WO2025149479A1 - Synthesis of modified nucleoside triphosphates - Google Patents

Abstract

The invention relates to a new diastereoselective synthesis method for highly modified nucleoside triphosphates with two conjugable groups. The method works in liquid phase with conventional laboratory equipment as used in any chemical laboratory or production. For this, a new chiral P(V) phosphorylation reagent has been designed that allows highly efficient diastereoselective synthesis under mild conditions. Overall, a highly efficient, diastereoselective, high quality, cost-effective synthetic method of modified nucleoside triphosphates has been developed.

Description

SYNTHESIS OF MODIFIED NUCLEOSIDE TRIPHOSPHATES

FIELD OF THE INVENTION

The present invention relates generally to the synthesis of nucleoside triphosphates, and in particular nucleoside triphosphates with a 5’-phosphoramidate. The invention also relates to reagents and intermediates used in such synthesis.

BACKGROUND

Over the last two decades, biological membranes have emerged as an important tool in a variety of biomedical applications. This includes the use of lipid bilayer membranes in nanopore based sequencing applications, where nanopores provide a constant and reproducible physical aperture, through which a target molecule can be directed and sequenced.

One approach for nanopore-based sequencing of, for example, nucleic acids involves a sequencing-by-expansion approach by transcribing the sequence of nucleic acids into a simple to measure polymer molecule called an Xpandomer. Much like with polymerase chain reaction (PCR), Xpandomer synthesis is based on the natural function of DNA replication where expandable nucleoside triphosphates (XNTPs) act as substrates for replication.

Xpandomer synthesis is based on four easily differentiated XNTPs that include High Signal-to-Noise Reporters, one for each DNA base. Engineered polymerases incorporate these modified nucleotides into Xpandomers, producing a copy of the target nucleic acid template from the library. As the Xpandomer molecule transits through the nanopore, the distinct electrical signal of each base reporter is easily identifiable to enable highly accurate and high throughput nanoporebased nucleic acid sequencing. See, e.g., U.S. Pat. No. 7,939,259, titled “High Throughput Nucleic Acid Sequencing by Expansion;” and PCT publication WO 2020/236526 Al, titled “Translocation control elements, reporter codes, and further means for translocation control for use in nanopore sequencing”, both of which are hereby incorporated herein in their entirety.

Nucleoside triphosphates with two clickable (e.g. terminal alkyne) groups, such as dNTP- 2c, are used as building blocks for reagents in nanopore sequencing, especially within the technology of sequencing by expansion. Within this technology, such building blocks are typically clicked to tether molecules that contain reporter and translocation control elements in order to generate XNTPs. Structures and previous processes of sequencing by synthesis and reagents used therein are disclosed in WO 2016/081871, WO 2020/236526 and WO 2020/172479. Exemplary nucleoside triphosphate molecules with two terminal alkyne groups are shown in Fig. 1.

The synthesis of nucleoside triphosphates with a 5’ phosphoramidate and two clickable groups has previously been conducted by solid-phase synthesis using commercially available DNA/RNA-synthesizers. The a-phosphoramidate in such nucleoside triphosphates (and XNTPs derived therefrom) is stereogenic, and the nucleoside triphosphates are obtained by the process of the art as 1 : 1 diastereomeric mixtures of two isomers with different configurations at the a- phosphoramidate. One configuration (“active” diastereomer) provides the desired functional performance in sequencing by expansion due to better acceptance and incorporation by the polymerase compared to the “inactive” diastereomer. It is thus desirable to remove the “inactive” diastereomer from the final solution used in a polymerase reaction, or even to avoid its production in the first place.

Overall, the manufacturing process for nucleoside triphosphate with two clickable groups according to WO 2016/081871 via solid-phase synthesis provides suboptimal yields and purity, is relatively expensive, is not diastereoselective and cannot be readily scaled up (see Fig. 2 and 6B). There is thus a need for an improved synthesis method for nucleoside triphosphates that can be used e.g. in sequencing by expansion. The present invention addresses these needs and others.

SUMMARY OF THE INVENTION

To address the shortcomings of the prior art, a new synthetic method has been developed that works in liquid phase with conventional laboratory equipment as used in any chemical laboratory or production, and can be readily scaled up. For this, a new chiral P(V) phosphorylation reagent has been designed that allows highly efficient synthesis under mild conditions. By using the chiral phosphorylation reagent in enantiopure form or with a high enantiomeric excess, diastereoselective synthesis is possible.

The method is based on phosphorylation of a 5 ’-amino-5’ -deoxynucleoside with a chiral phosphorylation reagent that comprises two leaving groups (LG¹ and LG²) with different properties. LG¹ can be selectively substituted in the reaction with the amine at the 5’ position of the 5 ’-amino-5 ’-deoxynucleoside in an Sx2-type reaction mechanism on the phosphorus (SN2P). A monophosphoramidate intermediate product (a nucleoside monophosphoramidate) is formed thereby that still contains LG². Subsequently, the nucleoside monophosphoramidate is selectively converted in another SN2P reaction with a pyrophosphate to provide a nucleoside triphosphate. The chiral phosphorylation reagent further comprises residue R⁴. The type of nucleoside triphosphate obtained by the method thus depends on the type of 5’ -amino-5’ deoxynucleoside used as reactant as well as on the nature of R⁴, and is not particularly limited in this respect. For example, the nucleoside triphosphate (as well as intermediates in the production thereof), can have two conjugable, such as clickable, groups, one attached to the a-phosphoramidate, and one attached to the nucleobase.

If the chiral phosphorylation reagent is used as a racemic mixture, the nucleoside monophosphoramidate and the corresponding nucleoside triphosphate are obtained as a diastereomeric mixture (see scheme in Fig. 3). If the chiral phosphorylation reagent is used in enantiopure form or with a high enantiomeric excess, one of the diastereomers of the nucleoside monophosphoramidate and the corresponding nucleoside triphosphate can be selectively formed (see path A and path B, in Fig. 4). Generally, diastereoselective synthesis will selectively produce the “active” diastereomer (path B). The chiral information is transferred in S\2-type reactions when the nucleoside monophosphoramidate is formed, and when the nucleoside monophosphoramidate is converted with a pyrophosphate to the nucleoside triphosphate. The nucleoside triphosphate can be further modified by linking the a-phosphoramidate to the nucleobase using a tether molecule. The resulting nucleoside triphosphates with an intramolecular tether are useful in e.g. sequencing by expansion.

Moreover, the (diastereoselective) synthesis of the nucleoside monophosphoramidate and the corresponding nucleoside triphosphate can be performed in one pot without isolation or workup of the intermediate compound.

The scheme in Fig. 5 shows the synthesis and enantioselective separation of the phosphorylation reagent.

