3'-o-aminooxymethyl blocked nucleotides and use thereof in enzymatic synthesis of polynucleotides

Abstract

This invention relates to a 3'-O-blocked nucleotide having an aminooxymethyl blocking group. It further relates to an enzymatic method of synthesizing a polynucleotide, using this 3'-O-blocked nucleotide. This invention also pertains to a method for preparing this compound and to nucleotide and nucleoside intermediates obtained during its synthesis. This invention further relates to a kit for synthesizing a polynucleotide comprising one or more vials of synthesis reagents, at least one of which contains an effective amount of this 3'-O- blocked nucleotide or a precursor thereof.

Description

[0001] 3'-O-AMINOOXYMETHYL BLOCKED NUCLEOTIDES AND USE THEREOF IN ENZYMATIC SYNTHESIS OF POLYNUCLEOTIDES

[0002] TECHNICAL FIELD

[0003] This invention relates to a 3’-O-blocked nucleotide having an aminooxy methyl blocking group. It further relates to an enzymatic method of synthesizing a polynucleotide, using this 3’-O-blocked nucleotide. This invention also pertains to a method for preparing this compound and to nucleotide and nucleoside intermediates obtained during its synthesis. This invention further relates to a kit for synthesizing a polynucleotide comprising one or more vials of synthesis reagents, at least one of which contains an effective amount of this 3'-O-blocked nucleotide or a precursor thereof.

[0004] BACKGROUND

[0005] Interest in enzymatic approaches to polynucleotide synthesis has increased not only because of increased demand for synthetic polynucleotides in many areas, such as synthetic biology (e.g. synthesis of insulin for the treatment of diabetes), CRISPR-Cas9 applications, and next generation sequencing (such as “sequencing by synthesis” or SBS, and “sequencing by binding” or SBB), but also because of the limitations of chemical approaches to polynucleotide synthesis, such as upper limits on product length, the use of moisturesensitive monomers and the use of environmentally unfriendly solvents (Jensen et al, Biochemistry, 57: 1821-1832, 2018).

[0006] Currently, most enzymatic approaches for both DNA and RNA synthesis employ polymerases that are used to implement repeated cycles of elongating an initiator polynucleotide by coupling with a 3’-O-protected nucleotide, such as 3'-O-amino nucleotide, then deprotecting the protected growing strand thus obtained, until an elongated polynucleotide of the desired sequence is obtained. For example, TdT variants have been engineered (WO96 / 07669) that efficiently incorporate into growing polynucleotide strands reversibly protected 3’-O-amino nucleoside triphosphate monomers developed by Steven Benner (US-10,752,887, W02020 / 099451, US-7,544,794, US-8,034,923, US-8,212,020, US-10,472,383; and Hutter et al, 2010). The utility of the -ONH2 chemical moiety as a protecting group rests in its ability to reversibly mask the 3'-OH group on 2-deoxyribose or ribose nucleoside. Additionally, the small size of the -ONH2 chemical moiety makes it a better blocking group for enzymatic incorporation, e.g. polymerases, compared to native 3'- OH nucleotides. However, several valuable modifications for DNA backbone, such as 2'-O-(2- methoxyethyl), 2’-O-methyl or LNA, have proven to be incompatible with 3'-ONH2 chemistry. In addition, this blocking group is not suitable for protecting the 3'-0 group of ribonucleotides, which makes mixed DNA and RNA synthesis not affordable with the same 3’-O-cleavable group and deprotection method. It would thus be desirable to find a blocking group orthogonal to 3'-ONH2 and which thus could be cleaved with the same deprotection method.

[0007] In view of the interest in extending the application of enzymatic synthesis of polynucleotides or sequencing applications, it would be desirable to provide a simple solution in order to overcome the above problems and, in particular, which affords obtaining blocked nucleotides in a reduced number of steps and with a higher yield. The blocking group should also allow deprotecting the oligonucleotide under conditions sufficiently mild to leave the nucleobases intact. Finally, it would be desirable to provide a 3’-O-blocking group which can be applied not only to ribonucleotides but also to deoxyribonucleotides and which could allow mixed DNA / RNA synthesis using both this blocking group and the conventional 3’-ONH2 chemistry.

[0008] In this context, the inventors have synthetized nucleotides comprising a 3-0’- aminooxymethyl blocking group. To the best of the inventors’ knowledge, CH2ONH2 has never been implemented as reversible terminator. Rather, the prior art (such as US- 7,544,794) taught away from selecting a blocking group having at least three non-hydrogen atoms, since such a group is thought to be too large to fit into the active site of polymerases.

[0009] So far, 3’-O-CH2ONH2 deoxynucleotides were used for conjugation with peptide molecules and biotin (De, Groaz, & Herdewijn, 2014; De, Groaz, Maiti, et al., 2014). In addition, derivatized 2’-O-CH2ONH2 as Schiff bases was mentioned as a protective group for RNA phosphoramidite synthesis and conjugation with aldehydes or carboxylic acids (Cieslak et al., 2012a; Cieslak et al., 2016; Cieslak et al., 2013). Moreover, 3’-OCH2ONH2 nucleosides have been mentioned by Zavgorodny et al., 1991. However, neither their synthesis nor any possible application were described.

[0010] SUMMARY OF THE INVENTION

[0011] The inventors have found that the reversible terminator of this invention allows overcoming the deficiencies of the NH2 terminator. In particular, the protected nucleotides of this invention may be easily prepared with rather high yield and fewer process steps compared to 3’-O-NH2 nucleotides. Moreover, the -CH2ONH2 blocking group proved to be useful for protecting the 3 ’-OH group of ribonucleotides and to provide blocked modified nucleotides, such as LNA and 2’-0Me deoxyribonucleotides.

[0012] A first object of this invention is thus a compound having the following formula (I) : where:

[0013] B is a nucleobase;

[0014] X is -CH2-O-NH2;

[0015] R9 is H, halogen or O-Z where Z is H or alkyl or a methylene group bridging the 2'-0 position and the 4'-C position of the compound of formula (I);

[0016] Rio is -PO3-Y or -P(S)O2-Y, where Y is either -PO3-PO3; and wherein the nucleobase optionally bears fluorescent labels, possibly attached thereto via linkers.

[0017] This invention also pertains to a method for extending a DNA primer by templatedependent synthesis or template-independent synthesis, comprising contacting said primer and optionally said template with a polymerase and a compound of formula (I) above.

[0018] According to another aspect, this invention pertains to a method of synthesizing a polynucleotide, the method comprising the steps of:

[0019] (a) providing initiators which are polynucleotides having each a free 3’-hydroxyl;

[0020] (b) repeating in a reaction mixture, until the polynucleotide is formed, cycles of (i) contacting under elongation conditions the initiators or elongated fragments having free 3’- hydroxyls with a compound of formula (I) above and with a polymerase, so that the initiators or elongated fragments are elongated by incorporation of the compound of formula (I) to form 3’-O-blocked elongated fragments, and (ii) deprotecting the elongated fragments to form elongated fragments having free 3 ’-hydroxyls. Intermediates which may be used for preparing the compound of formula (I) are another aspect of this invention, which is thus also directed to a compound having the following formula (I’) : where :

[0021] B is a nucleobase;

[0022] Re has formula (Ila) or (lib):

[0023] (lib) R

[0024] (Ila)4

[0025] Ri and R2 are each independently alkyl or a protective group, or Ri and R2 form together, with the adjacent nitrogen atom, a heterocycle having one or more cycles which are independently saturated or unsaturated, substituted or unsubstituted;

[0026] R3 and R4 are each independently alkyl;

[0027] Rs is H, -PO3-Y or -P(S)O2-Y where Y is either -PO3-PO3 or a protecting group;

[0028] R7 is H, halogen or O-Z where Z is H or alkyl or a methylene group bridging the 2'-0 position and the 4'-C position of the compound of formula (I) or a -SiYs group where each Y is independently a linear or branched alkyl group having from 1 to 6 carbon atoms or a phenyl group; and wherein the nucleobase optionally bears fluorescent labels, possibly attached thereto via linkers, with the exception of the compounds of formula (I’A):

[0029] wherein B is a nucleobase, especially the compound of formula (I’a):

[0030] This invention further relates to a kit for enzymatic synthesis of a polynucleotide comprising one or more vials of synthesis reagents, at least one of which contains an effective amount of a compound of formula (I) above or a precursor thereof having formula (I’) above.

