Reagents used for deprotection of 3'-O-aminopolynucleotides

Abstract

The present invention relates to an enzymatic method for synthesizing polynucleotides, comprising a deprotection step using a specific phosphonate compound as a deprotective agent. The present invention also relates to a method for deprotecting a 3'-O-amino extension fragment of a polynucleotide in an enzymatic method for synthesizing polynucleotides, comprising a step of contacting the extension fragment with the phosphonate compound. The present invention further relates to a kit for synthesizing polynucleotides, comprising one or more vials of synthetic reagents, at least one of which contains an effective amount of a phosphonate compound, and a specific method for preparing the phosphonate compound.

Description

[Technical Field]

[0001] The present invention relates to an enzymatic method for synthesizing polynucleotides, comprising a deprotection step using a specific phosphonate compound as a deprotective agent. The present invention also relates to a method for deprotecting a 3'-O-amino extension fragment of a polynucleotide in an enzymatic method for synthesizing polynucleotides, comprising a step of contacting the extension fragment with the phosphonate compound. The present invention further relates to a kit for synthesizing polynucleotides containing an effective amount of the phosphonate compound, and a specific method for preparing the phosphonate compound. [Background technology]

[0002] The growing interest in enzymatic approaches to polynucleotide synthesis stems not only from the increasing demand for synthetic polynucleotides in many areas, such as synthetic biology (e.g., insulin synthesis for treating diabetes), CRISPR-Cas9 applications, and next-generation sequencing ("synthetic sequencing" or SBS, and "binding sequencing" or SBB, etc.), but also from the limitations of chemical approaches to polynucleotide synthesis, including the upper limit on product length, the use of moisture-sensitive monomers, and the use of environmentally unfriendly solvents (Jensen et al., Biochemistry, 57: pp. 1821-1832, 2018).

[0003] Currently, most enzymatic approaches to the synthesis of both DNA and RNA utilize polymerases, which are used to elongate initiator polynucleotides by coupling them with 3'-O protected nucleotides such as 3'-O aminonucleotides, and then to deprotect the resulting protected growth chain. This cycle is repeated until the desired elongated polynucleotide sequence is obtained. For example, the TdT variant has been engineered (International Publication No. 96 / 07669) to efficiently incorporate a reversibly protected 3'-O-aminonucleoside triphosphate monomer developed by Steven Benner into a growing polynucleotide chain (Champion et al., U.S. Patent No. 10,752,887 and International Publication No. 2020 / 099451; Benner et al., U.S. Patents No. 7,544,794, 8,034,923, 8,212,020 and 10,472,383, and Hutter et al., Nucleosides, Nucleotides and Nucleic Acids, 29 (11): pp. 879-895, 2010). Its usefulness as a protecting group for the -ONH2 chemical moiety is based on its ability to reversibly mask the 3'-OH group of 2-deoxyribose or ribose nucleosides. In addition, the smaller-sized -ONH2 chemical moiety makes a better substrate for enzymes such as polymerases.

[0004] The deprotecting agent used to convert the 3'-ONH2 group of the protected oligonucleotide formed in each cycle to a 3-OH group is an oxidizing agent, such as hypochlorite, nitric oxide, or sodium nitrite buffered to approximately 5.5 pH (Daniel Hutter et al., Nucleosides, Nucleotides and Nucleic Acids, 29 (11): pp. 879-895, 2010), it has been proposed that the following can be selected: nitrite at pH 4-5, nitrite esters at pH 7-8, iodates, periodates, perchlorates, N-bromosuccinimide, N-bromoacetamide, or potassium ironate; reducing agents, e.g., dihydrogen in the presence of a Pd or Pt catalyst; electrophiles, e.g., maleimide, nitrobenzene, nitroolefin, or quinones, e.g., naphthoquinone (U.S. Patent No. 7,544,794; International Publication No. 2020 / 165334; U.S. Patent Application Publication No. 2021 / 214382). Currently, this process is almost entirely carried out using sodium nitrite, which has been found to alter the structure of nucleic acid bases, particularly the oxidative deamination of adenine, guanine, and cytosine (Hutter et al., 2010; U.S. Patent Application Publication No. 20180066295A1 - Deamination of Organophosphorus-Nucleosides). These structural changes lead to mutations, thereby limiting the practicality of the 3'-O-amino protecting group, especially in 3'-ONH2-NTP-based enzymatic DNA synthesis for biological applications. Sodium nitrite is also unstable in aqueous media, even at weakly acidic pH, thereby releasing nitrogen dioxide over time. This can also cause damage to residual polymerase proteins. A solution to reduce these side effects of sodium nitrite is presented in U.S. Patent No. 11,505,815, which consists of using azide-masked nitrogen-containing heterocycles in enzymatic DNA synthesis. [Prior art documents] [Patent Documents]

[0005] [Patent Document 1] International Publication No. 96 / 07669 [Patent Document 2] U.S. Patent No. 10,752,887 [Patent Document 3] International Publication No. 2020 / 099451 [Patent Document 4] U.S. Patent No. 7,544,794 [Patent Document 5] U.S. Patent No. 8,034,923 [Patent Document 6] U.S. Patent No. 8,212,020 [Patent Document 7] U.S. Patent No. 10,472,383 [Patent Document 8] International Publication No. 2020 / 165334 [Patent Document 9] U.S. Patent Application Publication No. 2021 / 214382 [Patent Document 10] U.S. Patent Application Publication No. 20180066295A1 [Patent Document 11] U.S. Patent No. 11,505,815 [Patent Document 12] U.S. Patent Application Publication No. 6657076B1 [Patent Document 13] International Publication No. 2019 / 135007 [Patent Document 14] U.S. Patent No. 5763594 [Patent Document 15] International Publication No. 2021 / 198040 [Patent Document 16] International Publication No. 2017 / 216472 [Patent Document 17] International Publication No. 2021 / 018919 [Patent Document 18] U.S. Patent Application Publication No. 2019 / 0078065 [Patent Document 19] U.S. Patent Publication Gazette No. 2019 / 0078126 [Patent Document 20] U.S. Patent No. 9045573

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[0007] Given the growing interest in the enzymatic synthesis or sequencing applications of polynucleotides, it would be desirable to provide a simple solution to overcome the aforementioned problems, particularly one that brings about deprotection of oligonucleotides under conditions mild enough to preserve nucleic acid bases intact, thereby improving the purity / accuracy of polynucleotides in DNA / RNA sequencing. These deprotecting agents should also enable the cleavage of protected extended polynucleotides in high yield, allowing for the incorporation of subsequent nucleotides.

[0008] α-carbonylphosphonates are known for their ability to cleave O-NH2 functional groups (Khomich, O. et al. 2017. "On the Reaction of Carbonyl Diphosphonic Acid with Hydroxylamine and O-Alkylhydroxylamines: Unexpected Degradation of PCP Bridge." Molecules 2017, Vol. 22, p. 1040 22 (7):1040). However, to the best of the applicant's knowledge, they have never been used for the deprotection of polynucleotides, much less to overcome the aforementioned drawbacks of other deprotecting agents such as sodium nitrite. [Means for solving the problem]

[0009] In this regard, the inventors have found that certain phosphonate compounds can overcome the above problem because they do not exhibit significant activity toward deamination, oxidation, depurination, and other undesirable reactions toward nucleosides, oligodeoxynucleotides, or fluorescent / non-fluorescent tags. In addition, they can be used under conditions mild enough to preserve nucleic acid bases intact, thereby increasing the purity / precision of polynucleotides in DNA / RNA sequences, and to retain the same polymerase for several cycles. The inventors have also shown that these compounds yield higher cleavage yields than sodium nitrite.

[0010] Therefore, the present invention is a method for synthesizing polynucleotides, (a) A step of preparing initiators which are polynucleotides each having a free 3'-hydroxyl group, (b) In the reaction mixture until a polynucleotide is formed, (i) under extension conditions, an initiator or extension fragment having a free 3'-hydroxyl group is contacted with 3'-O-NH2 nucleoside triphosphate and polymerase, thereby extending the initiator or extension fragment by the incorporation of 3'-O-aminonucleoside triphosphate to form a 3'-O-amino extension fragment, and (ii) the extension fragment is deprotected to form an extension fragment having a free 3'-hydroxyl group, and the cycle is repeated. Includes, Deprotection of the extended fragment is given by the following equation (I): TIFF2026516019000001.tif11170[In the formula, Each M is independently H; a monovalent or divalent metal atom; HNR 6 3 + or NR 6 4+(in the formula, each R 6(1) independently represents H or a linear or branched alkyl group having 1 to 6 carbon atoms; a protonated organic base; a linear or branched alkyl group having 1 to 6 carbon atoms; and three Si(R4) groups (wherein each R4 is independently selected from an aryl group and a linear or branched alkyl group having 1 to 6 carbon atoms), R is -CO-R1 (wherein R1 is selected from (i) a linear or branched alkyl group having 1 to 6 carbon atoms, (ii) an aryl group, and (iii) a -P(=O)(OM)OM group). The method involves contacting at least one phosphonate compound having [a specific characteristic].