Figure 6A shows the synthetic path of a dGTP-2c as an example. Figure 6B shows a comparison of analytical HPLC chromatograms of crude dGTP-2c products using the conventional solid-phase synthesis method (top), the new liquid phase method using racemic phosphorylation reagent (middle), and the new diastereoselective method using enantiopure phosphorylation reagent (bottom). Overall, the invention makes a two-step (convergent) method for producing nucleoside triphosphates possible. In contrast, previous solid-phase synthesis required ~15 steps plus additional phosphite reagent synthesis. Overall yields improved from 1-2% (solid phase synthesis) to 40% or even more when starting with the reduction of 5 ’-azido-5’ -deoxynucleosides to 5 ’-amino-5 ’-deoxynucleosides. A synthesis starting with 5 ’-amino-5 ’-deoxynucleosides can be performed with unprecedented conversion rates (>=80 area-%) and isolated yields of >60% for a chemical nucleoside triphosphate synthesis. The old solid-phase synthetic method required approximately 6-8 weeks manufacturing time to yield ~20 pmol dNTP-2c. With the new method several hundred pmol can be prepared in 1-2 weeks on a lab scale. When used as a diastereoselective method, an isomer of choice can be exclusively generated. Thereby, the total amount of the expensive 5 ’-azido-5’ -deoxynucleoside starting material can be used for synthesis of an isomer of choice. The new process is scalable, as it can be done in liquid-phase and the difficult separation of the two comparably labile triphosphate diastereomers is not required anymore. The products contain lower and fewer impurities with the new method. Purities of 98- 99 area% can be typically achieved now, which is beneficial for performance when the nucleoside triphosphates are used for sequencing applications.

Overall, a highly efficient, high purity, cost-effective synthetic method of nucleoside triphosphates has been developed that can be performed in diastereoselective mode if needed.

Exemplary embodiments of the disclosure are described by the following items:

[1] A chiral compound having the following structure: wherein LG¹ is a conjugate base of a strong acid (H-LG¹); LG² is a conjugate base of a weak acid (H-LG²); and R⁴ comprises or consists of a hydrocarbon; and G², when present, represents a terminal conjugable group, preferably a clickable group.

[2] The chiral compound of [1], wherein LG² is a substituted or unsubstituted cyclic or heterocyclic compound that is linked to the phosphorus via an exocyclic or an endocyclic group.

[3] A chiral compound having the following structure: wherein LG¹ is a conjugate base of a strong acid; LG² is a substituted or unsubstituted cyclic or heterocyclic compound that is linked to the phosphorus via an exocyclic or an endocyclic group; R⁴ comprises or consists of a hydrocarbon; and G², when present, represents a terminal conjugable group, preferably a clickable group.

[4] The chiral compound of [2] or [3], wherein the substituted or unsubstituted cyclic or heterocyclic compound is 5-membered or 6-membered. [5] The chiral compound of any one of [2]-[4], wherein the substituted or unsubstituted cyclic or heterocyclic compound is aromatic.

[6] The chiral compound of any one of [2]-[5], wherein the exocyclic group is selected from oxygen, sulfur and nitrogen.

[7] The chiral compound of any one of [2]-[6], wherein the endocyclic group is a nitrogen.

[8] The chiral compound of any one of the preceding items, wherein LG² is a phenolate, an imidazole, a hydroxypyridine, such as 4-hydroxypyridine, a thiophenolate, a naphtholate, such as 2-naphtholate, a benzimidazole, a hydroxy-quinoline or hydroxy-isoquinoline, or an imidazopyridines, optionally with one or more substituents.

[9] The chiral compound of any one of the preceding items, wherein LG² is a phenolate, optionally with one or more substituents, preferably one or more electron-withdrawing substituents.

[ 10] The chiral compound of any one of the preceding items, wherein LG² is a phenolate, having the following structure: wherein R, when present, represents one or more substituents, preferably electron-withdrawing substituents.

[11] The chiral compound of any one of the preceding items, wherein LG² is a phenolate with one or more substituents selected from a halide, a nitro, a nitroso group, a sulfonyl group, sulfonamide group, cyano group, a halogenated alkyl group, a carboxyester, and a substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group or cyclic or heterocyclic (aromatic or non-aromatic) group comprising 1-10, such as 1-6 or 1-3, carbon atoms (such as a methyl, ethyl or isopropyl group), which substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group or cyclic or heterocyclic group optionally includes one or more oxygen, nitrogen, phosphorus or sulfur heteroatoms.

[12] The chiral compound of any one of the preceding items, wherein LG² is a phenolate with one or more substituents selected from a halide, a nitro group, a nitroso group, a sulfonyl group, a sulfonamide group, a cyano group, a halogenated alkyl group, and a carboxyester, and optionally with one or more additional substituents that are a substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group or cyclic or heterocyclic (aromatic or non- aromatic) group comprising 1-10, such as 1-6 or 1-3, carbon atoms (such as a methyl, ethyl or isopropyl group), which substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group or cyclic or heterocyclic group optionally includes one or more oxygen, nitrogen, phosphorus or sulfur heteroatoms.

[13] The chiral compound of any one of the preceding items, wherein LG¹ and LG² are linked.

[14] The chiral compound of any one of item [13], wherein the chiral compound has the structure: wherein R, when present, represents one or more substituents, preferably electron-withdrawing substituents.

[15] The chiral compound of any one of any one of items [1]-[13], wherein LG² is 2,4,6- trifluorophenolate, 2,6-difluorophenolate, 4-nitrophenolate, 2-methyl-4-nitrophenolate, or 3,5- bis(methoxycarbonyl)phenolate, preferably 2,4,6-trifluorophenolate.

[16] The chiral compound of any one of the preceding items, wherein the conjugate acid of LG¹ has a pKa of 3.5 or lower, such as a pKa lower than 0.

[17] The chiral compound of any one of the preceding items, wherein the conjugate acid of LG² has a pKa of more than 3.5 to 10.5, such as a pKa of 5 to 10, or a pKa of 6 to 9, or a pKa of 7 to 8.

[ 18] The chiral compound of any one of the preceding items, wherein the conjugate acid of LG² has a Ka that is at least 10 times lower than the Ka of the conjugate acid of LG¹.

[19] The chiral compound of any one of items [ 1 ]-[ 12] or [ 15]-[ 18], wherein LG¹ is a halide, preferably chloride.

[20] The chiral compound of any one of items [ 1 ]-[ 12] or [ 15]-[ 18], wherein LG¹ is chloride and the conjugate acid of LG² has a pKa of 5 to 10.5, a pKa of 5 to 10, a pKa of 6 to 9, or a pKa of 7 to 8.

[21] The chiral compound of any one of item [20], wherein LG¹ is chloride, and LG² is 2,4,6- trifluorophenolate, 2,6-difluorophenolate, 4-nitrophenolate, 2-methyl-4-nitrophenolate, or 3,5- bis(methoxycarbonyl)phenolate, preferably 2,4,6-trifluorophenolate.

[22] The chiral compound of any one of the preceding items, wherein R⁴ comprises or consists of a branched, linear, cyclic or heterocyclic, substituted or unsubstituted, saturated or unsaturated hydrocarbon, optionally including one or more heteroatoms, optionally selected from nitrogen, oxygen, phosphorus and sulfur.

[23] The chiral compound of any one of the preceding items, wherein R⁴ consists of a branched or linear, substituted or unsubstituted, saturated or unsaturated hydrocarbon, optionally including one or more heteroatoms, optionally selected from nitrogen, oxygen, phosphorus and sulfur.

[24] The chiral compound of any one of the preceding items, wherein R⁴ comprises 1-100 carbon atoms, preferably 1-30 carbon atoms or 1-20 carbon atoms, such as 1-15 carbon atoms, 3- 15 carbon atoms, 3-10 carbon atoms or 3-6 carbon atoms, preferably 4 carbon atoms.

[25] The chiral compound of any one of the preceding items, wherein R⁴ consists of a linear or branched, unsubstituted, saturated hydrocarbon, optionally including one or more heteroatoms, such as nitrogen, oxygen, phosphorus or sulfur.