[0031] In still another aspect, this invention pertains to a method for preparing the compound of Formula (I) above, comprising reacting a compound of Formula (F) above, in one or two steps, with at least one organic or inorganic base, such as: ammonia; a primary amine such as methylamine; O-benzylhydroxylamine; methoxylamine; an ammonium salt such as ammonium fluoride or alkylammonium fluoride; and mixtures thereof.

[0032] BRIEF DESCRIPTION OF THE DRAWINGS

[0033] Figures 1 to 7 show reaction schemes for preparing 3’-O-blocked nucleotides according to this invention.

[0034] Figure 1: 3 ’-OCH2ONH2 thymidine

[0035] Figure 2: 3’-OCH2ONH2 uridine Figure3 : 2’-OMe-3’-OCH2ONH2 thymidine

[0036] Figure 4: LNA S’-OC hON h thymidine

[0037] Figure 5: 3 -OCH2ONH2 deoxy cytidine

[0038] Figure 6: S’-OC hON h deoxy guanosine

[0039] Figure 7: S’-OC hON h deoxy adenosine

[0040] Figure 8 illustrates the steps of the template-free enzymatic method of synthesizing a polynucleotide according to this invention.

[0041] DETAILED DESCRIPTION OF THE INVENTION

[0042] Definitions

[0043] Although polynucleotides typically have at least 100 nucleotide units, “polynucleotide” or “oligonucleotide” are used interchangeably in this description and each mean a linear polymer of nucleotide monomers or analogs thereof. Monomers making up polynucleotides and oligonucleotides are capable of specifically binding to a natural polynucleotide by way of a regular pattern of monomer-to-monomer interactions, such as Watson-Crick type of base pairing, base stacking, Hoogsteen or reverse Hoogsteen types of base pairing, or the like. Such monomers and their intemucleosidic linkages may be naturally occurring or may be analogs thereof, e.g. naturally occurring or non-naturally occurring analogs. Non-naturally occurring analogs may include PNAs, phosphorothioate intemucleosidic linkages, bases containing linking groups permitting the attachment of labels, such as fluorophores, or haptens, and the like. Whenever the use of an oligonucleotide or polynucleotide requires enzymatic processing, such as extension by a polymerase, ligation by a ligase, or the like, one of ordinary skill would understand that oligonucleotides or polynucleotides in those instances would not contain certain analogs of intemucleosidic linkages, sugar moieties, or bases at any or some positions. Polynucleotides typically range in size from a few monomeric units, e.g. 5-40, when they are usually referred to as “oligonucleotides,” to several thousand monomeric units. Unless otherwise noted the terminology and atom numbering conventions will follow those disclosed in Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York, 1999). Thus, "A" denotes deoxyadenosine, "C" denotes deoxycytidine, "G" denotes deoxyguanosine, and "T" denotes thymidine, “I” denotes deoxyinosine, “U” denotes uridine. Usually polynucleotides comprise the four natural nucleosides (e.g. deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine for DNA or their ribose counterparts for RNA) linked by phosphodiester linkages; however, they may also comprise non-natural nucleotide analogs, e.g. including modified bases, sugars, or intemucleosidic linkages. It is clear to those skilled in the art that where an enzyme has specific oligonucleotide or polynucleotide substrate requirements for activity, e.g. single stranded DNA, RNA / DNA duplex, or the like, then selection of appropriate composition for the oligonucleotide or polynucleotide substrates is well within the knowledge of one of ordinary skill, especially with guidance from treatises, such as Sambrook et al, Molecular Cloning, Second Edition (Cold Spring Harbor Laboratory, New York, 1989), and like references. Likewise, the oligonucleotide and polynucleotide may refer to either a single stranded form or a double stranded form (i.e. duplexes of an oligonucleotide or polynucleotide and its respective complement). It will be clear to one of ordinary skill which form or whether both forms are intended from the context of the terms usage.

[0044] The present invention pertains to new 3’-O-blocked nucleotides of formula (I) that will be described hereafter and which may be used in a method for synthesizing a polynucleotide.

[0045] Typically, this method comprises the steps of:

[0046] (a) providing initiators which are polynucleotides having each a free 3 ’-hydroxyl;

[0047] (b) repeating in a reaction mixture, until the polynucleotide is formed, cycles of (i) contacting under elongation conditions the initiators or elongated fragments having free 3 ’-hydroxyls with a compound of formula (I) and with a polymerase, so that the initiators or elongated fragments are elongated by incorporation of the compound of formula (I) to form 3’-O- blocked elongated fragments, and (ii) deprotecting the elongated fragments to form elongated fragments having free 3 ’-hydroxyls.

[0048] According to a preferred embodiment of this invention, the polymerase used in this method is a template-independent polymerase. The method of this invention is then preferably an enzymatic DNA synthesis or an enzymatic RNA synthesis or a mixed enzymatic DNA and RNA synthesis.

[0049] These methods comprise repeated cycles of steps (illustrated in Fig. 8) in which a predetermined 3 ’-0 -protected nucleotide is (i) coupled to a primer (initiator or elongated chain) in each cycle and (ii) deprotected. The general elements of template-free enzymatic synthesis of polynucleotides are described in the following references: WO2019 / 135007; US-5,763,594; and Jensen et al, 2018). In a less preferred embodiment of this invention, the polymerase is a templatedependent polymerase. The method of this invention is then preferably a method for sequencing DNA by synthesis, for example, sequencing-by-synthesis (SBS) and sequencing- by-binding (SBB).

[0050] According to an embodiment, the sequencing-by-synthesis method comprises the steps of:

[0051] (a) providing initiators which are polynucleotides having each a free 3 ’-hydroxyl;

[0052] (b) repeating in a reaction mixture, until the polynucleotide is formed, cycles of (i) contacting under elongation conditions the initiators or elongated fragments having free 3 ’-hydroxyls with a fluorescently labelled 3’-O-CH2-O-NH2 nucleotide and a polymerase, so that the initiators or elongated fragments are elongated by incorporation of a fluorescently labelled 3’-O-CH2-O-NH2 nucleotide to form 3’-O-CH2-O-NH2 elongated fragments, (ii) washing away excess of non-incorporated nucleotides, (iii) reading the fluorescence signal to know the nucleotide incorporated, (iv) removing the fluorescent label and 3’-O-CH2-O-NH2 group to form elongated fragments having free 3 ’-hydroxyls, and (v) repeating steps (i) to (iv) until the end of sequencing.

[0053] According to an embodiment, the sequencing-by -binding method comprises the steps of:

[0054] (a) providing initiators which are polynucleotides having each a free 3 ’-hydroxyl;

[0055] (b) repeating in a reaction mixture, until the polynucleotide is formed, cycles of (i) contacting under elongation conditions the initiators or elongated fragments having free 3 ’-hydroxyls with a 3’-O-CH2-O-NH2 nucleotide and a polymerase, so that the initiators or elongated fragments are elongated by incorporation of 3’ -O-CH2-O-NH2 nucleotide to form 3’-O-CH2- O-NH2 elongated fragments, (ii) contacting under elongation conditions the 3’-O-CH2-O- NH2 elongated fragments with a fluorescently labelled nucleotide and a polymerase, so that the fluorescently labelled nucleotide will be bound in the active site of the polymerase, (iii) washing away unbound fluorescent labelled nucleotides, (iv) reading the fluorescence signal to know the nucleotides bound in the active site, (v) washing away the bound nucleotides, (vi) deprotecting the 3’-O-CH2-O-NH2 polynucleotide, and (vii) repeating steps (i) to (vi) until the end of sequencing. With reference to Figure 8, initiator polynucleotides (100) are provided, for example, attached to solid support (102), which have free 3’-hydroxyl groups (103). To the initiator polynucleotides (100) (or elongated initiator polynucleotides in subsequent cycles) are added a 3’-O-protected-dNTP or 3’-O-protected-rNTP and a polymerase, such as a TdT or variant thereof usually for DNA synthesis (e.g. Ybert et al, WO / 2017 / 216472; Champion et al, WO2019 / 135007) or a polyA polymerase (PAP) or polyU polymerase (PUP) or variant thereof usually for RNA synthesis (e.g. Heinisch et al, W02021 / 018919) under conditions effective for the enzymatic incorporation of the 3’-O-protected-NTP onto the 3’ end of the initiator polynucleotides (100) (or elongated initiator polynucleotides). This reaction produces elongated initiator polynucleotides whose 3 ’-hydroxyls are protected (106). If the elongated sequence is not complete, then another cycle of addition is implemented (108). If the elongated initiator polynucleotide contains a completed sequence, then the 3’-O- protection group may be removed, or deprotected, and the desired sequence may be cleaved from the original initiator polynucleotide (110). Such cleavage may be carried out using any of a variety of single strand cleavage techniques, for example, by inserting a cleavable nucleotide at a predetermined location within the original initiator polynucleotide. An exemplary cleavable nucleotide may be a uracil nucleotide which is cleaved by uracil DNA glycosylase. If the elongated initiator polynucleotide does not contain a completed sequence, then the 3’-O-protection groups are removed by a deprotecting agent to expose free 3’- hydroxyls (103) and the elongated initiator polynucleotides are subjected to another cycle of nucleotide addition and deprotection.