[0011] The present invention also relates to a kit for synthesizing polynucleotides, comprising a polymerase and a vial of an effective amount of a phosphonate compound of formula (I).

[0012] The present invention further relates to a method for deprotecting a 3'-O-amino extension fragment of a polynucleotide in a method for synthesizing a polynucleotide, the method comprising the step of contacting the extension fragment with at least one phosphonate compound of formula (I).

[0013] The present invention also relates to a method for preparing a compound of formula (I) which is a carbonyl bisphosphonate, 1) A step of mixing tetraalkylmethylenediphosphonate with a sodium hypochlorite solution at room temperature until a first precipitate is formed, 2) A step of recovering the precipitate and dissolving it in a boiling halogenated organic solvent (such as dichlorobenzene or tetrachloroethylene) to obtain a mixture, 3) A step of gradually adding this mixture to the reactor and refluxing the contents of the reactor, thereby resulting in the generation of propene gas and the formation of a second precipitate containing dihalomethylenediphosphonate, 4) A step of cooling the reactor and recovering the second precipitate, 5) A step of mixing the precipitate with an aqueous solution of an inorganic base and refluxing until a third precipitate is obtained, 6) The step of cooling the mixture and recovering the third precipitate containing carbonyl bisphosphonate This includes methods. [Brief explanation of the drawing]

[0014] [Figure 1] This figure shows the reaction scheme used for preparing tetrapotassium carbonylbisphosphonate, a deprotective agent according to the present invention. [Figure 2] This figure shows the steps of a template-free enzymatic method for synthesizing polynucleotides according to the present invention. [Figure 3] This graph shows the conversion yield from 3'-ONH2 to 3'-OH based on the reaction of dTTP-ONH2 with 2 equivalents of carbonyl bisphosphonate at different pH and reaction times. [Figure 4A] The figure shows the HPLC profiles of dTTP-ONH2 treated with sodium nitrite under the following conditions: 1. pH 5.0 / incubation 5 min, 2. pH 6.0 / incubation 5 min, 3. pH 5.0 / incubation 20 min, 4. pH 6.0 / incubation 20 min, 5. pH 5.0 / incubation 60 min, and 6. pH 6.0 / incubation 60 min, followed by quenching with acetone: A. 2 equivalents, B. 10 equivalents, and C. 50 equivalents. [Figure 4B] The figure shows the HPLC profiles of dTTP-ONH2 treated with 2 equivalents of tetrasodium carbonylbisphosphonate and subsequently quenched with acetone under the following conditions: 1-4: incubation for 5 minutes / pH=5, 6, 7, 8; 5-8: incubation for 20 minutes / pH=5, 6, 7, 8; 9-12: incubation for 60 minutes / pH=5, 6, 7, 8. [Figure 5] This figure shows a reaction scheme used in the deprotection study of various 3'-O-NH2 nucleotides using carbonyl bisphosphonates, which are used as deprotective agents according to the present invention. [Figure 6] This graph shows the time-dependent cleavage yield results for various dNTPs incubated at 25°C under several pH conditions with carbonyl bisphosphonate as the deprotective agent of the present invention. [Figure 7] This graph shows the time-dependent cleavage yield results for various dNTPs incubated at 25°C under several pH conditions in the presence of 20 mM magnesium sulfate, together with carbonyl bisphosphonate as a deprotective agent of the present invention. [Figure 8] This figure shows the HPLC profiles of deoxynucleosides incubated with 0.5 M sodium nitrite at pH 4.5, 5.0, or 5.5 for 72 hours. [Figure 9] This figure shows the HPLC profiles of deoxynucleosides incubated with 0.5M tetrasodium carbonylbisphosphonate at pH 4.5, 5.0, or 5.5 for 72 hours. [Figure 10] This figure shows the HPLC profiles of deoxynucleosides incubated with 0.5 M sodium nitrite at pH 4.5, 5.0, or 5.5 for 72 hours. [Figure 11] This figure shows the HPLC profiles of deoxynucleosides incubated with 0.5M tetrasodium carbonylbisphosphonate at pH 4.5, 5.0, or 5.5 for 72 hours. [Figure 12] This figure shows the HPLC profiles of deoxynucleosides incubated with 0.5 M sodium nitrite at pH 4.5, 5.0, or 5.5 for 72 hours. [Figure 13] This figure shows the HPLC profiles of deoxynucleosides incubated with 0.5M tetrasodium carbonylbisphosphonate at pH 4.5, 5.0, or 5.5 for 72 hours. [Figure 14] This figure shows the HPLC profiles of deoxynucleosides incubated with 0.5 M sodium nitrite at pH 4.5, 5.0, or 5.5 for 72 hours. [Figure 15] This figure shows the HPLC profiles of deoxynucleosides incubated with 0.5M tetrasodium carbonylbisphosphonate at pH 4.5, 5.0, or 5.5 for 72 hours. [Figure 16] This figure shows the reaction scheme used in the deprotection study of dTTP-3'-O-NH2 using acetylphosphonic acid, which is used as a deprotective agent according to the present invention. [Figure 17] This graph shows the conversion yield from 3'-ONH2 to 3'-OH and the formation of a stable Schiff base adduct, based on the reaction of dTTP-ONH2 with 2 equivalents of acetylphosphonic acid at different pH and reaction times. [Figure 18] This graph shows the results of a comparative analysis of the substitution rates of 52-mer oligodeoxynucleotides synthesized using either carbonyl bisphosphonate (new DB) or aqueous nitrite solution (standard DB) as a deprotecting agent. [Figure 19] This figure shows the reaction scheme used in the deprotection study of dTTP-3'-O-NH2 using heptanoylphosphonic acid as a deprotective agent according to the present invention. [Figure 20] This graph shows the conversion yield from 3'-ONH2 to 3'-OH and the formation of a stable Schiff base adduct, based on the reaction of dTTP-ONH2 with 2 equivalents of heptanoylphosphonic acid at different pH and reaction times. [Modes for carrying out the invention]

[0015] definition Polynucleotides typically have at least 100 nucleotide units, but in this description, "polynucleotide" and "oligonucleotide" are used interchangeably, each meaning a linear polymer of nucleotide monomers or their analogs. Monomers constituting polynucleotides and oligonucleotides can be specifically bound to native polynucleotides by regular patterns of monomer-to-monomer interactions, such as Watson-Crick base pairing, base stacking, and Hoogsteen or inverse Hoogsteen base pairing. Such monomers and their nucleoside linkages may be naturally occurring or analogs thereof, such as naturally occurring or non-naturally occurring analogs. Examples of non-naturally occurring analogs include PNA, phosphorothioate nucleoside linkages, bases containing linking groups that allow for the attachment of labels such as fluorophores, or haptens. If the use of oligonucleotides or polynucleotides requires enzymatic processing such as polymerase extension or ligation by ligase, it will be understood by those skilled in the art that the oligonucleotides or polynucleotides in such cases do not contain any or some positions of internucleoside linkages, sugar moieties, or specific analogues of bases. The size of polynucleotides typically ranges from a few monomer units, e.g., 5 to 40 (when they are usually called "oligonucleotides"), to several thousand monomer units. Unless otherwise stated, terminology and atomic numbering rules follow those disclosed in Strachan and Read, Human Molecular Genetics 2 (Wiley-Liss, New York, 1999). Thus, "A" represents deoxyadenosine, "C" represents deoxycytidine, "G" represents deoxyguanosine, "T" represents thymidine, "I" represents deoxyinosine, and "U" represents uridine.Typically, polynucleotides consist of four natural nucleosides linked by phosphodiester bonds (e.g., deoxyadenosine, deoxycytidine, deoxyguanosine, deoxythymidine in DNA, or their ribose counterparts in RNA), but may also include non-natural nucleotide analogs, such as modified bases, sugars, or nucleoside linkages. If an enzyme has specific oligonucleotide or polynucleotide substrate requirements for its activity, such as single-stranded DNA or RNA / DNA double helix, the selection of a suitable oligonucleotide or polynucleotide substrate composition is clearly within the scope of the knowledge of those skilled in the art, particularly based on guidance from papers such as Sambrook et al., Molecular Cloning, 2nd edition (Cold Spring Harbor Laboratory, New York, 1989) and similar references. Similarly, oligonucleotides and polynucleotides may refer to either single-stranded or double-stranded forms (i.e., double helixes of oligonucleotides or polynucleotides and their respective complements). Those skilled in the art will find it clear from the context in which the term is used which form is intended, or whether both forms are intended.

[0016] The term "alkyl" refers to saturated, linear or branched aliphatic groups, such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, or hexyl.

[0017] The term "aryl" corresponds to monocyclic or bicyclic aromatic hydrocarbons, such as phenyl, biphenyl, or naphthyl, preferably phenyl.