[26] The chiral compound of any one of the preceding items, wherein R⁴ consists of two or more hydrocarbons that are linked by an atom or group of atoms other than carbon, such as a nitrogen atom, a phosphorus atom, a sulfur atom and/or an oxygen atom.

[27] The chiral compound of any one of the preceding items, wherein G² is an alkyne group or an azide group, preferably an alkyne group.

[28] The chiral compound of any one of the preceding items, wherein R⁴ with G² has one of the following structures:

[29] The chiral compound of any one of the preceding items, having the following structure:

[30] The chiral compound of any one of the preceding items, which is enantiopure.

[31] The chiral compound of any one of [30], wherein the chiral compound has the structure: pound of any one of item [31], having the following structure:

[33] A composition comprising the chiral compound of any one of [l]-[32],

[34] The composition of [33], wherein at least 80%, preferably at least 90%, such as at least 95%, at least 99% or 100% of the chiral compound has the structure depicted in [31],

[35] A method for producing a chiral compound of any one of [l]-[32] or a composition of [33] or [34], comprising reacting a phosphoryl halide with H-LG² and with HO-R⁴-G².

[36] The method of [35], wherein the phosphoryl halide is phosphoryl chloride.

[37] The method of [35] or [36], wherein the H-LG² is 2,4,6-trifluorophenol, 2,6- difluorophenol, 4-nitrophenol, 2-methyl-4-nitrophenol, or 3,5-bis(methoxycarbonyl)phenol and/or HO-R⁴-G² is hex-5-yn-l-ol.

[38] The method of any one of [35]-[37], further comprising purifying the chiral compound obtained, for example by normal phase chromatography.

[39] The method of any one of [35]-[38], further comprising separating the two enantiomers of the chiral compound, for example by chiral chromatography.

[40] A nucleoside monophosphoramidate having the following structure: wherein LG² is a conjugate base of a weak acid; NB is a nucleobase; R¹, when present, comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 -ribose modification; R³ is H or any protecting group; R⁴ comprises or consists of a hydrocarbon; and G¹ and G², when present, independently represent terminal conjugable groups, preferably clickable groups.

[41] The nucleoside monophosphoramidate of [40], wherein LG² is a substituted or unsubstituted cyclic or heterocyclic compound that is linked to the phosphorus via an exocyclic or an endocyclic group.

[42] A nucleoside monophosphoramidate having the following structure: wherein LG² is a substituted or unsubstituted cyclic or heterocyclic compound that is linked to the phosphorus via an exocyclic or an endocyclic group; NB is a nucleobase; R¹, when present, comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 '-ribose modification; R³ is H or any protecting group; R⁴ comprises or consists of a hydrocarbon; and G¹ and G², when present, independently represent terminal conjugable groups, preferably clickable groups.

[43] The nucleoside monophosphoramidate of [41] or [42], wherein the substituted or unsubstituted cyclic or heterocyclic compound is 5-membered or 6-membered.

[44] The nucleoside monophosphoramidate of any one of [41 ]-[43 ], wherein the substituted or unsubstituted cyclic or heterocyclic compound is aromatic.

[45] The nucleoside monophosphoramidate of any one of [41]-[44], wherein the exocyclic group is selected from oxygen, sulfur and nitrogen.

[46] The nucleoside monophosphoramidate of any one of [41]-[45], wherein the endocyclic group is a nitrogen.

[47] The nucleoside monophosphoramidate of any one of [40]- [46], wherein LG² is a phenolate, an imidazole, a hydroxypyridine, such as 4-hydroxypyridine, a thiophenolate, a naphtholate, such as 2-naphtholate, a benzimidazole, a hydroxy-quinoline or hydroxy-isoquinoline, or an imidazopyridines, optionally with one or more substituents.

[48] The nucleoside monophosphoramidate of any one of [40]- [47], wherein LG² is a phenolate, optionally with one or more substituents, preferably one or more electron-withdrawing substituents.

[49] The nucleoside monophosphoramidate of any one of [40]- [48], wherein LG² is a phenolate, having the following structure: wherein R, when present, represents one or more substituents, preferably electron-withdrawing substituents.

[50] The nucleoside monophosphoramidate of any one of [40]- [49], wherein LG² is a phenolate with one or more substituents selected from a halide, a nitro group, a nitroso group, a sulfonyl group, a sulfonamide group, a cyano group, a halogenated alkyl group, a carboxyester, and a substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group or cyclic or heterocyclic (aromatic or non-aromatic) group comprising 1-10, such as 1-6 or 1-3, carbon atoms (such as a methyl, ethyl or isopropyl group), which substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group or cyclic or heterocyclic group optionally includes one or more oxygen, nitrogen, phosphorus or sulfur heteroatoms.

[51] The nucleoside monophosphoramidate of any one of [40]-[50], wherein LG² is a phenolate with one or more substituents selected from a halide, nitro group, a nitroso group, a sulfonyl group, a sulfonamide group, a cyano group, a halogenated alkyl group, and a carboxyester, and optionally with one or more additional substituents that are a substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group or cyclic or heterocyclic (aromatic or non- aromatic) group comprising 1-10, such as 1-6 or 1-3, carbon atoms (such as a methyl, ethyl or isopropyl group), which substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group or cyclic or heterocyclic group optionally includes one or more oxygen, nitrogen, phosphorus or sulfur heteroatoms.

[52] The nucleoside monophosphoramidate of any one of [40]-[51], wherein the conjugate acid of LG² has a pKa of more than 3.5 to 10.5, such as a pKa of 5 to 10, or a pKa of 6 to 9, preferably 7 to 8.

[53] The nucleoside monophosphoramidate of any one of [40]- [52], wherein LG² is a phenolate with one or more electron-withdrawing substituents.

[54] The nucleoside monophosphoramidate of any one of [40]- [53], wherein LG² is a phenolate with one or more substituents selected from a halide, a nitro group, a carboxyester, and a substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group comprising 1-10, such as 1-6 or 1-3, carbon atoms (such as a methyl, ethyl or isopropyl group), which optionally includes one or more oxygen, nitrogen, phosphorus or sulfur heteroatoms.

[55] The nucleoside monophosphoramidate of any one of [40]- [54], wherein LG² is a phenolate with one or more substituents selected from a halide, a nitro group, a carboxyester, and optionally one or more additional substituents that are a substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group comprising 1-10, such as 1-6 or 1-3, carbon atoms (such as a methyl, ethyl or isopropyl group), which optionally includes one or more oxygen, nitrogen, phosphorus or sulfur heteroatoms.

[56] The nucleoside monophosphoramidate of any one of [40]-[55], wherein LG² is 2,4,6- trifluorophenolate, 2,6-difluorophenolate, 4-nitrophenolate, 2-methyl-4-nitrophenolate or 3,5- bis(methoxycarbonyl)phenolate, preferably 2,4,6-trifluorophenolate.

[57] The nucleoside monophosphoramidate of any one of [40]-[56], wherein LG² is a phenolate with two or three fluoride substitutions.

[58] The nucleoside monophosphoramidate of any one of [40]-[57], wherein R⁴ comprises or consists of a branched, linear, cyclic or heterocyclic, substituted or unsubstituted, saturated or unsaturated hydrocarbon, optionally including one or more heteroatoms, optionally selected from nitrogen, oxygen, phosphorus and sulfur.