[0056] As used herein, the term “protected” or “blocked” in reference to specified groups, such as, a 3 ’-hydroxyls of a nucleotide or a nucleoside is intended to mean a moiety which is attached covalently to the specified group that prevents a chemical change to the group during a chemical or enzymatic process. Whenever the specified group is a 3’-hydroxyl of a nucleoside triphosphate, or an elongated fragment in which a 3 ’-protected (or blocked)- nucleoside triphosphate has been incorporated, the prevented chemical change is a further, or subsequent, extension of the elongated fragment by an enzymatic coupling reaction.

[0057] As used herein, an “initiator” refers to a short oligonucleotide sequence with a free 3 ’-hydroxyl at its end, which can be further elongated by a polymerase, such as TdT. In one embodiment, the initiator is a DNA initiator. In an alternative embodiment, the initiator is an RNA initiatior. In some embodiments, an initiator possesses between 3 and 100 nucleotides, in particular between 3 and 20 nucleotides. In some embodiments, the initiator is singlestranded. In alternative embodiments, the initiator may be double-stranded. In some embodiments, an initiator oligonucleotide may be attached to a synthesis support by its 5 ’end; and in other embodiments, an initiator oligonucleotide may be attached indirectly to a synthesis support by forming a duplex with a complementary oligonucleotide that is directly attached to the synthesis support, e.g. through a covalent bond. In some embodiments a synthesis support is a solid support which may be a discrete region of a planar solid, or may be a bead.

[0058] In some embodiments, an initiator may comprise a non-nucleic acid compound having a free hydroxyl to which a TdT may couple a 3’-O-protected dNTP, e.g. Baiga, U.S. patent publications US2019 / 0078065 and US2019 / 0078126.

[0059] Synthesis supports to which initiators are attached may comprise polymers, porous or non-porous solids, including beads or microspheres, planar surfaces, such as a glass slide, membrane, or the like. In some embodiments, a solid support, or synthesis support, may comprise magnetic beads, particle-based resins, such as agarose, or the like.

[0060] Synthesis supports include, but are not limited to, soluble supports, such as, polymer supports, including polyethylene glycol (PEG) supports, dendrimer supports and the like; non-swellable solid supports, such as, polystyrene particles; swellable solid supports, such as resins or gels, including agarose. Synthesis supports may also form part of reaction chambers, such as, the filter membrane of a filter plate. Guidance for selecting soluble supports is found in references Bonora et al, Nucleic Acids Research, 212(5): 1213-1217 (1993); Dickerson et al, Chem. Rev. 102: 3325-3344 (2002); Fishman et al, J. Org. Chem, 68: 9843-9846 (2003); Gavert et al, Chem. Rev. 97: 489-509 (1997); Shchepinov et al, Nucleic Acids Research, 25(22): 4447-4454 (1997): and like references. Guidance for selecting solid supports is found in Brown et al, Synlett 1998(8): 817-827; Maeta et al, U.S. patent 9045573; Beaucage and Iyer, Tetrahedron, 48(12): 2223-2311 (1992); and the like. Guidance for attaching oligonucleotides to solid supports is found in Amdt-Jovin et al, Eur. J. Biochem, 54: 411-418 (1975); Ghosh et al, Nucleic Acids Research, 15(13): 5353-5372 (1987); Integrated DNA Technologies, “Strategies for attaching oligonucleotides to solid supports,” 2014(v6); Gokmen et al, Progress in Polymer Science 37: 365-405 (2012); and like references.

[0061] In some embodiments, the solid-phase support will typically be comprised of porous beads or particles in the form of a resin or gel. Numerous materials are suitable as solidphase supports for the synthesis of polynucleotides. As used herein, the term "particle" includes, without limitation, a "microparticle" or "nanoparticle" or "bead" or "microbead" or "microsphere." In some embodiments, a porous resin support derivatized with initiators has average pore diameters of at least 10 nm, or at least 20 nm, or at least 50 nm. In other embodiments, such porous resin support has an average pore diameter in the range of from 10 nm to 500 nm, or in the range of from 50 nm to 500 nm.

[0062] In some embodiments, initiators are attached to planar supports for massively parallel synthesis of oligonucleotides, e.g. via inkjet delivery of reagents, such as described by Horgan et al, International patent publication W02020 / 020608, which is incorporated herein by reference. In some embodiments such planar supports comprise a uniform coating of initiators with protected 3 ’-hydroxyls, wherein, for example, discrete reaction sites may be defined by delivering deprotection solution to discrete locations. In other embodiments, such planar supports comprise an array of discrete reaction sites each containing initiators, which, for example, may be formed on a substrate by photolithographic methods of Brennan, U.S. patent 5474796; Peck et al, U.S. patent 10384189; Indermuhle et al, U.S. patent 10669304; Fixe et al, Materials Research Society Symposium Proceedings. Volume 723, Molecularly Imprinted Materials - Sensors and Other Devices. Symposia (San Francisco, California on April 2-5, 2002); or like references.

[0063] After synthesis is completed polynucleotides with the desired nucleotide sequence may be released from initiators and the solid supports by cleavage. A wide variety of cleavable linkages or cleavable nucleotides may be used for this purpose. In some embodiments, cleaving the desired polynucleotide leaves a natural free 5 ’-hydroxyl on a cleaved strand; however, in alternative embodiments, a cleaving step may leave a moiety, e.g. a 5 ’-phosphate, that may be removed in a subsequent step, e.g. by phosphatase treatment. Cleaving steps may be carried out chemically, thermally, enzymatically or by photochemical methods. In some embodiments, cleavable nucleotides may be nucleotide analogs such as deoxyuridine or 8-oxo-deoxyguanosine that are recognized by specific glycosylases (e.g. uracil deoxyglycosylase followed by endonuclease VIII, and 8-oxoguanine DNA glycosylase, respectively). In some embodiments, cleavage may be accomplished by providing initiators with a deoxyinosine as the penultimate 3’ nucleotide, which may be cleaved by endonuclease V at the 3 ’ end of the initiator leaving a 5 ’ -phosphate on the released polynucleotide, e.g. as taught by Cretan, International patent publication WO / 2020 / 165137. Returning to Fig. 8, in some embodiments, an ordered sequence of nucleotides are coupled to an initiator nucleic acid using a polymerase, such as TdT, in the presence of 3 ’-0 -protected NTPs in each synthesis step. The above method may also include a washing step after each reaction, or extension, step, as well as after each de-protecting step. When the sequence of polynucleotides on a synthesis support includes reverse complementary subsequences, secondary intra-molecular or cross-molecular structures may be created by the formation of hydrogen bonds between the reverse complementary regions. In some embodiments, base protecting moieties for exocyclic amines are selected so that hydrogens of the protected nitrogen cannot participate in hydrogen bonding, thereby preventing the formation of such secondary structures. That is, base protecting moieties may be employed to prevent the formation of hydrogen bonds, such as are formed in normal base pairing, for example, between nucleosides A and T and between G and C. At the end of a synthesis, the base protecting moieties may be removed and the polynucleotide product may be cleaved from the solid support, for example, by cleaving it from its initiator.

[0064] In addition to providing 3’ -O-protected NTP monomers with base protection groups, elongation reactions may be performed at higher temperatures using thermal stable polymerases. For example, athermal stable template-free polymerase having activity above 40°C may be employed; or, in some embodiments, athermal stable template-free polymerase having activity in the range of from 40-85°C may be employed; or, in some embodiments, a thermal stable template-free polymerase having activity in the range of from 40-65 °C may be employed.

[0065] In some embodiments, elongation (or coupling) conditions may include adding solvents to an elongation reaction mixture that inhibit hydrogen bonding or base stacking. Such solvents include water miscible solvents with low dielectric constants, such as dimethyl sulfoxide (DMSO), methanol, and the like. Likewise, in some embodiments, elongation conditions may include the provision of chaotropic agents that include, but are not limited to, n-butanol, ethanol, guanidinium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, urea, and the like. In some embodiments, elongation conditions include the presence of a secondarystructure-suppressing amount of DMSO. In some embodiments, elongation conditions may include the provision of DNA binding proteins that inhibit the formation of secondary structures, wherein such proteins include, but are not limited to, single-stranded binding proteins, helicases, DNA glycolases, and the like.