[0018] The alkyl and aryl groups defined above also include their corresponding monosubstituted or polysubstituted forms. Examples of substituents, though not limited to them, include (C1-C6) alkyl and (C3-C) alkyl groups. 12 )Cycloalkyl, (C6~C 14 ) Aryl, F, Cl, Br, I, CN, NO2, CF3, R 7 O-, R8 S-, R 9 NH-, and R 10 R 11 N- is exemplified, and R 7 , R 8 , R 9 , R 10 , and R 11 are each independently, (C1-C6) alkyl, (C3-C 12 ) cycloalkyl, and (C6-C 14 ) aryl selected from.

[0019] As described above, the method of the present invention for synthesizing polynucleotides is (a) using an initiator which is a polynucleotide each having a free 3'-hydroxyl; and (b) in the reaction mixture until a polynucleotide is formed, (i) under extension conditions, contacting an initiator or an extension fragment having a free 3'-hydroxyl with a 3'-O-NH2 nucleoside triphosphate and a polymerase, whereby the initiator or the extension fragment is extended by incorporation of a 3'-O-amino nucleoside triphosphate to form a 3'-O-amino extension fragment, and (ii) repeating the cycle of deprotecting the extension fragment to form an extension fragment having a free 3'-hydroxyl including.

[0020] This method uses a specific type of deprotecting agent, namely, the following formula (I): TIFF2026516019000002.tif11170[wherein, each M is independently, H; a monovalent or divalent metal atom; HNR 6 3 + or NR 6 4+ (wherein each R 6Each of the following independently represents H or a linear or branched alkyl group having 1 to 6 carbon atoms (such as tetramethylammonium or tetraethylammonium); a protonated organic base; a linear or branched alkyl group having 1 to 6 carbon atoms; and selected from the group consisting of three Si(R4) groups (wherein each R4 is independently selected from an aryl group and a linear or branched alkyl group having 1 to 6 carbon atoms); R is -CO-R1 (wherein R1 is selected from (i) a linear or branched alkyl group having 1 to 6 carbon atoms, (ii) an aryl group, and (iii) a -P(=O)(OM)OM group). The method is characterized by using at least one phosphonate compound having [a specific characteristic].

[0021] According to a preferred embodiment, the phosphonate compound of formula (I) is such that R1 is selected from a linear or branched alkyl group having 1 to 6 carbon atoms and a -P(=O)(OM)OM group, preferably R1 is a -P(=O)(OM)OM group.

[0022] According to a preferred embodiment, M is selected from H, monovalent or divalent metal atoms, such as Li, Na, K, Cs, Ca, Mg, Cu, and Zn, and protonated organic bases, preferably M is selected from alkali metals, such as sodium, lithium, or potassium, and organic bases, such as pyridine, N-methylpyridine, dimethylaminopyridine, aniline, dimethylaniline, imidazole, N-methylimidazole, diisopropylethylamine, diisopropylamine, or triethylamine.

[0023] More preferably, the phosphonate compound of formula (I) is such that R1 is a -P(=O)(OM)OM group and M is H or an alkali metal, preferably sodium or potassium. These compounds will be referred to herein as carbonyl bisphosphonates and their salts.

[0024] In another embodiment, the phosphonate compound of formula (I) is such that R1 is methyl and M is H.

[0025] In yet another embodiment, the phosphonate compound of formula (I) is such that R1 is n-hexyl and M is H.

[0026] Examples of other phosphonate compounds of formula (I) are shown below:

[0027] [ka]

[0028] The deprotecting agent is used in the present invention in an effective amount. As used herein, the term “effective amount” means an amount or concentration sufficient to cleave the 3'-ONH2 polynucleotide when in contact with it. Those skilled in the art will understand that the effective amount of the deprotecting agent can be easily determined by conventional techniques, such as those shown in the examples. In some embodiments, for example, when carbonyl phosphonates and their salts are used as the deprotecting agent, the effective amount is given by a concentration in the range of 0.1 to 500 mM, or in other embodiments, in the range of 0.1 to 200 mM, or in other embodiments, in the range of 0.1 to 100 mM. Unless otherwise stated, the molar ratio of polynucleotide to deprotecting agent is typically in the range of 1:1 to 1:1000, preferably 1:1 to 1:100.

[0029] The deprotecting agent is typically supplied in an aqueous solution (i.e., buffer) buffered to a pH of 4 to 8, preferably 5 to 7, and the buffer may be selected from citrates, phosphates, e.g., sodium phosphate, acetates, e.g., sodium acetate, or bicarbonates. As shown in the examples, some of the deprotecting agents of the present invention, such as carbonyl bisphosphonates, are more effective at a pH of around 5, while others, such as heptanoylphosphonic acid, are more effective at a pH of around 8. Those skilled in the art will be able to select the appropriate deprotecting agent depending on the pH of the intended synthesis.

[0030] According to a preferred embodiment of the present invention, the buffer may further contain 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, particularly at higher pH. This compound may be present in the buffer in an amount ranging from 1 to 100 equivalents, preferably 1 to 10 equivalents, relative to the compound of formula (I).

[0031] In addition to water, the buffer may further contain at least one organic solvent miscible with water, particularly 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 contain one or more denaturing agents, such as formamide, urea, dimethylformamide, or dimethyl sulfoxide (up to about 25%).

[0032] The synthesis of tetrasodium salts of carbonyl bisphosphonates is described in the literature (Khomich, OA; Yanvarev, DV; Novikov, RA; Kornev, AB; Puljulla, E.; Vepsalainen, J.; Khomutov, AR; Kochetkov, SN On the Reaction of Carbonyl Diphosphonic Acid with Hydroxylamine and O-Alkylhydroxylamines: Unexpected Degradation of PCP Bridge. Molecules 2017, Vol. 22, p. 1040 2017, 22 (7), 1040).

[0033] The inventors have found that this compound can be obtained by various means starting from tetraisopropyl(dichloromethylene)bis(phosphonate). This compound itself can be prepared as taught in Quimby, OT; Prentice, JB; Nicholson, DA. Tetrasodium Carbonyldiphosphonate. Synthesis, Reactions, and Spectral Properties. Journal of Organic Chemistry 1967, 32 (12), pp. 4111-4114. In the first step, the compound can be hydrolyzed in dioxane in the presence of TMSBr, or by continuous heating with an 18% aqueous hydrochloric acid solution (Purdie, M. Process for Preparing Methylene Bisphosphonic and Salts. U.S. Patent Application Publication No. 6657076B1, September 11, 2000). However, both of these procedures are time-consuming and require aggressive corrosive reagents. Quantitative deesterification can be more easily achieved by thermally decomposing a solid tetraisopropyl derivative at a temperature above 200°C to obtain dichloromethylenebisphosphonic acid. For better safety, percussion deesterification may be performed in boiling tetrachloroethane, as described by Quimby, OT (see above). The inventors have found that this process can be carried out even faster by adding the tetraisopropyl ester to a boiling solution in small amounts in a halogenated organic solvent such as dichlorobenzene. This thermal decomposition process is exothermic and is autocatalyzed by the formed bisphosphonic acid, immediately generating gas. Therefore, the starting ester must be added to the reactor slowly in small amounts. Next, the basic hydrolysis of the dichloromethylene derivative can be carried out by refluxing it with an aqueous solution of sodium hydroxide.

[0034] Naturally, other carbonyl bisphosphonates may be prepared by following a similar strategy, changing the base used in the final step to another organic or inorganic base, such as triethylamine, pyridine, or potassium hydroxide. This may be further extended to starting materials having alkyl groups other than isopropyl groups.

[0035] In addition, asymmetric carbonyl phosphonates with different substituents are generally RC(O)Cl and P(OR 1 )3 reacts with RC(O)P(O)(OR 1 )2 is obtained, followed by R 1 It can be prepared by acidic or basic hydrolysis depending on the conditions. Examples of synthetic routes are shown in ZY Peng et al., Biochemical Pharmacology, Vol. 49, No. 1, pp. 105-113 (1995); R. Karaman et al., J. Chem. Soc. Perkin Trans. 1, pp. 765-774 (1989); and CE McKENNA et al., J. Chem. Soc., Chem. Commun., pp. 246-247 (1989). These experimental conditions may, of course, be optimized by those skilled in the art.

[0036] Therefore, the method for preparing the compound of formula (I) according to the present invention, which is a carbonyl bisphosphonate, is as follows: 1) A step of mixing tetraalkylmethylenediphosphonate with a sodium hypochlorite solution at room temperature until a first precipitate is formed, 2) A step of recovering the precipitate and dissolving it in a boiling halogenated organic solvent (such as dichlorobenzene or tetrachloroethylene) to obtain a mixture, 3) A step of gradually adding this mixture to the reactor and refluxing the contents of the reactor, thereby resulting in the generation of propene gas and the formation of a second precipitate containing dihalomethylenediphosphonate, 4) A step of cooling the reactor and recovering the second precipitate, 5) A step of mixing the precipitate with an aqueous solution of an inorganic base and refluxing until a third precipitate is obtained, 6) The step of cooling the mixture and recovering the third precipitate containing carbonyl bisphosphonate Includes.