[59] The nucleoside monophosphoramidate of any one of [40]-[58], wherein R⁴ consists of a branched or linear, substituted or unsubstituted, saturated or unsaturated hydrocarbon, optionally including one or more heteroatoms, optionally selected from nitrogen, oxygen, phosphorus and sulfur.

[60] The nucleoside monophosphoramidate of any one of [40]-[59], wherein R⁴ comprises 1- 100 carbon atoms, preferably 1-30 carbon atoms or 1-20 carbon atoms, such as 1-15 carbon atoms, 3-15 carbon atoms, 3-10 carbon atoms or 3-6 carbon atoms, preferably 4 carbon atoms.

[61] The nucleoside monophosphoramidate of any one of [40]-[60], wherein R⁴ consists of a linear or branched, unsubstituted, saturated hydrocarbon, optionally including one or more heteroatoms, such as nitrogen, oxygen, phosphorus or sulfur.

[62] The nucleoside monophosphoramidate of any one of [40]-[61], wherein R⁴ is acyclic.

[63] The nucleoside monophosphoramidate of any one of [40]- [62], wherein R⁴ comprises or consists of two or more hydrocarbons that are linked by an atom or group of atoms other than carbon, such as a nitrogen atom, a phosphorus atom, a sulfur atom and/or an oxygen atom.

[64] The nucleoside monophosphoramidate of any one of [40]- [63], wherein R⁴ with G² has one of the following structures: [65] The nucleoside monophosphoramidate of any one of [40]- [64], wherein R⁴ with G² is an alkyl group with a terminal alkyne, such as a hex-5 -yn-l-yl group.

[66] The nucleoside monophosphoramidate of any one of [40]-[65], wherein NB has one of the following structures:

R⁵ = H or protective group, preferably R⁵ = H

[67] The nucleoside monophosphoramidate of any one of [40]-[66], wherein R¹ comprises or consists of a branched, linear, cyclic or heterocyclic, substituted or unsubstituted, saturated or unsaturated hydrocarbon, optionally including one or more heteroatoms, optionally selected from nitrogen, oxygen, phosphorus and sulfur.

[68] The nucleoside monophosphoramidate of any one of [40]-[67], wherein R¹ consists of a branched or linear, substituted or unsubstituted, saturated or unsaturated hydrocarbon, optionally including one or more heteroatoms, optionally selected from nitrogen, oxygen, phosphorus and sulfur.

[69] The nucleoside monophosphoramidate of any one of [40]-[68], wherein R¹ comprises 1- 100 carbon atoms, preferably 1-30 carbon atoms or 1-20 carbon atoms, such as 1-15 carbon atoms, 3-15 carbon atoms, 3-10 carbon atoms, preferably 6 or 8 carbon atoms.

[70] The nucleoside monophosphoramidate of any one of [40]-[69], wherein R¹ consists of a linear or branched, unsubstituted, saturated hydrocarbon, optionally including one or more heteroatoms, such as nitrogen, oxygen, phosphorus or sulfur.

[71] The nucleoside monophosphoramidate of any one of [40]-[70], wherein R¹ is acyclic.

[72] The nucleoside monophosphoramidate of any one of [40]- [71], wherein R¹ with G¹ is a octa-1, 7-diynyl or a deca-l,9-diynyl group.

[73] The nucleoside monophosphoramidate of any one of [40]-[72], wherein each of G¹ and G² is an alkyne group or an azide group, preferably an alkyne group.

[74] The nucleoside monophosphoramidate of any one of [40]- [73], wherein the nucleoside monophosphoramidate has one of the following structures:

[75] The nucleoside monophosphoramidate of any one of [40]-[74], wherein the nucleoside monophosphoramidate has one of the following structures: [76] The nucleoside monophosphoramidate of [40] or [75], which is diastereomerically pure with regard to the phosphorus stereocenter.

[77] The nucleoside monophosphoramidate of [40]-[76] having the following structure:

[78] A method for producing a nucleoside monophosphoramidate according to any one of [40]- [77], comprising reacting the chiral compound of any one of [l]-[32] or a composition of [33] or

[34] with a 5 ’-amino-5’ -deoxynucleoside, and optionally purifying the product of this reaction. [79] The method of [78], comprising the following step:

[80] The method of [78] or [79], that is for diastereoselectively producing a nucleoside monophosphoramidate, comprising the following step:

[81] The method of any one of [78]-[80], further comprising purifying the nucleoside monophosphoramidate obtained.

[82] The method of [81], wherein the purification comprises separating the two diastereomers (with regard to the phosphorus stereocenter), for example by HPLC.

[83] A nucleoside monophosphoramidate produced by the method of any one of [78]-[82],

[84] A composition comprising the nucleoside monophosphoramidate of any of [40]-[77] or [83],

[85] The composition of [84], wherein at least 80%, preferably at least 90%, such as at least 95%, at least 99%, or 100% of the nucleoside monophosphoramidate has the structure depicted in [77],

[86] A method for producing a nucleoside triphosphate, comprising reacting the nucleoside monophosphoramidate of any one of [40]-[77] or [83] or the composition of [84] or [85] with a pyrophosphate, preferably with a pyrophosphate salt, such as Tris(tetrabutylammononium) hydrogen pyrophosphate.

[87] The method of [86], comprising the following step: wherein R⁶ is H or any organic or inorganic cation; and Z⁺ is H⁺ or any (monovalent or bivalent) cation.

[88] The method of [86] or [87] that is for diastereoselectively producing a nucleoside triphosphate, comprising the following step: wherein R⁶ is H or any organic or inorganic cation; and Z⁺ is H⁺ or any (monovalent or bivalent) cation.

[89] The method of any one of [86]-[88], comprising: (a) reacting the chiral compound of any one of [l]-[32] or the composition of [33] or [34] with a

5 ’-amino-5’ -deoxynucleoside, and

(b) reacting the product of step (a) with a pyrophosphate.

[90] The method of any one of [86]-[89], comprising the following step: [91] The method of any one of [86]-[90] that is for diastereoselectively producing a nucleoside triphosphate, comprising the following step:

[92] The method of any one of [86]-[91 ], wherein the reactions are SN2-type reactions.

[93] The method of any one of [ 86] -[92] , which is performed in liquid phase.

[94] The method of any one of [86]- [93], further comprising linking the a-phosphoramidate to the nucleobase.

[95] The method of [94], wherein R¹ is linked to R⁴ via a tether molecule.

[96] The method of [94] or [95], wherein a tether molecule is attached via click chemistry reactions.

[97] The method of any one of [94]- [96], comprising reacting G¹ and G² with terminal conjugable groups, preferably clickable groups, attached to a tether molecule.

[98] The method of any one of [94]-[97], wherein an expandable NTP is produced.

[99] The method of any one of [94]- [98], wherein linking the a-phosphoramidate to the nucleobase comprises the following step: wherein NB is a nucleobase; R¹ comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 -ribose modification; R³ is H or any protecting group; R⁴ comprises or consists of a hydrocarbon; G¹ and G² independently represent terminal conjugable groups, preferably clickable groups; G^la and G^2a independently represent terminal conjugable groups, preferably clickable groups; L¹ and L² independently represent linking groups formed by reacting G¹ with G^la and G² with G^2a, preferably 1,2, 3 -triazoles; and T is a tether molecule; and Z⁺ is H⁺ or any (monovalent or bivalent) cation.

[100] The method of [94]-[99] that is diastereoselective, wherein linking the a-phosphoramidate to the nucleobase comprises the following step:

[101] The method of [100], wherein the G¹ and G² are alkyne groups and G^la and G^2a are azide groups.