[0066] The above method may also include one or more capping steps in addition to washing steps after the coupling (or elongation) step. A first capping step may cap, or render inert to further elongations, unreacted 3 ’-OH groups on partially synthesized polynucleotides. Such capping step is usually implemented after a coupling step, and whenever a capping compound is used, it is selected to be unreactive with protection groups of the monomer just coupled to the growing strands. In some embodiments, such capping steps may be implemented by coupling (for example, by a second enzymatic coupling step) a capping compound that renders the partially synthesized polynucleotide incapable of further couplings, e.g. with TdT. Such capping compounds may be a dideoxynucleoside triphosphate.

[0067] The 3’-O-blocked nucleotides of this invention, which may be used in the above methods, are selected from compounds having the following formula (I) : where:

[0068] B is a nucleobase;

[0069] X is -CH2-O-NH2;

[0070] R.9 is H, halogen or O-Z where Z is H or alkyl or a methylene group bridging the 2'-0 position and the 4'-C position of the compound of formula (I);

[0071] Rio is -PO3-Y or -P(S)O2-Y, where Y is either -PO3-PO3, preferably Rio is -PO3-PO3-PO3. and wherein the nucleobase optionally bears fluorescent labels, possibly attached thereto via linkers.

[0072] These nucleotides may be obtained according to methods which are detailed in the following Examples.

[0073] The intermediate compounds obtained during the synthesis of the compound of formula (I) are another object of this invention and correspond to compounds having the following formula (T) :

[0074] O’) where :

[0075] B is a nucleobase;

[0076] Ri and R2 are each independently alkyl or a protective group, or Ri and R2 form together, with the adjacent nitrogen atom, a heterocycle having one or more cycles which are independently saturated or unsaturated, substituted or unsubstituted;

[0077] R3 and R4 are each independently alkyl;

[0078] Rs is H, -PO3-Y or -P(S)O2-Y where Y is either -PO3-PO3 or a protecting group;

[0079] Re has formula (Ila) or (lib):

[0080] (li b) R (Ha)4

[0081] R.7 is H, halogen or O-Z where Z is H or alkyl or a methylene group bridging the 2'-0 position and the 4'-C position of the compound of formula (I) or a -SiYs group where each Y is independently a linear or branched alkyl group having from 1 to 6 carbon atoms or a phenyl group; and wherein the nucleobase optionally bears fluorescent labels, possibly attached thereto via linkers, with the exception of the compounds of formula (I’A): wherein B is a nucleobase, especially the compound of formula (I’a):

[0082]

[0083] Examples of protecting groups that may be comprised within Formula (I’) are: silyl ether groups, e.g., trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl (TBDMS), tertbutyldiphenylsilyl, dimethylphenylsilyl, and diphenylmethylsilyl: benzy l carbonates; trityl, monomethoxytrityl, and dimethoxytrityl; esters, for example, acetate, benzoate, and the like; pixyl; tert-butyloxy carbonyl, 9-fluorenylmethoxy carbonyl (Fmoc); and tetrahydropyranyl,; preferably benzoate or TBDMS .

[0084] The term “alkyl” refers to a saturated, linear or branched aliphatic group such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, or hexyl. Methyl is preferred in this invention for Ri and R2.

[0085] In a preferred embodiment of this invention, the compound of formula (F) is selected from those wherein:

[0086] - Ri and R2 form together, with the adjacent nitrogen atom, the heterocycle of formula (III):

[0087] (III),

[0088] R3 and R4 are both methyl.

[0089] Preferably Rs is -PO3-PO3-PO3.

[0090] In addition, R7 is preferably H, OH or O-Z where Z is a methylene group bridging the 2'-0 position and the 4'-C position of the compound. In a specific embodiment, R7 is O- Z where Z is a methylene group bridging the 2'-0 position and the 4'-C position of the compound of formula (F). For preparing the compound of Formula (I), a compound of Formula (F) is typically reacted, in one or two steps, with at least one organic or inorganic base, such as: ammonia; a primary amine such as methylamine or O-benzylhydroxylamine; methoxylamine; an ammonium salt such as ammonium fluoride or alkylammonium fluoride; and mixtures thereof.

[0091] For instance, in case the compound of Formula (I’) comprises an oxime moiety, i.e. when Rs and R4 are both methyl, conversion of the oxime group into -O-NH2 group may be obtained by reacting the compound of Formula (F) with O-benzylhydroxylamine (BOX). If the compound of Formula (F) is a nucleoside, a phosphorylation of this compound is first carried out, for instance under Yoshikawa conditions, before performing the conversion step, in order to arrive at the compound of Formula (I).

[0092] When the compound of Formula (F) includes a phthalimide moiety of Formula (III), the phthalimide may be converted to a -O-NH2 group in two steps, the first of which comprises converting the phthalimide group to an oxime group, by reacting the compound of Formula (I’) with an ammonium salt such as ammonium fluoride or alkylammonium fluoride, or with a primary amine such as methylamine, in a polar protic solvent such as methanol or ethanol, for instance as described by Cieslak et al., 2010. The oxime group may then be converted to a -O-NH2 group as described above.

[0093] Alternatively, simultaneous deprotection and conversion of the phthalimide moiety may be obtained in a single step by reacting the compound of Formula (F) with ammonia or methoxylamine, in a polar protic solvent such as methanol or ethanol.

[0094] The compound of Formula (I’) may itself be prepared according to known procedures, starting from commercial nucleosides.

[0095] Typically, the nucleoside is first provided with a protecting group (corresponding to Rs and optionally Z), for instance with a silyl group as described by Hakimelahi et al., 1982 or De Groaz and Herdewijn, 2014. As a silylating agent, mention can be made of tert-butyl dimethyl silyl chloride. Alternatively, a benzoyl ester may be used as a protecting agent as described by Nishino et al., 2006. Then the 3’-OH group is substituted with a methylthiomethyl group, using for instance the procedure described by De Groaz & Herdewijn, 2014 or Pechenov et al., 2000, before converting it to a chloromethylether group with sulfuryl chloride which is then reacted with N-hydroxyphthamide according to Cieslak et al., 2012b, for instance, to give the compound of Formula (I’) comprising a moiety of Formula (III).

[0096] The deprotecting agents used in the methods for synthetizing a polynucleotide described above, in order to remove the Re group of the compound of Formula (I), may be selected, e.g. among those typically used with 3-O'-NH2 blocked nucleotides, such as: oxidants such as hypochlorite, nitric oxide, sodium nitrite buffered to a pH of about 5.5 (Daniel Hutter et al., Nucleosides, Nucleotides and Nucleic Acids, 29:879-895, 2010), nitrous acid at pH 4-5, nitrite esters at pH 7-8, iodates, periodates, perchlorates, N- bromosuccinimide, N-bromoacetamide or potassium ferrates; reducing agents such as dihydrogen in the presence of a Pd or Pt catalyst; electrophiles such as maleimide, nitrobenzene, nitroolefins or quinones such as naphthoquinones, for example (US-7,544,794; W02020 / 165334).

[0097] According to a preferred embodiment, however, the deprotecting agent may comprise sodium nitrite or at least one phosphonate compound having the following formula (IV):

[0098] R-P(=0)(0M)0M

[0099] (IV) in which:

[0100] M is selected from the group consisting of: H, , a monovalent or divalent metal atom (such as Li, Na, K, Cs, Ca, Mg, Cu, Zn, etc.), HNR63+or NR64+wherein each R6independently designates H or a linear or branched alkyl group having from 1 to 6 carbon atoms (such as tetramethylammonium or tetraethylammonium), an organic base (such as pyridine, triethylamine, aniline, dimethylaniline, diisopropylethylamine, diisopropylamine, dimethylaminopyridine, N-methylimidazole and imidazole), a linear or branched alkyl group having from 1 to 6 carbon atoms and a Si(R4)3 group wherein each R4 is independently selected from a -COOH group, an aryl group, a -CN group or a linear or branched alkyl group having from 1 to 6 carbon atoms;

[0101] R is selected from the following groups: -CO-Ri wherein Ri is selected from OH, a linear or branched alkyl group having from 1 to 6 carbon atoms, an aryl group or a - P(=0)(0M)0M group; a -C(=CH-R2)-X-R3 group wherein X is O or -NRaRb, wherein Raand Rbare independently selected from alkyl groups having from 1 to 6 carbon atoms, R2 is a linear or branched alkyl group having from 1 to 6 carbon atoms, R3 is M or a Si FUh group; a -C(Y)(OH)-Rs group wherein Y is -CN or -SCL" and Rs is a linear or branched alkyl group having from 1 to 6 carbon atoms. According to a preferred embodiment, the phosphonate compound of formula (IV) is such that R is selected from -CO-Ri groups wherein Ri is selected from H, -COOH, -CN, a linear or branched alkyl group having from 1 to 6 carbon atoms, an aryl group or a - P(=0)(0M)0M group, preferably Ri is selected from -COOH, a methyl group, a phenyl group which is optionally substituted with one or more OH groups, and a -P(=0)(0M)0M group, more preferably Ri is selected from -COOH, a methyl group and a -P(=0)(0M)0M group.