[0037] In one embodiment, the inorganic base used in step (5) is sodium hydroxide. In this embodiment, the process may include a further step of substituting the sodium ion of the carbonyl bisphosphonate with a protonated organic base. This further step makes it possible to improve the solubility of the deprotecting agent. In another embodiment, the inorganic base used in step (5) is potassium hydroxide. In this embodiment, ion exchange is not required, but if desired, the potassium ion of the carbonyl bisphosphonate may be substituted with a protonated organic base. The deprotecting agent in the form of a potassium salt thus obtained has been shown to have high solubility and to result in improved yield and purity. In these ion exchange steps, which can be carried out under conditions easily adjustable by those skilled in the art, the organic base may be selected from pyridine, N-methylpyridine, dimethylaminopyridine, aniline, dimethylaniline, imidazole, N-methylimidazole, diisopropylethylamine, diisopropylamine, and triethylamine, preferably N-methylimidazole.

[0038] The above-mentioned deprotective agents are used in methods for synthesizing polynucleotides.

[0039] According to a first aspect of the present invention, the polymerase used in this method is a template-independent polymerase. In this case, the method of the present invention is preferably enzymatic DNA synthesis or enzymatic RNA synthesis.

[0040] These methods involve repeating cycles of the process (shown in Figure 2), in which a given 3'-O-protected nucleotide is (i) coupled to an initiator or growth chain in each cycle, and (ii) deprotected. General elements of template-free enzymatic synthesis of polynucleotides are described in the following references: Champion et al., International Publication No. 2019 / 135007; Hiatt et al., U.S. Patent No. 5763594; and Jensen et al., Biochemistry, 57: pp. 1821-1832 (2018). 3'-O-NH2 nucleoside triphosphates that can be used as 3'-O-protected nucleotides in this process can be obtained, for example, as described in International Publication No. 2020 / 165334 or International Publication No. 2021 / 198040.

[0041] In another aspect of the present invention, the polymerase is a template-dependent polymerase. In this case, the method of the present invention is preferably a method for sequencing DNA by synthesis, such as synthetic sequencing (SBS) and binding sequencing (SBB).

[0042] According to one embodiment, the sequencing method by synthesis is (a) A step of preparing initiators which are polynucleotides each having a free 3'-hydroxyl group, (b) in the reaction mixture until a polynucleotide is formed, (i) under extension conditions, an initiator or extension fragment having a free 3'-hydroxyl group is contacted with a fluorescently labeled 3'-O-NH2 nucleotide and polymerase, thereby extending the initiator or extension fragment by the incorporation of the fluorescently labeled 3'-O-aminonucleotide to form a 3'-O-amino extension fragment, (ii) excess nucleotides that were not incorporated are washed away, (iii) the fluorescent signal is read to determine that the nucleotides have been incorporated, (iv) the fluorescent label is removed and the 3'-O-amino group is removed with a phosphonate compound of formula (I) to form an extension fragment having a free 3'-hydroxyl group, and (v) steps (i) to (iv) are repeated until sequencing is complete, and the cycle is repeated. Includes.

[0043] According to one embodiment, the sequencing method by coupling is (a) A step of preparing initiators which are polynucleotides each having a free 3'-hydroxyl group, (b) In the reaction mixture until a polynucleotide is formed, (i) under extension conditions, an initiator or extension fragment having a free 3'-hydroxyl group is contacted with a 3'-O-NH2 nucleotide and polymerase, thereby extending the initiator or extension fragment by the incorporation of a 3'-O-aminonucleotide to form a 3'-O-amino extension fragment, (ii) under extension conditions, the 3'-O-amino extension fragment is contacted with a fluorescently labeled nucleotide and polymerase, thereby binding the fluorescently labeled nucleotide to the active site of the polymerase, (iii) the unbound fluorescently labeled nucleotide is washed away, (iv) the fluorescence signal is read to determine that a nucleotide has been bound to the active site, (v) the bound nucleotide is washed away, (vi) the 3'-O-NH2 polynucleotide is deprotected with a phosphonate compound of formula (I), and (vii) steps (i) through (vi) are repeated until sequencing is complete. Includes.

[0044] A specific embodiment of the process for synthesizing polynucleotides according to the present invention will be described below with reference to Figure 2.

[0045] The initiator polynucleotide (100) is prepared, for example, by being attached to a solid support (102) and has a free 3'-hydroxyl group (103). An initiator polynucleotide (100) (or an initiator polynucleotide extended in a subsequent cycle) is added under conditions effective for the enzymatic incorporation of the 3'-O-protected NTP into the 3' end of the initiator polynucleotide (100) (or an initiator polynucleotide extended in a subsequent cycle) with a polymerase, e.g., TdT or its variant for normal DNA synthesis (e.g., Ybert et al., International Publication No. 2017 / 216472; Champion et al., International Publication No. 2019 / 135007), or poly(A) polymerase (PAP) or poly(U) polymerase (PUP) or its variant for normal RNA synthesis (e.g., Heinisch et al., International Publication No. 2021 / 018919). This reaction produces an extended initiator polynucleotide with a protected 3'-hydroxyl group (106).

[0046] If the extended sequence is incomplete, another addition cycle is performed (108). The 3'-O-protecting group is removed by the deprotecting agent of the present invention to expose the free 3'-hydroxyl (103), and the extended initiator polynucleotide is subjected to another cycle of nucleotide addition and deprotection.

[0047] If the extended initiator polynucleotide contains the complete sequence, the 3'-O-protecting group may be removed, i.e., deprotected, and the desired sequence may be cleaved from the original initiator polynucleotide (110). Such cleavage may be carried out using any of the various single-strand cleavage techniques, for example, by inserting a cleavable nucleotide at a given location within the original initiator polynucleotide. An example of a cleavable nucleotide may be a uracil nucleotide cleaved by uracil DNA glycosylase.

[0048] As used herein, the term “protected” with respect to a particular group, such as the 3'-hydroxyl of a nucleotide or nucleoside, is intended to mean a portion covalently bonded to the particular group that prevents chemical change to that group during a chemical or enzymatic process. Whenever the particular group is the 3'-hydroxyl of a nucleoside triphosphate or an extended fragment incorporating a 3'-protected (or blocked) nucleoside triphosphate, the chemical change prevented is any further or subsequent extension of the extended fragment by an enzymatic coupling reaction.

[0049] As used herein, “initiator” refers to a short oligonucleotide sequence having a free 3'-hydroxyl group at its terminus and which can be further extended by a polymerase such as TdT. In one embodiment, the start fragment is a DNA start fragment. In an alternative embodiment, the start fragment is an RNA start fragment. In some embodiments, the start fragment has 3 to 100 nucleotides, particularly 3 to 20 nucleotides. In some embodiments, the start fragment is single-stranded. In an alternative embodiment, the start fragment may be double-stranded. In some embodiments, the initiator oligonucleotide may be attached to the synthetic support by its 5' end, and in other embodiments, the initiator oligonucleotide may be attached to the synthetic support indirectly by forming a double helix with a complementary oligonucleotide that is directly attached to the synthetic support, for example, by covalent bonding. In some embodiments, the synthetic support is a solid support, which may be a separate region of a planar solid or a bead.

[0050] In some embodiments, the initiator may include a non-nucleic acid compound having a free hydroxyl group that can couple TdT to a 3'-O-protected dNTP. For example, Baiga, U.S. Patent Application Publication 2019 / 0078065 and U.S. Patent Application Publication 2019 / 0078126.

[0051] The synthetic support to which the initiator is attached may include polymers, porous or non-porous solids including beads or microspheres, planar surfaces such as glass slides, films, etc. In some embodiments, the solid support or synthetic support may include magnetic beads, particle-based resins such as agarose, etc.

[0052] Examples of synthetic supports, though not limited to them, include soluble supports, such as polymer supports (including polyethylene glycol (PEG) supports, dendrimer supports, etc.); non-swelling solid supports, such as polystyrene particles; and swelling solid supports, such as resins or gels (including agarose). The synthetic support may also form part of the reaction chamber, such as the filter film of a filter plate. Guidelines for selecting soluble supports can be found in the following references: Bonora et al., Nucleic Acids Research, 212(5): pp. 1213-1217 (1993); Dickerson et al., Chem. Rev. 102: pp. 3325-3344 (2002); Fishman et al., J. Org. Chem., 68: pp. 9843-9846 (2003); Gavert et al., Chem. Rev. 97: pp. 489-509 (1997); Shchepinov et al., Nucleic Acids Research, 25(22): pp. 4447-4454 (1997); and similar references. Guidelines for selecting solid supports can be found in Brown et al., Synlett 1998(8): pp. 817-827; Maeta et al., U.S. Patent No. 9045573; Beaucage and Iyer, Tetrahedron, 48(12): pp. 2223-2311 (1992); etc. Guidelines for attaching oligonucleotides to solid supports can be found in Arndt-Jovin et al., Eur. J. Biochem., 54: pp. 411-418 (1975); Ghosh et al., Nucleic Acids Research, 15(13): pp. 5353-5372 (1987); Integrated DNA Technologies, "Strategies for attaching oligonucleotides to solid supports", 2014(v6); Gokmen et al., Progress in Polymer Science 37: pp. 365-405 (2012); and similar references.