[102] The method of any one of [86]- [ 101 ], wherein R¹ with G¹ is a octa-1, 7-diynyl or a deca- 1,9-diynyl group, and wherein R⁴ with G² has one of the following structures:

[103] The method of any one of [86]- [102], further comprising purifying the nucleoside triphosphate obtained.

[104] The method of [103], wherein the purification comprises separating the two diastereomers (with regard to the phosphorus stereocenter), for example by HPLC.

[105] The method of any one of [86]-[104], further comprising, when R³ is a protecting group, removing the protecting group.

[106] The method of any one of [86]-[105], further comprising, when R⁵ is a protective group, removing the protective group.

[107] A method for producing a nucleoside diphosphate or oligophosphate, such as a tetraphosphate, comprising reacting the nucleoside monophosphoramidate of any one of [40]-[77] or [83] or the composition of [84] or [85] with a monophosphate or oligophosphate, preferably with a monophosphate or oligophosphate salt, wherein the oligophosphate is optionally a triphosphate or tetraphosphate.

[108] A 5 ’-amino-5’ -deoxynucleoside having the following structure: wherein NB is a nucleobase; R¹ comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 -ribose modification; R³ is H or any protecting group; G¹ represents terminal conjugable group, preferably clickable group. [109] A method for producing a 5 ’-amino-5’ deoxynucleoside of [108], comprising selective reduction of a 5 ’-azido-5’ -deoxynucleoside.

[110] The method of [ 109] comprising the following step :

[H l] The method of [109] or [110], wherein NB has one of the following structures: wherein R⁵ = H or protective group, preferably R⁵ = H.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1: Structures of exemplary nucleoside triphosphate molecules.

FIG. 2: Analytical HPLC results with crude products from the prior art synthesis method for exemplary nucleoside triphosphates. Acquisition wavelengths: dATP-2c - 281 nm; C8-dTTP-2c - 292 nm; dGTP-2c - 294 nm; C6pC6-dCTP-2c - 299 nm. X-axis shows response [mAU], Y-axis shows time [min]. FIG. 3: Exemplary reaction scheme according to the present disclosure when starting from a 5’- azido-5’ -deoxynucleoside, using racemic mixture of the phosphorylation reagent.

FIG. 4: Exemplary reaction scheme according to the present disclosure when starting from a 5’- amino-5’ -deoxynucleoside, using enantiopure phosphorylation reagent. By using enantiopure phosphorylation reagent of one or the other configuration at the phosphorus stereocenter (path A and B, respectively), nucleoside triphosphates can be produced in diastereomerically pure form.

FIG. 5: (A) Exemplary synthesis scheme for the phosphorylation reagent; (B) comparison of different LG²; (C) structure of a TRIPP reagent.

FIG. 6: (A) Exemplary synthesis scheme using TRIPP. (B) The anal. HPL chromatogram on the top shows the crude product of previous solid-phase synthesis method (ahead of purification). The anal. HPL chromatogram in the middle shows the crude product using racemic TRIPP. The anal. HPL chromatogram on the bottom shows the crude product using enantiopure TRIPP. Yields and purities are related to isolated compounds after subsequent HPLC purification. <10%, -44% and -81% indicate area%.

FIG: 7: Results from chiral separation of the two TRIPP enantiomers. (A) Small scale experiment: The “active” configuration of the phosphorylation reagent (i.e. the configuration that gives rise to the active nucleoside triphosphate isomer by the diastereoselective methods disclosed herein) is represented in the first peak eluting at 10:00 min. (B) Upscaled: the “active” TRIPP configuration is represented in the first peak eluting at 9: 19 min

FIG. 8: (A) Exemplary 31P{ 1H} NMR spectrum of enantiopure TRIPP reagent; TRIPP: 1.48 ppm; (B) Exemplary LC-MS chromatogram of enantiopure TRIPP reagent after quenching with MeOH (quenching 1 pL of reagent solution (50 mg/mL in 2-MeTHF) in 100 pL MeOH and analyzing the MeOH-adduct); 4.999 min: hydrolyzed TRIPP (part from reaction with water in MeOH, part from reaction with the eluent in the LC-MS); 6.400 min: methanolized TRIPP (the percentage of this peak should be highest).

Fig. 9: Phosphorylation reagent in which LG² is 2,6-difluorophenolate. (A) Results from chiral separation of the two enantiomers. The “active” phosphorylation reagent configuration is represented in the first peak eluting at -13 :00 min. (B) Analytical HPLC results with crude product from an exemplary nucleoside triphosphate synthesis using the racemic phosphorylation reagent. Fig. 10: Phosphorylation reagent in which LG² is 4-nitrophenolate. (A) Results from chiral separation of the two enantiomers. The “active” phosphorylation reagent configuration is represented in the first peak eluting at -9:30 min. (B) Analytical HPLC results with crude product from an exemplary nucleoside triphosphate synthesis using the racemic phosphorylation reagent. Fig. 11: Phosphorylation reagent in which LG² is 2-methyl-4-nitrophenolate. Analytical HPLC results with crude product from an exemplary nucleoside triphosphate synthesis using the racemic phosphorylation reagent.

Fig. 12: Phosphorylation reagent in which LG² is 3,5-bis(methoxycarbonyl)phenolate. (A) Results from chiral separation of the two enantiomers. The “active” phosphorylation reagent configuration is represented in the first peak eluting at ~9:30min. (B) Analytical HPLC results with crude product from an exemplary nucleoside triphosphate synthesis using the racemic phosphorylation reagent. Fig. 13: Phosphorylation reagent in which LG² is 2,6-dimethylphenolate. Analytical HPLC results with crude product from an exemplary nucleoside triphosphate synthesis using the racemic phosphorylation reagent.

Fig. 14: Phosphorylation reagent in which LG² is 2-ethylphenolate. Analytical HPLC results with crude product after 6 days at 4 °C from an exemplary nucleoside triphosphate synthesis using the racemic phosphorylation reagent.

Fig. 15: Phosphorylation reagent in which LG² is 2-isopropylphenolate. Analytical HPLC results with crude product from an exemplary nucleoside triphosphate synthesis using the racemic phosphorylation reagent.

DETAILED DESCRIPTION OF THE INVENTION

The invention will now be described in detail by way of reference only using the following definitions and examples. All patents and publications, including all sequences disclosed within such patents and publications, referred to herein are expressly incorporated by reference.

Unless defined otherwise herein, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Singleton (Singleton et al., Dictionary of microbiology and molecular biology, 2nd ed., 1994, John Wiley and Sons, New York), Hale (Hale and Marham, The Harper Collins dictionary of biology, 1991, Harper Perennial, NY) and Walker (Walker and Cox, The Language of Biotechnology: A Dictionary of Terms. 1988, American Chemical Society, Washington, D.C. ISBN-0-8412-1499-1) provide one of skill with a general dictionary of many of the terms used in this invention. Practitioners are particularly directed to Sambrook (Sambrook et al., Molecular cloning: A laboratory manual, 1989, Cold Spring Harbor Laboratory Press), and Ausubel (Ausubel et al., Current protocols in molecular biology, 1993, John Wiley & Sons, Inc.), for definitions and terms of the art. It is to be understood that this invention is not limited to the particular methodology, protocols, and reagents described, as these may vary. As used herein, the singular forms “a,” “an” and “the” include plural referents unless the context clearly dictates otherwise.