[0102] Still preferably, the phosphonate compound of formula (IV) is such that R is selected from -CO-Ri groups wherein Ri is a -P(=0)(0M)0M group and M is H or an alkali metal, preferably sodium. These compounds will be referred to in this description as carbonylbisphosphonate and its salts.

[0103] In another embodiment, the phosphonate compound of formula (IV) is such that R is selected from -CO-Ri groups wherein Ri is methyl and M is H.

[0104] Examples of phosphonate compounds of formula (IV) are provided below:

[0105] The deprotecting agent is used in this invention in an effective amount. As used herein, the term an “effective amount” means an amount or concentration sufficient to cleave a 3'-ONH2 polynucleotide when contacted with the latter. It is understood that one of ordinary skill could readily determine an effective amount of the deprotecting agent by conventional techniques, as provided in the Examples. In some embodiments, e.g. employing carbonylphosphonate and its salts as deprotecting agents, an effective amount is provided by a concentration in the range of from 0.1 to 500 mM, or in other embodiments in the range of from 0.1 to 200 mM, or in other embodiments in the range of from 0.1 to 100 mM. Stated otherwise, the molar ratio between the polynucleotides and the deprotecting agent typically ranges from 1 : 1 to 1 : 1000 and preferably from 1:1 to 1 : 100.

[0106] The deprotecting agent is typically provided in an aqueous solution buffered at a pH of from 5 to 7 (i.e. buffer), wherein the buffering agent may be selected from citrates, phosphates such as sodium phosphate, acetates such as sodium acetate or bicarbonates.

[0107] According to a preferred embodiment of this invention, the buffer may further include at least one inorganic salt of a divalent metal such as magnesium, calcium, zinc or copper, preferably magnesium sulfate, which has been found to increase the cleavage yield, especially at higher pH. This compound may be comprised within the buffer in an amount ranging from 1 to 100 equivalents, preferably from 1 to 10 equivalents, with respect to the compound of formula (IV)

[0108] In addition to water, the buffer may further include at least one organic solvent which is miscible with water, especially a polar protic solvent, such as methanol or ethanol (up to about 50% v / v), or a polar aprotic solvent, such as tetrahydrofuran or dioxane (up to about 40% v / v). Alternatively or in addition to these organic solvents, the buffer may also include one or more denaturants, such as formamide, urea, dimethylformamide or dimethylsulfoxide (up to about 25%).

[0109] The synthesis of tetrasodium salt of carbonylbisphosphonate is described in the literature (Khomich, O. A.; Yanvarev, D. V.; Novikov, R. A.; Kornev, A. B.; Puljulla, E.; Vepsalainen, J.; Khomutov, A. R.; Kochetkov, S. N. On the Reaction of Carbonyl Diphosphonic Acid with Hydroxylamine and O- Alkylhydroxylamines: Unexpected Degradation of P-C-P Bridge. Molecules 2017, Vol. 22, Page 10402017, 22 (7), 1040).

[0110] This compound may be obtained by various means starting from tetraisopropyl(dichloromethylene)bis(phosphonate) which may itself be prepared as taught by Quimby, O. T.; Prentice, J. B.; Nicholson, D. A. Tetrasodium Carbonyldiphosphonate. Synthesis, Reactions, and Spectral Properties. Journal of Organic Chemistry 1967, 32 (12), 4111-4114). In a first step, this compound may be hydrolyzed in presence of TMSBr in dioxane or by continuous heating with 18% aqueous hydrochloric acid (Purdie, M. Process for Preparing Methylene Bisphosphonic and Salts. US6657076B1, September 11, 2000). However, both these procedures are time consuming and require aggressive corrosive reagents. Quantitative deesterification may be achieved much easier by pyrolysis of the solid tetraisopropyl derivative at a temperature above 200°C to obtain dichloromethylene bisphosphonic acid. For better safety, percussion deesterification might be performed in boiling tetrachloroethane as described by Quimby, O.T. (see above). The inventors have found that this step may be performed even faster in an halogenated organic solvent such as dichlorobenzene by adding of small portions of tetraisopropyl ester to the boiling solution. The pyrolysis step is exothermic and auto-catalyzed by formed bisphosphonic acid leading to instant gas evolving. Accordingly, loading of the reactor with the starting ester has to be done slowly portion wise. Basic hydrolysis of dichloromethylene derivative may then be performed by refluxing with aqueous solution of sodium hydroxide. Of course, a similar strategy may be followed to prepare other carbonylbisphophonate salts, by changing the base used in the last step by other organic or inorganic bases such as tri ethyl amine, pyridine or potassium hydroxide. It may further be extended to starting materials bearing other alkyl groups than isopropyl groups.

[0111] In addition, non-symmetrical carbonylphosphonates bearing different substituents may generally be prepared by Arbuzov reaction between RC(O)C1 and PfOR'h to obtain RC(O)P(O)(OR1)2 followed by acidic or basic hydrolysis depending on R1. Examples of synthesis routes are provided by Z.Y. Peng et al., Biochemical Pharmacology, Vol. 49, No. 1, 105-113 (1995); R. Karaman et al., J. Chem. Soc. Perkin Trans. 1, 765-774 (1989); C.E. McKENNA et al., J. Chem. Soc., Chem. Commun.. 246-247 (1989).

[0112] The method for preparing a compound of formula (IV) which is a carbonylbisphosphonate salt thus comprises the following steps:

[0113] 1) mixing tetraalkyl methylenediphosphonate with a sodium hypochlorite solution at room temperature until a first precipitate has formed,

[0114] 2) recovering the precipitate and dissolving it in a boiling halogenated organic solvent (such as dichlorobenzene or tetrachloroethylene) to obtain a mixture,

[0115] 3) adding this mixture stepwise into a reactor and refluxing the content of the reactor, thus resulting in propene gas evolvement and the formation of a second precipitate comprising dihalomethylene diphosphonate,

[0116] 4) cooling down the reactor and recovering said second precipitate,

[0117] 5) mixing said precipitate with an aqueous solution of an inorganic base and refluxing until obtaining a third precipitate, and

[0118] 6) cooling down this mixture and recovering said third precipitate, which comprises a carbonylbisphosphonate salt.

[0119] A variety of kits may be provided for implementing the method of this invention. In one aspect, kits may comprise one or more containers (or bottles, or vials) of synthesis reagents at least one of which contains an effective amount of a compound of formula (I). In some embodiments, kits comprise a vial of polymerase and an effective amount of a compound of formula (I) or a precursor thereof having formula (I’), especially the compound of formula (I’) in which R3 = R4 = methyl. This precursor indeed appears to be more suitable for long-term storage than the compound of Formula (I) and can be readily converted to the latter before use, for instance with buffered aqueous methoxyl amine. In some embodiments, kits may include one or more of the following items, either separately or together with the above-mentioned items: (i) one or more containers comprising at least one compound of formula (I) or a precursor thereof having formula (I’), (ii) solid supports with initiators attached thereto, (iii) cleavage reagents for releasing completed polynucleotides from solid supports, (iv) wash reagents or buffers for removing unreacted compounds of formula (I) at the end of an enzymatic addition or coupling step, and (v) postsynthesis processing reagents, such as purification columns, desalting reagents, eluting reagents, and the like.

[0120] The following examples are provided merely for illustrative purposes and are not intended to reduce the scope of this invention which is defined by the attached claims.