[0053] In some embodiments, the solid support will typically consist of porous beads or particles in the form of a resin or gel. Numerous materials are suitable as solid supports for the synthesis of polynucleotides. As used herein, the term “particles” includes, but is not limited to, “fine particles” or “nanoparticles” or “beads” or “microbeads” or “microspheres.”

[0054] In some embodiments, the porous resin support derivatized with the initiator has an average pore size of at least 10 nm, or at least 20 nm, or at least 50 nm. In other embodiments, such a porous resin support has an average pore size in the range of 10 nm to 500 nm, or in the range of 50 nm to 500 nm.

[0055] In some embodiments, the initiator is attached to a planar support for large-scale parallel synthesis of oligonucleotides, which is done via inkjet delivery of reagents, for example, as described in Horgan et al., International Publication No. 2020 / 020608 (which is incorporated herein by reference). In some embodiments, such a planar support comprises a uniform coating of initiators having protected 3'-hydroxyls, where distinct reaction sites may be defined, for example, by delivering a deprotection solution to separate locations. In other embodiments, such planar supports may comprise an array of distinct reaction sites, each containing an initiator, which may be formed on a substrate by photolithography, for example, Brennan, U.S. Patent No. 5,474,796; Peck et al., U.S. Patent No. 1,0384,189; Indermuhle et al., U.S. Patent No. 1,066,9304; Fixe et al., Materials Research Society Symposium Proceedings, Vol. 723, Molecularly Imprinted Materials - Sensors and Other Devices, Symposium (San Francisco, California, April 2-5, 2002); or similar references.

[0056] After synthesis is complete, the polynucleotide having the desired nucleotide sequence can be released from the initiator and solid support by cleavage. For this purpose, a wide variety of cleavable ligatures or cleavable nucleotides can be used. In some embodiments, cleavage of the desired polynucleotide leaves a native free 5'-hydroxyl group in the cleaved chain, while in alternative embodiments, the cleavage step may leave a portion, such as a 5'-phosphate, which may be removed in a subsequent step, for example, by phosphatase treatment. The cleavage step can be carried out by chemical, thermal, enzymatic, or photochemical methods. In some embodiments, the cleavable nucleotide may be a nucleotide analog such as deoxyuridine or 8-oxo-deoxyguanosine, which 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 carried out by supplying deoxyinosine to the initiator as the second-to-last 3' nucleotide, which can be cleaved at the 3' end of the initiator with endonuclease V, as taught, for example, by Creton, International Publication 2020 / 165137, leaving a 5'-phosphate in the released polynucleotide. Returning to Figure 2, in some embodiments, in each synthesis step, the ordered nucleotide sequence is coupled to the initiator nucleic acid in the presence of a 3'-O-protected NTP using a polymerase such as TdT. The above method may also include a washing step after each reaction or extension step, and after each deprotection step.

[0057] When the sequence of polynucleotides on a synthetic support contains reverse-complementary subsequences, the formation of hydrogen bonds between the reverse-complementary regions may create intramolecular or intermolecular secondary structures. In some embodiments, a base-protected moiety for the extracyclic amine is selected so that the hydrogen of the protected nitrogen cannot participate in hydrogen bonding, thereby preventing the formation of such secondary structures. That is, the base-protected moiety can be used to prevent the formation of hydrogen bonds, such as those formed between nucleosides A and T, and between G and C, as is typical. At the end of synthesis, the base-protected moiety can be removed, and the polynucleotide product can be cleaved from the solid support, for example, by cleaving its initiator. In addition to imparting a base-protecting group to the 3'-O-protected NTP monomer, the extension reaction may be carried out at a higher temperature using a thermally stable polymerase. For example, a thermally stable template-free polymerase active above 40°C may be used, or in some embodiments, a thermally stable template-free polymerase active in the range of 40 to 85°C may be used, or in some embodiments, a thermally stable template-free polymerase active in the range of 40 to 65°C may be used.

[0058] In some embodiments, the extension (or coupling) conditions may include adding a solvent that inhibits hydrogen bonding or base stacking to the extension reaction mixture. Examples of such solvents include water-miscible solvents with low dielectric constant, such as dimethyl sulfoxide (DMSO) and methanol. Similarly, in some embodiments, the extension conditions may include supplying a chaotropic agent, which is not limited to n-butanol, ethanol, guanidium chloride, lithium perchlorate, lithium acetate, magnesium chloride, phenol, 2-propanol, sodium dodecyl sulfate, thiourea, and urea. In some embodiments, the extension conditions include the presence of a secondary structure-inhibiting amount of DMSO. In some embodiments, the extension conditions may include supplying a DNA-binding protein that inhibits secondary structure formation, which is not limited to single-strand binding proteins, helicases, and DNA glycoses.

[0059] Unprotected 3'-O-amino-dNTPs may be purchased from commercial suppliers or synthesized using publicly available techniques, e.g., Benner, U.S. Patent Nos. 7,544,794 and 8,212,020.

[0060] If a base-protected dNTP is used, the method may further include step (e) of removing the base-protected moiety, which may include treatment with concentrated ammonia (for example) if it is an acyl or amidine protecting group.

[0061] The above method may also include one or more capping steps in addition to the washing step after the coupling (or extension) step. The first capping step can cap the unreacted 3'-OH group of the partially synthesized polynucleotide or inactivate it for further extension. Such a capping step is usually performed after the coupling step, and if a capping compound is used, it is always selected so as not to react with the protecting group of the monomer that has just been coupled to the grown chain. In some embodiments, such a capping step can be performed by coupling (e.g., by a second enzymatic coupling step) a capping compound that makes further coupling (e.g., with TdT) of the partially synthesized polynucleotide impossible. Such a capping compound may be a dideoxynucleoside triphosphate.

[0062] Various kits for carrying out the method of the present invention may be provided. In one embodiment, the kit may comprise one or more containers (or bottles, or vials) of synthetic reagents, at least one of which contains an effective amount of a phosphonate compound of formula (I). In some embodiments, the kit comprises a vial of polymerase and a vial of an effective amount of at least one phosphonate compound of formula (I).

[0063] In some embodiments, the kit may include, separately or together with the articles described above, one or more of the following articles: (i) one or more containers containing 3'-O-amino-dNTPs, (ii) a solid support to which the initiator is attached, (iii) a cleavage reagent for releasing the completed polynucleotide from the solid support, (iv) a washing reagent or buffer for removing unreacted 3'-O-amino-dNTPs at the end of the enzymatic addition or coupling step, and (v) one or more post-synthesis treatment reagents such as a purification column, desalting reagent, or elution reagent. [Examples]

[0064] (Example 1) Synthesis of tetrasodium carbonylbisphosphonate The NMR spectrum was recorded using a 243 MHz (31P) Bruker Avance 600 DRX spectrometer (Bruker, Karlsruhe, Germany). 31P NMR of tetrasodium carbonylbisphosphonate (D2O), δ - 0.4 (s, 2P).

[0065] Tetraisopropyl(dichloromethylene)bis(phosphonate). This compound was prepared according to the same method as described in Quimby, OT; Curry, JD; Allan Nicholson, D.; Prentice, JB; Roy, CH Metalated Methylenediphosphonate Esters. Preparation, Characterization and Synthetic Applications. J Organomet Chem 1968, 13 (1), pp. 199-207.

[0066] Specifically, tetraisopropylmethylenediphosphonate (3.45 g, 10 mmol) was added to 40 mL (approximately 0.27 mol) of a 5 wt% sodium hypochlorite solution at pH 13.0 and stirred at room temperature. After a while, a white solid precipitate formed, and stirring was continued for a further 2 hours. The reaction mixture was then filtered off, the precipitate was washed several times with water, air-dried, and then dried in a vacuum dryer. Tetraisopropyldichloromethylenediphosphonate was obtained as a white solid (4.05 g, 98%), and was used in the next step without any further purification. 1 ¹H NMR (600 MHz, chloroform-d): δ 4.94 (dqq, J = 9.5, 6.2, 6.2 Hz, 4H), 1.40 (d, J = 6.4 Hz, 24H); 13 ¹³C NMR (151 MHz, chloroform-d): δ 75.29 (t, J = 3.6 Hz), 24.47 (s), 23.64 (t, J = 3.3 Hz); 31 1P NMR (243 MHz, chloroform-d) δ 6.71 (s, 2P).