In the structures shown herein, when not all natural valencies of an atom are filled by named groups, it should be understood that the unfilled valencies are filled by hydrogen. When a wavy line in a structure intersects a bond, then the intersected bond is the location where the structure joins to the remainder of a molecule.

When a structure depicts a molecule with one or more negatively charged oxygens in a phosphate group, the structure likewise encompasses the molecule with the oxygen(s) in conjunction with H⁺ and/or any organic or inorganic cations. When a structure depicts a molecule with one or more hydroxyl groups in a phosphate group, the structure likewise encompasses the molecule with the oxygen(s) from the hydroxyl group(s) in conjunction with H⁺ and/or any organic or inorganic cations.

Reference throughout this specification to "one embodiment" or "an embodiment" and variations thereof means that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment. Thus, the appearances of the phrases "in one embodiment" or "in an embodiment" in various places throughout this specification are not necessarily all referring to the same embodiment. Furthermore, the particular features, structures, or characteristics may be combined in any suitable manner in one or more embodiments.

The headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be had by reference to the specification as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the specification as a whole.

I. Terms

Phosphate: A “phosphate” includes an “organophosphate” as well as variants thereof, such as an “amidophosphate” (which is a synonym for “phosphoramidate”) or a “thiophosphate”. A phosphate can include a side chain, such as -R⁴-G² in the nucleoside monophosphoramidates or triphosphates disclosed herein. The first, second and third phosphate counted from the 5’ end of a nucleoside are also referred to as a-phosphate, P-phosphate and y-phosphate, respectively (or, in case the a-phosphate is a phosphoramidate, it can also be referred to as “a-phosphoramidate”). The type of a given phosphate is also derivable from the structures provided herein.

Expandable NTP: An “expandable NTP” or “XNTP” refers to a 5' a-phosphate nonnatural nucleoside triphosphate (NTP) molecule (typically a non-natural 2 ’-deoxynucleoside triphosphate molecule) compatible with template-dependent enzymatic polymerization. Each XNTP has two distinct functional regions, i.e., a selectively cleavable bond (e.g. a phosphoramidate bond) linking the 5’ a-phosphate to a sugar comprised in a nucleoside and a tether molecule that is attached within the XNTP at positions that allow for controlled expansion by cleavage of the cleavable bond (e.g. a tether linking the 5’ a-phosphate and the nucleobase). An XNTP can thus be present in a constrained configuration (when the cleavable bond is still intact) or in an expanded configuration (when the cleavable bond has been cleaved, e.g. via acid treatment). dNTP-2c: An “dNTP-2c” refers to a 5' a-phosphate modified non-natural dNTP molecule that can serve as an intermediate in the synthesis of XNTPs. A dNTP-2c comprises two clickable groups, such as terminal alkynes, one as part of a modification at the 5’ a-phosphate, and one as part of a modification at the nucleobase. The two clickable groups allow addition of a tether between the a-phosphate and the nucleobase via click reactions to form an XNTP.

Strong acid: A “strong acid” as used herein includes particularly strong, very strong, strong and moderately strong acids. A particularly strong acid can be, for example, an acid with a pKa lower than -3.5; a very strong, strong or moderately strong acid can be, for example, an acid with a pKa of -3.5 to 3.5 (see Chapter 3.2.1 ofHollemann and Wiberg, Lehrbuch der anorganischen Chemie, 102^nd edition, 2007).

Weak acid: A “weak acid” can be, for example, an acid with a pKa of more than 3.5 to 10.5 (see Chapter 3.2.1 of Hollemann and Wiberg, Lehrbuch der anorganischen Chemie, 102^nd edition, 2007).

Xpandomer: An “Xpandomer” refers to a molecule consisting of at least two monomers derived from XNTPs. An Xpandomer is obtainable, for example, by polymerase-mediated synthesis of a complementary strand to a template nucleic acid using XNTPs as polymerase substrates. An expanded configuration of the Xpandomer can be obtained by cleavage of the cleavable bond in the XNTPs, e.g. via acid treatment.

IL Phosphorylation reagent

The present disclosure provides a chiral compound for the phosphorylation of nucleosides, and in particular for the 5’ phosphorylation of 5 ’-amino-5’ -deoxynucleosides. The chiral compound has the following structure: wherein LG¹ is a conjugate base of a strong acid; LG² is a conjugate base of a weak acid; and R⁴ comprises or consists of a hydrocarbon; and G², when present, represents a terminal conjugable group, preferably a clickable group. The compound is chiral (i.e. with a phosphorus stereocenter).

The chiral compound comprises two leaving groups LG¹ and LG². The leaving ability of a leaving group inversely correlates with its basicity (see Chapter 6.7 of Vollhardt and Schore, Organic Chemistry, 5^th edition, 2011). For example, chloride is a weaker base than fluoride and therefore has a better leaving ability. In other words, conjugated bases of strong acids have a better leaving ability than conjugated bases of weak acids.

A measure of acidity is the acid dissociation constant Ka. Acidity is commonly indicated as the -logio value of the Ka, termed pKa. The pKa (or the Ka) of a given chemical entity can be readily determined e.g. by titration, e.g. in water. Determination can be done e.g. at 25 °C. The acids with a pKa as indicated in Table 1 and 2 below (such as pKa 7.2, 7.5 and 10 of 2,4,6- trifluorophenol, 2,6-difluorophenol and phenol, respectively) can be used as reference points, for example.

Examples of acids and conjugate bases with corresponding pKa values of the conjugate acids are given in Table 1 and Table 2.

Table 1: Strong acids

Table 2: Weak acids

PhOH = phenol; PhO- = phenolate

For (diastereo)selective two-step nucleophilic substitutions with a 5’-amino-5’- deoxynucleoside and a pyrophosphate salt, two leaving groups with different properties have been designed:

LG¹ can be a conjugate base of a strong acid (allowing a fast, selective and quantitative S\2-type reaction with a 5 ’-amino-5’ -deoxynucleoside). The conjugate acid of LG¹ (H-LG¹) can have a pKa of 3.5 or lower, preferably a pKa of 3.2 or lower, and most preferably a pKa lower than 0. In preferred embodiments, the conjugate acid of LG¹ has a pKa that is the same as or lower than the pKa of HF, most preferably a pKa that is the same or lower than the pKa of HNO3 (see Table 1).

LG¹ is preferably a conjugate base of a mineral acid, such as a halide (e.g. chloride, bromide, iodide or fluoride), preferably chloride, bromide or iodide, and most preferably chloride. LG¹ can also be a conjugate base of an organic acid such as a phenolate (e.g. 2,4,6- trinitrophenolate) .

LG² can be a conjugate base of a weak acid (allowing an SN2-type reaction with a pyrophosphate salt). The conjugate acid of LG² (H-LG²) can have a pKa of more than 3.5 to 10.5, such as a pKa of 5 to 10, a pKa of 6 to 9, preferably a pKa of 7 to 8. In a preferred embodiment, the conjugate acid of LG² has a pKa that is the same as or higher than the pKa of 2,4,5- trichlorophenol, and that is the same as or lower than the pKa of 3,5-dichlorophenol (see Table 2).