[0121] EXAMPLES

[0122] Example 1: Synthesis of 3'-0-blocked (poly)nucleotide compounds of this invention

[0123] 1A- dTTP-3 ’-OCH2ONH2

[0124] The general route of synthesis is shown on Figure 1. lAa) Synthesis of 5’-0-(tert-ButyldimethyIsilyl)-3’-0-(methyIthiomethyI)-2’- deoxythymidine

[0125] 5’-TBDMS deoxythymidine 5.34 g (15 mmol) was dissolved in dry DMSO (50 mL) and then acetic anhydride (34 mL) and acetic acid (11 mL) were added in one portion. The mixture was stirred at room temperature for 48 hours. Then, the reaction mixture was slowly poured into 10% aqueous sodium carbonate solution (600 mL) with stirring. The quenched aqueous solution was transferred into a separating funnel and extracted with ethyl acetate (3x200 mL). The organic phases were combined, washed with sodium chloride brine, dried with sodium sulfate and concentrated in vacuo. Crude mixture were separated by flash column chromatography with a linear gradient of ethyl acetate in n-hexane (from 20% to 100%). The concentrated product was obtained as white solid foam 4.71 g (75%), dried in vacuum desiccator and stored over phosphorus pentoxide until the next step in a desiccator. lAb) Synthesis of 5’-0-(tert-butyldimethylsilyl)-3’-0-(phthalimidooxymethyl)-2’- deoxythymidine :

[0126] A flame-dried flask with a stirring bar was charged with 5’-O-(tert-butyldimethylsilyl)-3’- O-(methylthiomethyl)-2’-deoxythymidine (4.66 g, 11.2 mmol) in dry DCM (80 mL). 1 M sulfuryl chloride solution in DCM (13.4 mL, 13.4 mmol) was added to a stirred solution dropwise at 0°C. The reaction mixture was allowed to warm to room temperature over 2 h, then it was concentrated under reduced pressure and re-dissolved in dry DCM (40 mL). In a separate flask N-hydroxyphthalimide (5.49 g, 33.6 mmol) was suspended in dry DCM (40 mL), and DBU (4.4 mL, 29.4 mmol) was then added and stirred to a homogenous red solution. The obtained solution was added by drops to a stirring solution of the 3’-O- chloromethyl ether of nucleoside upon stirring. After 2 h, the solution was concentrated to a semi-dryness and taken in ethyl acetate (300 mL) and water (100 mL). The organic phase was separated, washed with saturated sodium bicarbonate solution (2x100 mL) dried with sodium sulfate and concentrated under reduced pressure. The crude product was separated by flash column chromatography on silica gel eluted with gradient of methanol in DCM from 0% to 5% to obtain 4.31 g (74%) as a white solid. lAc) Synthesis of 3’-D-(Isopropenyliminooxymethyl)-2’-deoxythymidine:

[0127] 5 ’ -<9-(tert-buty Idimethy Isily l)-3 ’ -O-(phthalimidooxymethyl)-2’ -deoxy thymidine (4.25 g, 8.0 mmol) and ammonium fluoride (2.88 g, 80 mmol) in methanol (40 mL) were stirred at +60°C until complete deprotection of both phtalimidyl and silyl groups, that was achieved in 6 h. The reaction mixture was chilled to an ambient temperature, diluted with acetone (80 mL) and stirred for 30 min. This mixture was then dry loaded with celite to dryness. The celite pad was dry loaded onto a top of silica gel column and eluted with linear gradient of methanol in DCM from 0% to 12% to give 2.09 g (81%) as a white solid. lAd) Synthesis of 3’-0-(aminooxymethyl)-2’-deoxythymidine triphosphate triethylammonium salt: A 10-mL flame dried flask with a magnetic stir bar was charged with 3’-O- (acetoxime)methylene dT (A3) (100 mg, 0.305 mmol), proton sponge (73 mg, 0.34 mmol) in 2 mL of trimethyl phosphate. The flask was plugged with a rubber septum and purged with argon. Then reaction was cooled to -15°C and POCh (47 pL, 0.49 mmol) was injected slowly to the mixture. The cooling bath was removed and the temperature was allowed to rise to 0°C, stirring was continued for 30 min at 0°C. A cocktail of tributylamine (235 pL, 1 mmol), tributyl ammonium pyrophosphate (353 mg, 0.62 mmol) in 3 mL of dry acetonitrile was prepared and injected in one portion to the reaction mixture at -30°C. Stirring was continued for Ih at 0°C. Then the reaction mixture was quenched with IM TEAB (10 mL) and stirred for Ih. This solution was concentrated in vacuum to the half of the volume, diluted to a 20 mL volume and extracted with MTBE (3x20mL). The aqueous solution was concentrated in vacuum and purified with AKTA pure chromatograph on ion-exchange CaptoQ column. Separation was performed by gradient elution with 50 mM TEAB (pH 8.5) to 300 mM TEAB in water. The pure fractions after HPLC-analysis were combined and concentrated at +35°C with rotovap to give a solution containing 170 pmol of thymidine triphosphate acetoxime according to UV-Vis measurement. Into the nucleoside triphosphate solution in 5mL of deionized water, O-benzyloxyamine (1.08 g 15 mmol) were added and the pH was adjusted to 6.5-6.6 with glacial acetic acid. The reaction was stirred overnight until complete conversion and IM aqueous sodium carbonate solution was added to reach pH 8.0. The reaction was extracted with MTBE (3x100 mL). Then the aqueous phase was acidified to pH 7.4 with acetic acid and concentrated under reduced pressure at +35°C. The purification was performed with preparative RP-HPLC with C18-silica gel as a stationary phase, eluted with a linear gradient of acetonitrile in 100 mM TEAB (pH 8.3) from 0% to 30% ACN. The final nucleoside triphosphate was separated as triethylammonium salt after lyophilization to obtain 163 pmol (53%) as a white solid according to UV-Vis measurement.

[0128] IB- rUTP-3 ’-OCH2ONH2

[0129] The first steps of the synthesis of the rU derivative were performed according to the scheme shown on Figure 2.

[0130] Specifically, selective 2’,5’-silylation of uridine was performed as described by Hakimelahi et al., 1982. The synthesis was performed at 6.1 g (25 mmol) scale and 2',5'-di-O- tertbutyldimethylsilyl uridine was separated with silica gel column chromatography with linear gradient of methanol in DCM (from 0% to 5%) to obtain 7.33 g (62%) of white solid compound. Steps similar to those described in Example 1A as la), lb) and 1c) were then successively performed.

[0131] The compound thus obtained was phosphorylated according to the following scheme:

[0132] Specifically, 3’-<9-(acetoxime)methylene rU (102 mg) was triphosphorylated according to a standard Yoshikawa’s procedure: 1.6 eq of POCh followed by 2 eq of TBA PPi, then quenching with TEAB, extraction and concentration in vacuo. The TEAB solution of triphosphate was adjusted to pH 7.0 and frozen for storage prior to chromatography separation.

[0133] The mixture was purified by ion-exchange CaptoQ and C18-Gemini (TEAB / ACN).

[0134] Deprotection was then performed according to the following scheme:

[0135] For this purpose, the rUTP acetoxime was diluted in miliQ water, O-benzylhydroxylamine (BOX) was added and pH was adjusted with acetic acid to 6.6. Few drops of ethanol were added to homogenize the mixture. The reaction mixture was stirred for 12h. Then the reaction was basified to pH 8.2 with sodium bicarbonate, worked up with MTBE ether (extracted 3 times). Then pH re-adjusted with acetic acid to 7.4 and concentrated in vacuo to minimal volume. The product was frozen and subjected to C18 HPLC purification next day.

[0136] 1C- 2 ’-OMe-3 ’-OCH2ONH2-dTTP The synthesis of the 2’-OMe-dT derivative were performed according to the scheme shown on Figure 3.

[0137] 5’-Silylation of 2’-0Me deoxythymidine (8 mmol) was done with bulky TBDPSC1 in DCM / imidazole. The product was precipitated with 5% citric acid and quantitatively isolated by filtration.

[0138] Steps similar to those described in Example 1A as la), lb) and 1c) were then successively performed.

[0139] The compound thus obtained was phosphorylated according to the following scheme:

[0140] 2’-0Me 3’-<9-(acetoxime)methylene dT (110 mg) was triphosphorylated according to a standard Yoshikawa’s procedure: 1.6 eq of POCh followed by 2 eq of TBA PPi, then quenching with TEAB, extraction and concentration in vacuo. The TEAB solution of triphosphate was adjusted to pH 7.0 and frozen for storage prior to chromatography separation.

[0141] The mixture was purified by ion-exchange CaptoQ and C18-Gemini (TEAB / ACN). Deprotection was then performed according to the following scheme:

[0142] The 2’-OMe acetoxime was diluted in miliQ water, O-benzylhydroxylamine (BOX) was added and pH was adjusted with acetic acid to 6.6. Few drops of ethanol were added to homogenize the mixture. The reaction mixture was stirred for 12h, a small portion diluted with ethanol was injected into Then the reaction was basified to pH 8.2 with sodium bicarbonate, worked up with MTBE ether (extracted 3 times). Then the pH was re-adjusted with acetic acid to 7.4 and concentrated in vacuo to minimal volume. The product was frozen and subjected to Cl 8 HPLC purification next day.