[0067] Dichloromethylenediphosphonate. A round-bottom flask equipped with a magnetic stirrer was fitted with a dropping funnel, a condenser connected to a bubble counter, and a nitrogen gas inlet. The reactor was placed in an oil bath and connected to a cylinder containing nitrogen. Tetraisopropyl ester (4.0 g, 9.7 mmol) was dissolved in dichlorobenzene (20 mL) and transferred to the dropping funnel. 1 mL of this mixture and a small portion of dichloromethylenediphosphonic acid were boiled in the reactor. As the mixture spontaneously decomposed, propene gas was immediately released, forming a gray substance. The remaining mixture was then added dropwise from the funnel to the reactor while refluxing and stirring, during which time the reactor was purged with a nitrogen stream and propene gas was released through the bubble counter. The addition of tetraisopropyl ester was completed in 20 minutes, after which reflux was continued for another 30 minutes. The reactor was removed from the heating bath, cooled to ambient temperature, and the contents were filtered off. The gray precipitate was rinsed several times with dichloromethylene on a filter, transferred to a vacuum dryer, and dried under vacuum using an oil pump to obtain 2.21 g (93%) of dichloromethylenediphosphonate as a gray hygroscopic powder. 1 H NMR (600 MHz, DMSO-d6) δ 9.47 (s, 4H); 13 C NMR (151 MHz, DMSO-d6) δ 40.06; 31 P NMR (243 MHz, DMSO-d6) δ 6.51.

[0068] Tetrasodium carbonylbisphosphonate Dichloromethylenediphosphonate (2.0 g, 8.8 mmol) was dissolved in deionized water (10 mL), and sodium hydroxide (2.1 g, 53 mmol) was slowly added in small amounts. The reaction mixture was stirred under reflux for 4 hours. After cooling to ambient temperature, anhydrous ethanol (20 mL) was added, and the formed precipitate was filtered off and washed with anhydrous ethanol to obtain the tetrasodium salt of carbonyl bisphosphonate. This crystalline material was dried in a vacuum dryer until a constant weight was obtained to obtain the dihydrate of carbonyl bisphosphonate (2.4 g, 88%). 13C NMR (151 MHz, heavy water) δ 244.8 (dd, J = 118.8 Hz); 31 P NMR (243 MHz, heavy water) δ -0.43.

[0069] (Example 2) Deprotection experiment A 1 mM solution (100 μL) of dTTP-3'-ONH2 dissolved in a 50 mM sodium acetate (pH 5.0), 50 mM sodium phosphate (pH 6.0), 50 mM sodium phosphate (pH 7.0), or 50 mM sodium bicarbonate (pH approximately 8, i.e., 8.25) buffer was shaken at 25°C in a sealed Eppendorf tube using a ThermoMixer, together with 2 equivalents of the carbonyl bisphosphonate prepared in Example 1. After incubation for 5 min / 20 min / 60 min, the corresponding reaction mixture was quenched with 5-fold excess hydroxylamine hydrochloride in 100 μL of deionized water, followed by quenching with excess 5% aqueous acetone. Thereafter, the unreacted residual dTTP-ONH2 was converted to the inactive acetoxime form - dTTP-OX. The quenched reaction mixture was analyzed by reverse-phase HPLC under the following conditions, as shown in Figure 3: C18 Xterra 3.5 μm, 4.6 × 50 mm column (Waters); Buffer A: 50 mM TEAB (pH 8.5), Buffer B: 100% ACN. Gradient elution method: Flow rate: 1.5 mL / min, 0-2 min - 0% ACN B, 5.3 min - 10% ACN, 8 min - 30% ACN, 8-10 min - 100% ACN.

[0070] As shown in Figure 3, after just 5 minutes, the conversion from 3'-ONH2 to 3'-OH reaches 100% at pH 5.

[0071] (Example 3) Deprotection comparative experiment A 1 mM solution of dTTP-3'-ONH2 (100 μL), dissolved in either 50 mM sodium acetate (pH 5.0) or 50 mM sodium phosphate (pH 6.0) buffer, was shaken at 25°C in a sealed Eppendorf tube using a ThermoMixer with 2 equivalents / 10 equivalents / 50 equivalents of sodium nitrite. After incubation for 5 min / 20 min / 60 min, the corresponding reaction mixture was quenched with 100 μL of 5% acetone in 100 mM sodium bicarbonate buffer (pH approximately 8, i.e., 8.25). This converted any unreacted residual dTTP-ONH2 into the inactive acetoxime form -dTTP-OX. The quenched reaction mixture was analyzed by reverse-phase HPLC under the following conditions, as shown in Figure 4: C18 Xterra 3.5 μm, 4.6 × 50 mm column (Waters); Buffer A: 50 mM TEAB (pH 8.5), Buffer B: 100% ACN. Gradient elution method: Flow rate: 1.5 mL / min, 0-2 min - 0% ACN B, 5.3 min - 10% ACN, 8 min - 30% ACN, 8-10 min - 100% ACN.

[0072] As shown in Figure 4A, sodium nitrite reacts with oxyamines several times slower than carbonyl bisphosphonates (Figure 4B). Even dTTP-ONH2 incubated with 50 equivalents of sodium nitrite at pH 5.0 / 60 minutes only achieved about 80% conversion to dTTP-OH.

[0073] (Example 4) Deprotection studies on various nucleotides The general scheme of the reaction carried out in this example is shown in Figure 5 (Step I: Deprotection; Step II: Conversion step, followed by HPLC).

[0074] Materials and methods: Screening tests were performed in a 96-well plate with a reaction volume of 100 μL. Nucleoside triphosphates (dATP-3'-ONH2, dTTP-3'-ONH2, dCTP-3'-ONH2, or dGTP-3'-ONH2) were dissolved at a final concentration of 1 mM in one of four 50 mM buffers (A: sodium acetate pH 5.0, B: sodium phosphate pH 6.0, C: sodium phosphate pH 7.0, D: sodium bicarbonate pH approximately 8, i.e., 8.25). Tetrasodium carbonylbisphosphonate was added to the dNTP solution at a final concentration of 2 mM, incubated for 5, 20, or 60 minutes, and then quenched with 2X hydroxylamine hydrochloride. The 96-well plate was shaken at 25°C or 40°C using a thermomixer. The catalytic effect of magnesium on cleavage was also investigated. For this purpose, the same experiment was performed in the presence of 20 mM magnesium sulfate. Prior to HPLC analysis, the reaction mixture was further quenched with acetone to convert 3'-ONH2 to the acetooxime form. The samples were analyzed by RP-HPLC with a stepwise gradient of acetonitrile in 50 mM TEAB buffer (pH 8.5). The cleavage rate was determined based on the ratio of dNTP-3'-OH to dNTP-3'-acetooxime.

[0075] HPLC conditions: Xterra RP C18 column: 4.6*50mm, (3.5um silica), Buffer A: 50mM TEAA (pH 7.0), Buffer B: 100% ACN. Method: 0-2 min - 0% ACN B, 6 min - 10% ACN, 8 min - 30% ACN, 8-10 min - 100% ACN. Flow rate: 1.5 mL / min, no loop insert.

[0076] result: As shown in Figures 6 and 7: • Effect of nucleic acid bases. All nucleotides exhibited very similar cleavage rates under all conditions. No significant heterocyclic activity was observed. • Effect of pH. The highest cleavage rate was achieved in a weakly acidic medium - pH 5.0 is optimal. • Effect of Mg(II). Magnesium salts bind to carbonyl bisphosphonates and precipitate. Magnesium significantly promotes cleavage yield at higher pH levels. • Purity. Undesirable peaks appeared only at higher pH levels (8.25). In the pH range of 5.0–7.0, the HPLC profile appeared fairly clean, with two or one major peak. No accumulation of by-products over time was observed.

[0077] (Example 5) Stability testing of nucleosides 10 mM dA, dT, dG, and dC nucleosides were incubated with 0.5 M NaNO2 and 0.5 carbonyl bisphosphonate in acetate buffer (pH 4.5, pH 5.0, pH 5.5) at pH 4.5, 5.0, and 5.5. After 72 hours of incubation, quenching was performed in sealed plates in bicarbonate buffer (pH approximately 8, i.e., 8.25) at 25°C in the dark.

[0078] Next, HPLC analysis was performed under the following conditions: C18-Gemini 5μm, 4.6×250mm (Phenomenex), A: miliQ water, B: 100% ACN. Flow rate 1mL / min. Gradient elution profile: 0% to 15% ACN in 5CV.

[0079] The results of these experiments are shown in Figures 8 to 15. The corresponding purity of residual nucleosides in the mixture is indicated.

[0080] As these figures show, for most of the nucleosides tested, the deprotective agents of the present invention (Figures 9, 11, 13, and 15) significantly reduce nucleoside degradation compared to sodium nitrite (Figures 8, 10, 12, and 14).

[0081] (Example 6) Synthesis of acetylphosphonic acid

[0082] [ka]

[0083] Acetylphosphonic acid was prepared as described in Karaman, R. et al., Journal of the Chemical Society, Perkin Transactions 1, 4, pp. 765-774 (1989). This synthesis generally consists of the Albuzov reaction of the corresponding chloroanhydride with trimethyl phosphite, followed by deesterification with trimethylsilyl bromide, as shown below.