Typically, the Ka of the conjugate acid of LG² will be at least 10 times or at least 100 times lower than the Ka of the conjugate acid of LG¹. Thus, for example, when the pKa of the conjugate acid of LG¹ is 3, the pKa of the conjugate acid of LG² is typically 4 or higher or 5 or higher. Preferably, the Ka of the conjugate acid of LG² is much lower than the Ka of the conjugate acid of LG¹, such as at least 10⁴ times, at least 10⁵ times, at least 10⁶ times, at least 10⁷ times, at least 10⁸ times, at least 10⁹ times, at least 10¹⁰ times, at least 10¹¹ times, at least 10¹² times, at least 10¹³ times or at least 10¹⁴ times lower. When LG¹ is chloride, the Ka of the conjugate acid of LG² is preferably at least 10¹² times lower, preferably at least 10¹³ times or at least 10¹⁴ times lower, than the Ka of HC1 (i.e. the conjugate acid of chloride). Thus, for example, when the pKa of the conjugate acid of LG¹ is -7 (as in the case of chloride as LG¹), the pKa of the conjugate acid of LG² is preferably 5 or higher (more preferably 6 or higher, or 7 or higher).

LG² is typically a substituted or unsubstituted cyclic or heterocyclic compound that is linked to the phosphorus via an exocyclic or an endocyclic group. Thus, the disclosure also provides a chiral compound having the following structure: wherein LG¹ is a conjugate base of a strong acid; LG² is a substituted or unsubstituted cyclic or heterocyclic compound that is linked to the phosphorus via an exocyclic or an endocyclic group; R⁴ comprises or consists of a hydrocarbon; and G², when present, represents a terminal conjugable group, preferably a clickable group.

A cyclic compound in this context means that the backbone of the cyclic compound consists only of carbon atoms. A heterocyclic compound in this context means that the backbone of the heterocyclic compound consists of carbon atoms and other atoms, such as nitrogen, oxygen or sulfur. The substituted or unsubstituted cyclic or heterocyclic compound can be 3-membered, 4-membered, 5-membered or 6-membered, for example, and is preferably 5-membered or 6- membered. The substituted or unsubstituted cyclic or heterocyclic compound can be aromatic. The substituted or unsubstituted cyclic or heterocyclic compound can be monocyclic, bicyclic or polycyclic, such as tricyclic. The substituted or unsubstituted cyclic or heterocyclic compound includes a phenolate, an imidazole, a hydroxypyridine, such as 4-hydroxypyridine, a thiophenolate, a naphtholate, such as 2-naphtholate, a benzimidazole, a hydroxy-quinoline or hydroxy-isoquinoline, or an imidazopyridines, for example, optionally with one or more substituents. Suitable substituents are described further below. The terms “endocyclic” and “exocyclic” are as commonly used in the art, i.e. an “endocyclic group” is part of a (hetero)cycle backbone (e.g., a nitrogen heteroatom), and an “exocyclic group” is connected (e.g. covalently bound) to but outside a (hetero)cycle backbone. An exocyclic group can be selected from oxygen, sulfur and nitrogen, for example. When the substituted or unsubstituted cyclic or heterocyclic compound is a phenolate, it can be linked to the phosphorus via the exocyclic oxygen derived from the phenol hydroxyl group, for example. When the substituted or unsubstituted cyclic or heterocyclic compound is an imidazole, it can be linked to the phosphorus via the endocyclic nitrogen, for example.

LG² is preferably a phenolate, optionally with one or more substituents, preferably with one or more electron- withdrawing substituents. Thus, LG² typically has the following structure: wherein R, when present, represents one or more substituents, preferably electron-withdrawing substituents.

The disclosure provides a chiral compound having the following structure: wherein LG¹ is a conjugate base of a strong acid; R⁴ comprises or consists of a hydrocarbon; and

G², when present, represents a terminal conjugable group, preferably a clickable group; and LG² has the following structure: wherein R, when present, represents one or more substituents, preferably electron-withdrawing substituents.

In a preferred embodiment, the chiral compound has the following structure: wherein R⁴ comprises or consists of a hydrocarbon; and G², when present, represents a terminal conjugable group, preferably a clickable group; and LG² has the following structure: wherein R, when present, represents one or more substituents, preferably electron-withdrawing substituents.

In some embodiments, LG¹ and LG² are linked. Such a linkage can be achieved by suitably selecting an entity with two functional groups having different pKas, such as substituted or unsubstituted salicylic acid. In some embodiments, the chiral compound thus has the structure: wherein R, when present, represents one or more substituents, preferably electron-withdrawing substituents.

R can be present or absent, and is preferably present. When present, R represents one or more substituents, preferably electron-withdrawing substituents. R represents 1, 2, 3, 4 or 5 substituents, preferably 1, 2 or 3 substituents. When R represents more than one substituent, the substituents can be the same or different. Electron-withdrawing substituents are well-known in the art. Examples of an electronwithdrawing substituent include a halide (F, Cl, Br or I, preferably F), a nitro group, a carboxyester, a cyano group, a nitroso group, a sulfonyl group, sulfonamide group, a sulfo group, or a halogenated (C1-C3, typically Ci) alkyl group, such as -a trifluoromethyl group. Thus, in some embodiments, LG² is a phenolate with one or more electron-withdrawing substituents selected from a halide (F, Cl, Br or I, preferably F), a nitro group, a carboxyester, a cyano group, a nitroso group, a sulfonyl group, sulfonamide group, a sulfo group or a halogenated (C1-C3, typically Ci) alkyl group, such as -CF3. It is known that electron-withdrawing substituents reduce the pKa of phenol and that the position relative to the -OH influences the strength of that effect of electronwithdrawing substituents. Thereby, the skilled person can readily provide phenolates with one or more (electron- withdrawing) substituents that is derived from a substituted phenol with a desired pKa. Exemplary phenols and corresponding phenolates are given in Table 2.

For example, the one or more substituents can be one, two or three halides (e.g. F, Cl, Br and/or I, preferably F, Cl and/or Br, more preferably F), a nitro group or one or more carboxyesters (such as carboxyesters at position 3 and 5). A phenolate with two or three halide substituents is preferably a difluorophenolate or trifluorophenolate, such as 2,6-difluorophenolate or 2,4,6- trifluorophenolate. A nitrophenolate is preferably 4-nitrophenolate. A carboxyester phenolate is preferably 3,5-bis(methoxycarbonyl)phenolate.

The one or more substituents can also be one or more substituents that are non-electron withdrawing (typically in addition to one or more electron-withdrawing substituents). Examples of a non-electron withdrawing substituent includes an substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group or cyclic or heterocyclic (e.g. aromatic) group comprising 1-10, such as 1-6 or 1-3, carbon atoms, which optionally includes one or more oxygen, nitrogen, phosphorus or sulfur heteroatoms. For example, the alkyl group can be a methyl, ethyl or isopropyl group. For example, a cyclic group can be a substituted or unsubstituted phenyl group (e.g. at the para position of a phenolate).

Thus, in some embodiments, the substituents are selected from a halide, a nitro group, a nitroso group, a sulfonyl group, a sulfonamide group, a cyano group, a carboxyester, and a substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group comprising 1-10, such as 1-6 or 1-3, carbon atoms, which optionally includes one or more oxygen, nitrogen, phosphorus or sulfur heteroatoms, such as a methyl, ethyl or isopropyl group.

In some embodiments, the substituents are selected from a halide, a nitro group, a nitroso group, a sulfonyl group, a sulfonamide group, a cyano group, a carboxyester, and optionally one or more additional substituents that are a substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group or cyclic or heterocyclic (e.g. aromatic) group comprising 1- 10, such as 1-6 or 1-3, carbon atoms, which substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group or cyclic or heterocyclic group optionally includes one or more oxygen, nitrogen, phosphorus or sulfur heteroatoms, such as a methyl, ethyl or isopropyl group.