[0143] ID- LNA 3 ’-OCH2ONH2-dT

[0144] The synthesis is shown on Figure 4.

[0145] Silylation was performed as described in Example 1C. The product was precipitated with ice-water (10X volume), filtrated and washed with n-hexane on a filter to afford grey powder. Steps similar to those described in Example 1A as la), lb) and 1c) were then successively performed.

[0146] The compound thus obtained was phosphorylated according to the following scheme:

[0147] LNA 3’-<9-(acetoxime)methylene dT (110 mg) was triphosphorylated according to a standard Yoshikawa’s procedure: 1.6 eq of POCh followed by 2 eq of TBA PPi, then quenching with TEAB, extraction and concentration in vacuo. The TEAB solution of triphosphate was adjusted to pH 7.0 and frozen for storage prior to chromatography separation.

[0148] The mixture was purified by ion-exchange CaptoQ and C18-Gemini (TEAB / ACN). Deprotection was then performed according to the following scheme:

[0149] The LNA acetoxime was diluted in miliQ water, BOX was added and pH was adjusted with acetic acid to 6.6. Few drops of ethanol were added to homogenize the mixture. The reaction mixture was stirred for 12h. Then the reaction was basified to pH 8.2 with sodium bicarbonate, work up with MTBE ether (extracted 3 times). Then the pH was re-adjusted with acetic acid to 7.4 and concentrated in vacuo to minimal volume. The product was frozen and subjected to Cl 8 HPLC purification next day.

[0150] IE- dCTP-3 ’-OCH2ONH2

[0151] The synthesis of this compound was performed according to the scheme shown on Figure 5. 5’-OH protection with benzoyl ester was performed as described by S. Nishino et al., 2006. The synthesis was done on scale of 20 mmol. Incomplete conversion observed after addition of l.l eq ofBzCl and Ih stirring at RT. Then 0.55 eq more ofBzCl were added followed by 1.5 eq of Et3N. After evaporation and work up with ethyl acetate / sodium bicarbonate 5’-Bz nucleoside was recrystallized from boiled 90% ethanol and collected by filtration. The mother liquid containing product was concentrated and separated by column chromatography.

[0152] Steps similar to those described in Example 1A as la) and lb) were then successively performed.

[0153] The final deprotection step was performed in 7M methanol ammonia upon stirring overnight at ambient temperature. The reaction medium was concentrated re-evaporated with acetone. The crude mixture was uploaded onto the silica gel column, and purified by gradient elution of methanol in DCM from 0 to 15%.

[0154] IF- dGTP-3 ’-OCH2ONH2

[0155] The synthesis is shown on Figure 6.

[0156] The first step is similar to that of Example IE. Steps similar to those described as steps la) and lb) in Example 1 A were then performed. dATP-3’-OCH2ONH2, dCTP-3 ’-OCH2ONH2, dGTP-3 ’-OCH2ONH2were synthesized according to the same procedure described for dTTP-3’-OCH2ONH2in two steps: triphosphorylation and deprotection with O-benzylhydroxylamine.

[0157] 1G- dATP-3 ’-OCH2ONH2

[0158] The synthesis is shown on Figure 7.

[0159] The first step is similar to that of Example IE. Steps similar to those described as steps la) and lb) in Example 1 A were then performed. The same last step as that described in Example 1G was then conducted. Example 2: Deprotection study

[0160] Materials and methods:

[0161] Screening tests were performed in a 96-well plate with a reaction volume of lOOpL. dTTP-3’-OCH2ONH2, was taken at 1 mM final concentration in one of four 50 mM buffers (A: sodium acetate pH 5.0, B: sodium phosphate pH 6.0, C: sodium phosphate 7.0, D: sodium bicarbonate pH 8.25). Tetrasodium carbonylbisphosphonate or NaNCh was added to the dTTP solutions in various concentrations (2, 10 and 50 eq.) with 0 or 20 mM of MgSOi and incubated during 5, 20 or 60 mins prior to quenching with 20 pL of hydroxylamine hydrochloride (0.2 M). The 96-well plate were agitated with Thermomixer at 25°C or 40°C. The catalytic effect of magnesium on cleavage rate was also investigated. Prior to HPLC analysis reaction, 100 pL of aliquotes were additionally quenched with acetone (10 pL). The samples were analyzed by RP-HPLC. The cleavage rate was determined based on the ratio between dTTP-3’-OH and dTTP-3’-O-methylacetoxime.

[0162] A comparative experiment was performed with the same nucleotide but bearing a -ONH2 protecting group instead of a -OCH2ONH2 protecting group. The cleavage rate was determined based on the ratio between dTTP-3’-OH and dTTP-3 ’-acetoxime.

[0163] HPLC conditions:

[0164] Xterra RP C18 Column: 4.6x50 mm, (3.5 pm silica), Buffer A: 50 mM TEAB (pH 8.5), Buffer B: 100% ACN. Method: 0-2 min - 0% ACN B, 6 min - 10% ACN, 8 min - 30% ACN, 8-10 min - 100% ACN. Flowrate: 1.5 mL / min.

[0165] Results:

[0166] The kinetics and pH-dependence of dTTP-ONH2 and dTTP-OCH2ONH2 are very similar.

[0167] Example 3: Enzymatic DNA Synthesis study

[0168] An enzymatic DNA synthesis study was performed by elongating an initiator DNA attached to a solid support, as described in WO2020 / 165137, except that the initiator bore a fluorine tag.

[0169] Two cycles of the following steps were performed:

[0170] • Elongation step by incubating 375 pmol of the initiator DNA (or elongated fragment) at 37°C for 30 mins in the presence of 25 pM of a Terminal Deoxynucleotidyl Transferase (TdT) variant (such as those disclosed in PCT / EP2024 / 080046), with 200 pM of dTTP-3’-O-CH2ONH2, in a 25 mM Tris pH8 buffer containing 1 mM MnCl2and 5% DMSO.

[0171] • Centrifugation for 30 sec at 500g, followed by washing with 50pL of a pH 6.4 200 mM cacodylate buffer (comprising 500mM LiCl and 8 mM EDTA) for 30 sec, followed by centrifugation for 30 sec at 500g.

[0172] • Deblocking step for 30 sec using 50 pL of a solution containing 20 mM carbonyl bisphosphonate at pH 5.0, followed by a centrifugation at 500g for 30sec, a second deblocking step of 30 sec duration.

[0173] • Centrifugation for 30 sec at 500g, followed by washing with 50pL of a pH 7.0, 30 mM, Tris buffer for 20 sec, followed by centrifugation for 30 sec at 500g

[0174] After the elongation steps are completed, the synthesized DNA was further washed and then enzymatically cleaved from the solid support according to the methods described in WO2020 / 165137. The samples were then run on a gel and the fluorescence of the gel bands was measured.

[0175] After completion of the two cycles of elongation, the average of 3 replicates showed a n+2 band (successful elongation by two nucleotides) in addition to the n+1 band (partial elongation) and the n band (no elongation).

[0176] In conclusion, the present example successfully demonstrates two cycles of elongation - deprotection using the novel reversible blocking group of the invention.