[0084] Dimethyl acetylphosphonate. Acetyl chloride (10 mmol) was dissolved in dry DCM (20 mL) and cooled in an ice bath. Trimethyl phosphite (11 mmol) was added dropwise to the mixture while stirring. After the addition was complete, the cooling bath was removed and stirring was continued at ambient temperature for 2 hours. The reaction mixture was concentrated in a rotary evaporator to obtain a colorless liquid, which was then subjected to vacuum distillation (1-2 mmHg) to collect the fraction of the target compound. Yield: 1.37 g (90%) as a colorless liquid.

[0085] Acetylphosphonic acid. Dimethylheptanoylphosphonate (1.35 g, 9 mmol) was dissolved in dry acetonitrile (25 mL), and trimethylsilyl bromide (2.95 mL, 22.5 mmol) was slowly added to the reaction mixture while stirring. The reaction mixture was stirred at ambient temperature for 3 hours, then evaporated under vacuum, and re-evaporated twice with methanol. Yield: 1.05 g (95%) as a viscous, colorless oil.

[0086] (Example 7) Deprotection experiment The general scheme of the reaction carried out in this example is shown in Figure 16 (Step I: Deprotection; Step II: Conversion step, followed by HPLC).

[0087] A 1 mM solution (100 μL) of dTTP-3'-ONH2 dissolved in 50 mM sodium acetate (pH 5.0), 50 mM sodium phosphate (pH 6.0), 50 mM sodium phosphate (pH 7.0), or 50 mM sodium bicarbonate (pH approximately 8, i.e., 8.25) buffer was shaken at 25°C in a sealed Eppendorf tube using a ThermoMixer with 2 equivalents of acetylphosphonic acid. After incubation for 5 min / 20 min / 60 min, the corresponding reaction mixture contained unreacted dTTP-3'-ONH2 and cleavage products (dTTP-3'-OH), as well as an adduct of dTTP-3'-ONH2 with acetylphosphonic acid (Schiff base) (dTTP-3'-ONC(CH3)PO3H2). The reaction mixture was quenched with 2 equivalents of hydroxylamine hydrochloride in 100 μL of deionized water, followed by quenching with an excess of 5% aqueous acetone. This converted the unreacted residual dTTP-ONH2 to the inactive acetoxime form - dTTP-OX. The quenched reaction mixture was analyzed by reverse-phase HPLC under the following conditions: C18 Xterra 3.5 μm, 4.6 × 50 mm column (Waters); Buffer A: 50 mM TEAB (pH 8.5), Buffer B: 100% ACN. Gradient elution method: Flow rate: 1.5 mL / min, 0-2 min - 0% ACN B, 5.3 min - 10% ACN, 8 min - 30% ACN, 8-10 min - 100% ACN.

[0088] As shown in Figure 17, the conversion from 3'-ONH2 to 3'-OH reaches approximately 60% at pH 6 or 7 after 60 minutes.

[0089] (Example 8) Enzymatic DNA synthesis experiment Oligodeoxyribonucleotide synthesis was performed in a 96-well filter plate according to a standard procedure using four deoxyribonucleotides (dTTP-ONH2, dATP-ONH2, dGTP-ONH2, dCTP-ONH2) protected with a TdT modifying enzyme and a 3'-ONH2 group. Both the extension and deprotection steps were performed under identical conditions for both weakly acidic pH nitrite and carbonyl bisphosphonate as deprotection buffers. Pools of 24 52-mer oligonucleotides (SEQ ID NOs: 1-24) were synthesized in two replicates, desalted by precipitation, and analyzed by next-generation sequencing (NGS). Nucleic acid base substitution values ​​for both types of deprotection buffers (carbonyl bisphosphonate and nitrite) are shown in Figure 18.

[0090] As shown in this figure, nitrite causes more changes to the structure of nucleic acid bases than the deprotective agent of the present invention, in particular, amino group exchange of cytosine (C), which leads to a higher error rate in NGS.

[0091] (Example 9) Synthesis of heptanoylphosphonic acid This synthesis was reproduced from Karaman, R. et al., Journal of the Chemical Society, Perkin Transactions 1, 4, pp. 765-774 (1989), with the following minor modifications to the procedure.

[0092] [ka]

[0093] Dimethyl heptanoylphosphonate. Heptanoyl chloride (10 mmol) was dissolved in dry DCM (20 mL) and cooled in an ice bath. Trimethyl phosphite (11 mmol) was added dropwise to the mixture while stirring. After the addition was complete, the cooling bath was removed and stirring was continued at ambient temperature for 2 hours. The reaction mixture was concentrated in a rotary evaporator to obtain a colorless oily substance, which was then subjected to vacuum distillation (0.2 mmHg) to collect the fraction of the target compound. Yield: 1.84 g (82%) as a viscous, colorless liquid.

[0094] Heptanolphosphonic acid. Dimethyl heptanoylphosphonate (1.8 g, 8.1 mmol) was dissolved in dry acetonitrile (25 mL), and trimethylsilyl bromide (2.67 mL, 20.3 mmol) was slowly added to the reaction mixture while stirring. The reaction mixture was stirred at ambient temperature for 3 hours, then evaporated under vacuum, and re-evaporated twice with methanol. The remaining oil was dissolved in water (50 mL) and ethyl acetate (50 mL) in a separatory funnel. The organic layer was separated and concentrated under vacuum. Yield: 1.08 g (69%) as a white solid.

[0095] (Example 10) Deprotection experiment The general scheme of the reaction carried out in this example is shown in Figure 19 (Step I: Deprotection; Step II: Conversion step, followed by HPLC).

[0096] Materials and methods: The screening test was performed in a 96-well plate with a reaction volume of 100 μL. Nucleoside triphosphate (dTTP-3'-ONH2) was dissolved at a final concentration of 1 mM in one of four 50 mM buffers (A: sodium acetate pH 5.0, B: sodium phosphate pH 6.0, C: sodium phosphate pH 7.0, D: sodium bicarbonate pH approximately 8, i.e., 8.25). Heptanoylphosphonic acid was added to the dTTP solution at a final concentration of 2 mM, incubated for 5, 20, or 60 minutes, and then quenched with hydroxylamine hydrochloride at twice the concentration. The 96-well plate was shaken at 25°C or 40°C using a thermomixer. Before HPLC analysis, the reaction mixture was further quenched with acetone to convert 3'-ONH2 to the acetooxime form. The samples were analyzed by RP-HPLC with a stepwise gradient of acetonitrile in 50 mM TEAB buffer (pH 8.5). The cleavage rate was determined based on the ratio of dNTP-3'-OH to dNTP-3'-acetoxime.

[0097] HPLC conditions: Xterra RP C18 column: 4.6*50mm, (3.5um silica), Buffer A: 50mM TEAA (pH 7.0), Buffer B: 100% ACN. Method: 0-2 min - 0% ACN B, 6 min - 10% ACN, 8 min - 30% ACN, 8-10 min - 100% ACN. Flow rate: 1.5 mL / min, no loop insert.

[0098] result: As shown in Figures 19 and 20: • The effect of pH. The highest cleavage rate was achieved when the pH was raised from 5 to approximately 8 - pH 8.25 is optimal. • Purity. Undesirable peaks appeared at lower pH levels (5-6). In the pH range of 7.0-8.25, the HPLC profile showed only two or one major peak.

