled together to give

[35] . Oxidation (e.g. with iodine or hydrogen peroxide) and release from the solid support gives desired oligonucleotide

[36] . Although the sequence shown in Scheme 7 above employs 2-deoxyribonucleosides, the skilled person will appreciate that the methodology is equally applicable to ribonucleosides. In fact, the use of ribonucleosides offers the possibility of tethering the nucleoside to the solid support via the 2’-hydroxyl group as illustrated below

[37] ,

[37] Xi, X2, and X3 are independently selected protecting groups. In an alternative embodiment, the second nucleotide residue may be attached to the solid support via an amine group of one of the nucleobases. Such an arrangement is 5 shown in Scheme 8.

[40] Scheme 8 = solid support X1, X2 = independently selected protecting groups 10 Linker = divalent linking group. Referring to Scheme 8 above, illustrated are three preferred embodiments wherein nucleosides are attached to the solid support via the amine group of the nucleobase. Preferred embodiments include those based on deoxyadenosine

[38] , deoxycytidine

[39] and deoxyguanosine

[40] . Suitable linkers for use in attaching the nucleosides and nucleotide fragments to the solid support include succinyl and -O-hydroquinone diacetyl. Methods are described in Current Protocols in Nucleic Acid Chemistry (2000) 3.2.1-3.2.23 (John Wiley &Sons, Inc.). In a further aspect, the inventive methods described herein may be used in the preparation of partial modified inverso and / or retro inverso nucleotides. A generalised sequence is shown in Scheme 9. Scheme 9 Referring to Scheme 9, according to the methodology hereinbefore described, resin support (black circle) coupled to 5’-terminus DMTr protected first nucleotide fragment [Naa]n and 5’-terminus DMTr protected second nucleotide fragment [Naa]m

[41] is prepared. DMTr protection is removed under mild acidic conditions to give

[42] , which is reacted with dicarboxylic acid

[43] or an anhydride thereof (q representing any natural non-zero number) under ester-bond forming conditions. First and second nucleotide fragments are connected at the 5’-termini

[44] , and the coupled product is cleaved from the resin support to give partial modified inverso nucleotide

[45] . The sequence shown in Scheme 9 employs a dicarboxylic acid; however, any difunctional reagent could be used to form an alternative linker between the two nucleotide fragments, such as a dihalide etc. This aspect of the invention (i.e. that illustrated in Scheme 9) in a preferred aspect may be used to prepare symmetrical partial modified inverso and / or retro inverso 5 nucleotide in which the first and second nucleotide fragments are identical. However, the skilled person will appreciate that non-identical (different) nucleotide fragments may be linked using a similar methodology. The partial modified inverso and / or retro inverso nucleotides obtainable by the methodologies described form a further aspect of the invention. 10

Claims

1. A process for the preparation of an oligonucleotide, comprising the steps of:a) providing a solid support, the solid support having attached thereto:i. a first nucleotide fragment having a free hydroxyl group; andii. a second nucleotide fragment having a phosphoramidite group; andb) coupling the free hydroxyl group of the first nucleotide fragment with the phosphoramidite group of the second nucleotide fragment to form a phosphite group connecting the first and second nucleotide fragments.

2. A process according to claim 1 wherein the free hydroxyl group of the first nucleotide fragment is present as the 5’ hydroxyl group of a nucleoside residue.

3. A process according to any preceding claim wherein the phosphoramidite group of the second nucleotide fragment is present at the 3’ position of a nucleotide residue.

4. A process according to any preceding claim wherein the first nucleotide fragment is protected at the 5’ terminus with a first protecting group, and the second nucleotide fragment is protected at the 5’-terminus with a second protecting group, wherein the first and second protecting groups are orthogonal.

5. A process according to claim 4 wherein the first protecting groups is selected from a trityl protecting group, preferably dimethoxytrityl and the second protecting group is selected from a silicon-containing protecting group, preferably tert-butyldimethylsilyl.

6. A process according to claim 4 or 5 wherein the first nucleotide fragment is assembled on the support using a stepwise phosphoramidite nucleotide synthesis protocol.

7. A process according to any of claims 4 to 6 wherein the second nucleotide fragment is assembled on the support using a stepwise phosphoramidite nucleotide synthesis protocol.

8. A process according to claim 2 wherein the free hydroxyl group of the first nucleotide fragment is the 5’-terminus.

9. A process according to any preceding claim wherein the first nucleotide fragment is bound to the support at the 3’ terminus.

10. A process according to any preceding claim wherein the second nucleotide is bound to the support via a linker attached to a primary amine group present on the heterocyclic base of a nucleotide residue.

11. A process according to claim 10 wherein the nucleotide bound to the support has a phosphoramidite group in the 3’ position.

12. A process according to any preceding claim comprising a further step of oxidation of the phosphite group to a phosphate group.

13. A process according to any preceding claim comprising a further step of cleaving the coupled product from the solid support.

14. A process according to claim 13 comprising a further step or steps of deprotecting the product of cleavage to give an oligonucleotide.

15. A process for the preparation of an oligonucleotide, comprising the steps of:a) providing a solid support, the solid support having attached thereto:i. a first nucleotide fragment having a free hydroxyl group; andii. a second nucleotide fragment having a free hydroxyl group; andb) coupling the free hydroxyl group of the first nucleotide fragment and the free hydroxyl group of the second nucleotide fragment with a divalent reagent.

16. A process according to claim 15 wherein the divalent reagent is a dicarboxylic acid.

17. A process according to claim 16 wherein the dicarboxylic acid is selected from oxalic acid, malonic acid, succinic acid, glutaric acid, adipic acid, pimelic acid and suberic acid.

18. A process according to any one of claims 15 to 17 comprising a further step of cleaving the coupled product from the solid support.

19. A process according to claim 18 comprising a further step or steps of deprotecting the product of cleavage to give an oligonucleotide.s

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