[0177] References:

[0178] Cieslak, J., Ausi'n. C., Grajkowski, A., & Beaucage, S. L. (2013). The 2-Cyano-2,2- dimethylethanimine-N-oxymethyl Group for the 2'-Hydroxyl Protection of Ribonucleosides in the Solid-Phase Synthesis of RNA Sequences. Chemistry - A European Journal, 19(\4), 4623-4632. https: / / doi.org / 10.1002 / CHEM.201204235

[0179] Cieslak, J., Grajkowski, A., Ausi'n. C., & Beaucage, S. L. (2016). Protection of the 2'- Hydroxy Function of Ribonucleosides as an Iminooxymethyl Propanoate and Its 2'-O- Deprotection through an Intramolecular Decarboxylative Elimination Process. European Journal of Organic Chemistry, 2016(35), 5817-5821. https: / / doi.org / 10.1002 / EJOC.201601308

[0180] Cieslak, J., Grajkowski, A., Ausi'n. C., Gapeev, A., & Beaucage, S. L. (2012a). Permanent or reversible conjugation of 2'-O- or 5'-O-aminooxymethylated nucleosides with functional groups as a convenient and efficient approach to the modification of RNA and DNA sequences. Nucleic Acids Research, 40(5), 2312-2329. https: / / doi.org / 10.1093 / NAR / GKR896

[0181] De, S., Groaz, E., & Herdewijn, P. (2014). Tailoring Peptide-Nucleotide Conjugates (PNCs) for Nucleotide Delivery in Bacterial Cells. European Journal of Organic Chemistry, 2014(W), 2322-2348. https: / / doi.org / 10.1002 / EJGC.201301781

[0182] De, S., Groaz, E., Maiti, M., Pezo, V., Marliere, P., & Herdewijn, P. (2014). Synthesis of new biocarrier-nucleotide systems for cellular delivery in bacterial auxotrophic strains. Tetrahedron, 70(46), 8843-8851. https: / / doi.Org / 10.1016 / J.TET.2014.09.096

[0183] Ducharme, Y., & Harrison, K. A. (2011). A versatile approach to the synthesis of oligonucleotide analogs containing neutral 5’-thioformacetal intemucleoside linkages. Https: / / Doi.Org / 10.1139 / V99-135 , 77(8), 1410-1418. https: / / doi.org / 10.1139 / V99-135

[0184] Flamme, M., Hanlon, S., tiling. H., Puentener, K., Sladojevich, F., & Ho enstein, M. (2021). Towards the enzymatic synthesis of phosphorothioate containing LNA oligonucleotides. Bioorganic & Medicinal Chemistry Letters, 48, 128242. https: / / doi.Org / 10.1016 / J.BMCL.2021.128242

[0185] Flamme, M., Katkevica, D., Pajuste, K., Katkevics, M., Sabat, N., Hanlon, S., Marzuoli, I., Piintener, K., Sladojevich, F., & Hohenstein, M. (2022). Benzoyl and pivaloyl as efficient protecting groups for controlled enzymatic synthesis of DNA and XNA oligonucleotides. Asian Journal of Organic Chemistry. https : / / doi . org / 10.1002 / AJOC .202200384 Hakimelahi, G. H., Proba, Z. A., Ogilvie-, K. K., F’roba, Z. A., & Ogilvie, K. K. (1982). New catalysts and procedures for the dimethoxytritylation and selective silylation of ribonucleosides. Https: / / Doi.Org / 10.1139 / V82-165 , 60(9). 1106-1113. https: / / doi.org / 10.1139 / V82-165

[0186] Hutter et al, Nucleosides Nucleotides Nucleic Acids , 29(11): 879-895, 2010

[0187] Jensen et al., Biochemistry, 57: 1821-1832, 2018

[0188] Nishino, S., Yamamoto, H., Nagato, Y., & Ishido, Y. (1986). Partial Protection of Carbohydrate Derivatives. Part 19. Highly Regioselective 5'-O-Aroylation of 2'- Deoxyribonucleosides in Terms of Dilution - Drop-by-Drop - Addition Procedure. Nucleosides, Nucleotides & Nucleic Acids , 5(2), 159-168. https: / / doi.org / 10.1080 / 07328318608068670

[0189] Pechenov, A. E., Zavgorodny, S. G., Shvets, V. I., & Miroshnikov, A. I. (2000). TheS,X- acetals in nucleoside chemistry: II. The synthesis of 3'-O-methylthiomethylribonucleosides. Russian Journal of Bioorganic Chemistry 200026:6, 26(6), 407-413. https: / / doi.org / 10.1007 / BF02758669

[0190] Zavgorodny, S. G., Pechenov, A. E., Shvets, V. I., & Miroshnikov, A. I. (2000). S, X- acetals in nucleoside chemistry. III1. Synthesis of 2'- and 3'-O-azidomethyl derivatives of ribonucleosides. Nucleosides, Nucleotides and Nucleic Acids , 79(10-12), 1977-1991. https: / / doi.org / 10.1080 / 15257770008045472

Claims

CLAIMS1. A compound having the following formula (I) :where:B is a nucleobase;X is -CH2-O-NH2;R9 is H, halogen or O-Z where Z is H or alkyl or a methylene group bridging the 2'-0 position and the 4'-C position of the compound of formula (I);Rio is -PO3-Y or -P(S)O2-Y, where Y is either -PO3-PO3; and wherein the nucleobase optionally bears fluorescent labels, possibly attached thereto via linkers.

2. A method for extending a DNA primer by template-dependent synthesis or templateindependent synthesis, comprising contacting said primer and optionally said template with a polymerase and a compound of formula (I) as recited in claim 1.

3. A method of synthesizing a polynucleotide, the method comprising the steps of:(a) providing initiators which are polynucleotides having each a free 3’-hydroxyl;(b) repeating in a reaction mixture, until the polynucleotide is formed, cycles of (i) contacting under elongation conditions the initiators or elongated fragments having free 3’- hydroxyls with a compound of formula (I) as recited in claim 1 and with a polymerase, so that the initiators or elongated fragments are elongated by incorporation of the compound of formula (I) to form 3’-O-blocked elongated fragments, and (ii) deprotecting the elongated fragments to form elongated fragments having free 3 ’-hydroxyls.

4. The method of claim 3, wherein the polymerase is a template-independent polymerase and the method is an enzymatic DNA synthesis or an enzymatic RNA synthesis or a mixed enzymatic DNA and RNA synthesis.

5. The method of any one of claims 2 to 4, wherein deprotecting is performed by contacting the elongated fragments with sodium nitrite or with at least one compound having the following formula (IV) :R-P(=O)(OM)OM (IV) in which:M is selected from the group consisting of: H, a monovalent or divalent metal atom, HNR63+or NR64+wherein each R6independently designates H or a linear or branched alkyl group having from 1 to 6 carbon atoms, an organic base, a linear or branched alkyl group having from 1 to 6 carbon atoms and a Si (R4)s group wherein each R4 is independently selected from a -COOH group, a -CN group, an aryl group or a linear or branched alkyl group having from 1 to 6 carbon atoms;R is selected from the following groups: -CO-Ri wherein Ri is selected from H, -COOH, CN, a linear or branched alkyl group having from 1 to 6 carbon atoms, an aryl group or a - P(=O)(OM)OM group; a -C(=CH-R2)-X-R3 group wherein X is O or -NRaRb, wherein Raand Rbare independently selected from alkyl groups having from 1 to 6 carbon atoms, R2 is a linear or branched alkyl group having from 1 to 6 carbon atoms, R3 is M or a Si(R03 group; a -C(Y)(OH)-Rs group wherein Y is -CN or -SO?' and Rs is a linear or branched alkyl group having from 1 to 6 carbon atoms.

6. A compound having the following formula (I’) :where :B is a nucleobase;Re has formula (Ila) or (lib):(li b) R(I la)4Ri and R2 are each independently alkyl or a protective group, or Ri and R2 form together, with the adjacent nitrogen atom, a heterocycle having one or more cycles which are independently saturated or unsaturated, substituted or unsubstituted;R3 and R4 are each independently alkyl;Rs is H, -PO3-Y or -P(S)O2-Y where Y is either -PO3-PO3 or a protecting group;R7 is H, halogen or O-Z where Z is H or alkyl or a methylene group bridging the 2'-0 position and the 4'-C position of the compound of formula (I) or a -SiYs group where each Y is independently a linear or branched alkyl group having from 1 to 6 carbon atoms or a phenyl group; and wherein the nucleobase optionally bears fluorescent labels, possibly attached thereto via linkers, with the exception of the compounds of formula (I’A):wherein B is a nucleobase.

7. The compound of formula (I’) according to claim 6, which is selected from those wherein:- Ri and R2 form together, with the adjacent nitrogen atom, the heterocycle of formula (III):(III),R3 and R4 are both methyl.

8. The compound of formula (I’) according to claim 7, wherein R7 is O-Z where Z is a methylene group bridging the 2'-0 position and the 4'-C position of the compound.

9. A kit for enzymatic synthesis of a polynucleotide comprising one or more vials of synthesis reagents, at least one of which contains an effective amount of a compound of formula (I) as recited in claim 1 or a precursor thereof having formula (I’) as recited in claim 6, especially the compound of formula (I’) in which R3 = R4 = methyl.

10. A method for preparing the compound of Formula (I) according to claim 1, comprising reacting a compound of Formula (I’) as recited in claim 7 or 8, in one or two steps, with at least one organic or inorganic base, such as: ammonia; a primary amine such as methylamine or O-benzylhydroxylamine; methoxylamine; an ammonium salt such as ammonium fluoride or alkylammonium fluoride; and mixtures thereof.

Images