[0099] item This specification further includes the following: 1. A method for synthesizing polynucleotides, (a) A step of preparing initiators which are polynucleotides each having a free 3'-hydroxyl group, (b) in the reaction mixture until a polynucleotide is formed, (i) under extension conditions, an initiator or extension fragment having a free 3'-hydroxyl group is contacted with 3'-O-NH2 nucleoside triphosphate and polymerase, thereby extending the initiator or extension fragment by the incorporation of 3'-O-nucleoside triphosphate to form a 3'-O-amino extension fragment, and (ii) the extension fragment is deprotected to form an extension fragment having a free 3'-hydroxyl group, and the cycle is repeated. Includes, Deprotection of the extended fragment is performed using the following equation (I): [ka] [In the formula, M is (a) H, (b) monovalent or divalent metal atom, (c) HNR 6 3 +or NR 6 4 + (In the formula, each R 6 (1) independently represents H or a linear or branched alkyl group having 1 to 6 carbon atoms), (d) an organic base, (e) a linear or branched alkyl group having 1 to 6 carbon atoms, (f) three Si(R4) groups (wherein each R4 is independently selected from an aryl group or a linear or branched alkyl group having 1 to 6 carbon atoms); and (g) two PH groups, selected from the group. R is one of the following groups: (a)-CO-R1 (wherein R1 is selected from (i)H, (ii)-COOH, (iii)CN, (iv)a linear or branched alkyl group having 1 to 6 carbon atoms, (v)aryl group, or (vi)-P(=O)(OM)OM group); (b)-C(=CH-R2)-X-R3 group (wherein X is O or -NR) a R b (In the formula, R a and R b R1 is independently selected from alkyl groups having 1 to 6 carbon atoms, R2 is a linear or branched alkyl group having 1 to 6 carbon atoms, and R3 is an M or Si(R4)3 group) and (c)-C(Y)(OH)-R5 group (wherein Y is -CN or -SO3) - (where R5 is selected from linear or branched alkyl groups having 1 to 6 carbon atoms) This is carried out by contacting with at least one phosphonate compound having method. 2. A method for synthesizing polynucleotides, (a) A step of preparing initiators which are polynucleotides each having a free 3'-hydroxyl group, (b) in the reaction mixture until a polynucleotide is formed, (i) under extension conditions, an initiator or extension fragment having a free 3'-hydroxyl group is contacted with 3'-O-NH2 nucleoside triphosphate and polymerase, thereby extending the initiator or extension fragment by the incorporation of 3'-O-aminonucleoside triphosphate to form a 3'-O-amino extension fragment, and (ii) the extension fragment is deprotected to form an extension fragment having a free 3'-hydroxyl group, and the cycle is repeated. Includes, Deprotection of the extended fragment is given by the following equation (I): [ka] [In the formula, Each M is independently (a) H, (b) monovalent or divalent metal atom, (c) HNR 6 3 + or NR 6 4 + (In the formula, each R 6 (1) independently represents H or a linear or branched alkyl group having 1 to 6 carbon atoms), (d) a protonated organic base, (e) a linear or branched alkyl group having 1 to 6 carbon atoms, (f) three Si(R4) groups (wherein each R4 is independently selected from an aryl group or a linear or branched alkyl group having 1 to 6 carbon atoms); and (g) two PH groups, selected from the group. R is one of the following groups: (a)-CO-R1 (wherein R1 is selected from (i)H, (ii)-COOH, (iii)CN, (iv)a linear or branched alkyl group having 1 to 6 carbon atoms, (v)aryl group, or (vi)-P(=O)(OM)OM group); (b)-C(=CH-R2)-X-R3 group (wherein X is O or -NR) a R b (In the formula, R a and R b (c)-C(Y)(OH)-R5 group (wherein Y is -CN or -SO3)- (where R5 is selected from linear or branched alkyl groups having 1 to 6 carbon atoms) This is carried out by contacting with at least one phosphonate compound having method. [Explanation of symbols]

[0100] 100 Initiator Polynucleotides 102 Solid support 103 Free 3'-hydroxyl group 104 Template-Free Polymerase 106 Extended initiator polynucleotides 108 Repeated deprotection 110 Deprotection cleavage

Claims

1. A method for synthesizing polynucleotides, (a) A step of providing an initiator which is a polynucleotide having free 3'-hydroxyl groups, (b) In the reaction mixture until a polynucleotide is formed, (i) under extension conditions, an initiator or extension fragment having a free 3'-hydroxyl group is formed into 3'-O-NH 2 (ii) a process of contacting the nucleoside triphosphate with polymerase, thereby extending the initiator or extension fragment by incorporating 3'-O-aminonucleoside triphosphate to form a 3'-O-amino extension fragment, and repeating the cycle of (ii) deprotecting the extension fragment to form an extension fragment having a free 3'-hydroxyl group. Includes, Deprotection of the extended fragment is given by the following equation (I): 【Chemistry 1】 [In the formula, Each M is independently H; a monovalent or divalent metal atom; HNR 6 3 + or NR 6 4 + (wherein each R 6 independently represents H or a linear or branched alkyl group having 1 to 6 carbon atoms); a protonated organic base; a linear or branched alkyl group having 1 to 6 carbon atoms; and Si(R 4 ) 3 group (wherein each R 4 is independently selected from the group consisting of an aryl group or a linear or branched alkyl group having 1 to 6 carbon atoms), R is -CO-R 1 (In the formula, R 1 [The group is selected from (i) a linear or branched alkyl group having 1 to 6 carbon atoms, (ii) an aryl group, and (iii) a P(=O)(OM)OM group] This is carried out by contacting with at least one phosphonate compound having method.

2. The phosphonate compound of formula (I) is R 1 is a linear or branched alkyl group having 1 to 6 carbon atoms or a -P(=O)(OM)OM group, preferably R 1 The method according to claim 1, wherein is a -P(=O)(OM)OM group.

3. The method according to claim 1 or 2, wherein M is selected from H, monovalent or divalent metal atoms, such as Li, Na, K, Cs, Ca, Mg, Cu, and Zn, and protonated organic bases, preferably M is selected from alkali metals, such as sodium, lithium, or potassium, and organic bases, such as pyridine, N-methylpyridine, dimethylaminopyridine, aniline, dimethylaniline, imidazole, N-methylimidazole, diisopropylethylamine, diisopropylamine, or triethylamine.

4. The method according to any one of claims 1 to 3, wherein the effective amount is given by a concentration of the phosphonate compound in an aqueous solution buffered to a pH of 4 to 8, preferably 5 to 7, at a concentration of 0.1 to 500 mM, for example, 0.1 to 200 mM or 0.1 to 100 mM.

5. The method according to claim 4, wherein the buffer further comprises at least one inorganic salt of a divalent metal, such as magnesium, calcium, zinc, or copper, preferably magnesium sulfate.

6. The method according to any one of claims 1 to 5, wherein the polymerase is a template-independent polymerase.

7. The method according to claim 6, wherein the method is enzymatic DNA synthesis or enzymatic RNA synthesis.

8. The method according to any one of claims 1 to 5, wherein the polymerase is a template-dependent polymerase.

9. The method according to claim 8, wherein the method is a method of DNA sequencing by synthesis, for example, synthesis sequencing (SBS) or binding sequencing (SBB).

10. Synthetic sequencing method, (a) A step of providing an initiator which is a polynucleotide having free 3'-hydroxyl groups, (b) in the reaction mixture until a polynucleotide is formed, (i) under extension conditions, an initiator or extension fragment having a free 3'-hydroxyl group is fluorescently labeled with 3'-O-NH 2 (ii) bringing the nucleotide into contact with polymerase, thereby extending the initiator or extension fragment by the incorporation of a fluorescently labeled 3'-O-aminonucleotide to form a 3'-O-amino extension fragment; (iii) washing away excess, unincorporated nucleotides; (iv) removing the fluorescent label and removing the 3'-O-amino group with a phosphonate compound specified in any one of claims 1 to 5 to form an extension fragment having a free 3'-hydroxyl group; and (v) repeating steps (i) to (iv) until sequencing is complete. The method according to claim 9, including the method described in claim 9.

11. The sequencing method using coupling is (a) A step of providing an initiator which is a polynucleotide having free 3'-hydroxyl groups, (b) In the reaction mixture until a polynucleotide is formed, (i) under extension conditions, an initiator or extension fragment having a free 3'-hydroxyl group is added to the 3'-O-NH group. 2 (ii) Contacting the initiator or extension fragment with a nucleotide and polymerase, thereby extending the 3'-O-aminonucleotide to form a 3'-O-amino extension fragment; (ii) under extension conditions, contacting the 3'-O-amino extension fragment with a fluorescently labeled nucleotide and polymerase, thereby binding the fluorescently labeled nucleotide to the active site of the polymerase; (iii) washing away the unbound fluorescently labeled nucleotide; (iv) reading out a fluorescent signal to determine that a nucleotide has bound to the active site; (v) washing away the bound nucleotide; and (vi) 3'-O-NH with a phosphonate compound as defined in any one of claims 1 to 5. 2 The polynucleotide is deprotected, and the cycle is repeated, with steps (i) through (iv) being repeated until sequencing is complete. The method according to claim 9, including the method described in claim 9.

12. A kit for the enzymatic synthesis of polynucleotides, comprising a vial of polymerase and an effective amount of a vial of a phosphonate compound of formula (I) as defined in any one of claims 1 to 5.

13. A method for deprotecting a 3'-O-amino extension fragment of a polynucleotide in an enzymatic method for synthesizing a polynucleotide, comprising the step of contacting the extension fragment with at least one phosphonate compound of formula (I) as defined in any one of claims 1 to 5.

14. A method for preparing a phosphonate compound of formula (I) which is a carbonyl bisphosphonate, 1) A step of mixing tetraalkylmethylenediphosphonate with a sodium hypochlorite solution at room temperature until a first precipitate is formed, 2) A step of collecting the precipitate and dissolving it in a boiling halogenated organic solvent to obtain a mixture, 3) A step of gradually adding this mixture to the reactor and refluxing the contents of the reactor, thereby resulting in the generation of propene gas and the formation of a second precipitate containing dichloromethylenediphosphonate, 4) A step of cooling the reactor and recovering the second precipitate, 5) A step of mixing the precipitate with an aqueous solution of an inorganic base and refluxing until a third precipitate is obtained, 6) The step of cooling the mixture and recovering the third precipitate containing carbonyl bisphosphonate Methods that include...

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