Typically, the substituents will not comprise an amino group, a hydroxyl group, or a thiol group.

In a preferred embodiment, LG² is 2,4,6-trifluorophenolate, 2,6-difluorophenolate, 4- nitrophenolate, 2-methyl-4-nitrophenolate or 3,5-bis(methoxycarbonyl)phenolate, more preferably 2-methyl-4-nitrophenolate or 2,4,6-trifluorophenolate, and most preferably 2,4,6- trifluorophenolate.

Preferably, each substituent has a molecular weight (MW) of 300 g/mol or less, 200 g/mol or less, 100 g/mol or less, 80 g/mol or less, or 60 g/mol or less.

When bulky substituents (which are more sterically demanding as methyl or more sterically demanding as ethyl; e.g. electron poor groups and/or chiral auxiliaries) are used, they are preferably positioned in meta or para position to the phenolic OH-group. In some embodiments, bulky groups can be defined, for example, as having a MW of 40 g/mol or more.

One of the preferred structures of the chiral compound has a chloride as LG¹ and 2,4,6- trifluorophenolate as LG². Such a chiral compound is herein called TRIPP (trifluorophenyl phosphate).

The method also works with phenolates derived from phenols with a pKa lower than that of 2,4,6-trifluorophenol (3FP), such as 2,3,5,6-tetrafluorophenol (4FP) or 2, 3, 4,5,6- pentafluorophenol (5FP) (see Fig. 5). 4FP or 5FP as LG² lead to lower yields during isolation of the racemic phosphorylation reaction, though; and the synthesis of the nucleoside monophosphoramidate with 4FP or 5FP is lower yielding and leads to a lower stability compared to 3FP which can cause some loss of the chiral information via racemization events. The method also works with phenolates derived from phenols with a pKa higher than that of 2,4,6- trifluorophenol (such as 2,6-dimethylphenolate). In this case, the nucleoside monophosphoramidate is more stable and reacts with the pyrophosphate salt less efficiently, though.

The SN2P reactions described herein do not depend on the exact nature ofR⁴. R⁴ is therefore not particularly limited as long as a stereocenter is present at the phosphorus atom in the chiral compound. Moreover, -O-R⁴ should not interfere with the reaction of LG¹ and a 5’-amino-5’- deoxynucleoside and of LG² and a pyrophosphate. For example, HO-R⁴ can be such that it has a pKa of 15 or more, such as 20 or more, or 30 or more.

R⁴ comprises or consists of a hydrocarbon. In a preferred embodiment, R⁴ consists of a hydrocarbon. The hydrocarbon can be substituted or unsubstituted, preferably unsubstituted. The hydrocarbon can be saturated or unsaturated, preferably saturated. For example, R⁴ can comprise or consist of an alkyl, alkenyl, or alkynyl.

Typically, R⁴ comprises 1-100 carbon atoms, preferably 1-30 carbon atoms or 1-20 carbon atoms, such as 1-15 carbon atoms, 3-15 carbon atoms, 3-10 carbon atoms or 3-6 carbon atoms, such as 4 carbon atoms. Typically, R⁴ will be acyclic. Preferably, R⁴ is linear. In preferred embodiments, R⁴ comprises or consists of a linear alkyl. In some embodiments, the molecular weight of R⁴ is 1500 g/mol or less, 1000 g/mol or less, 500 g/mol or less, 200 g/mol or less, or 100 g/mol or less.

In some embodiments, R⁴ comprises or consists of a branched, linear, cyclic or heterocyclic, substituted or unsubstituted, saturated or unsaturated hydrocarbon, optionally including one or more heteroatoms, optionally selected from nitrogen, oxygen, phosphorus and sulfur. A cyclic or heterocyclic hydrocarbon can be 5-membered or 6-membered, for example. A cyclic or heterocyclic hydrocarbon can be aromatic, for example.

In some embodiments, R⁴ is a substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group comprising 1-100 carbon atoms, which optionally includes one or more oxygen, nitrogen, phosphorus or sulfur heteroatoms (e.g. to include an ether, a thioether, a phosphordiester or phosphortriester, or PEG, a heterocycle, such as a triazole or imidazole).

In some embodiments, R⁴ is -R^w-Z, wherein R^w is a substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group having between 1 and 100 carbon atoms, which optionally includes one or more oxygen, nitrogen, phosphorus or sulfur heteroatoms, and where Z is alkyl, alkenyl, alkynyl, acyl, -Het, or -CH2-Het, where "Het" is a substituted or unsubstituted 5- or 6-membered heterocyclic moiety.

In some embodiments, R⁴ does not comprise an amino group, a hydroxyl group, or a thiol group. In some embodiments, R⁴ can comprise a protected amino group, a protected hydroxyl group, or a protected thiol group or a protected or unprotected functional group that is compatible with the synthesis and usage of the phosphorylation reagent. Deprotection can then be done e.g. at the monophosphoramidate or triphosphate stage.

G² can be present or absent, and is preferably present. When present, G² represents a terminal conjugable group, preferably a clickable group. A clickable group can be any group that allows selective reaction with a complementary clickable group via click chemistry. Click chemistry and suitable pairs of clickable groups are well known in the art, see e.g. Fantoni et al., 2021, Chemical Reviews, 121 (12): 7122-7154; and Klbcker et al., 2020, Chem. Soc. Rev., 49:8749-8773. Examples of click reactions include alkyne + azide reactions (CuAAC), copper- free click strain promoted azide alkyne click (SPAAC) reactions (e.g. DBCO + azide), inverseelectron demand Diels-Alder cycloaddition (IEDDA). Thus, for example, a terminal clickable group can be a terminal alkyne or azide group, preferably an alkyne group (including a silyl- protected terminal alkyne). Further conjugable groups include, e.g. a dien or a dienophile. A conjugable group also includes protected forms of conjugable groups, such as a silyl-protected terminal alkyne. Thus, for example, a terminal clickable group can be a terminal alkyne or azide group, or a silyl-protected terminal alkyne group, preferably an alkyne group. A silyl protection may be, for example, a trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tri-iso- propylsilyloxymethyl, or triisopropylsilyl protection, preferably a triethylsilyl protection. Thus, a silyl-protected alkyne group may be a triethylsilyl-protected alkyne, for example.

In preferred examples, R⁴ is a n-butyl group. In most preferred examples, R⁴ with G² is a hex-5-ynyl group. Thus, as a preferred example, R⁴ with G² has the structure:

R⁴ can also comprise or consist of two or more hydrocarbons that are linked by an atom or group of atoms other than carbon, such as a phosphorus atom and/or an oxygen atom. For example, two hydrocarbons, such as alkyls, (each independently comprising 1-10, 2-10 or 2-6 carbon atoms) can be linked by an oxygen atom. In another example, two or three hydrocarbons, such as alkyls, (each independently comprising 1-10, 2-10 or 2-6 carbon atoms), one of which optionally being a beta-cyano-ethyl group, can be linked by a phosphate diester or a phosphate triester, respectively. Thus, in an embodiment, R⁴ with G² has the structure:

Thus, for example, R⁴ can consist of a) a linear hydrocarbon, or b) three linear hydrocarbons, one of which being a beta-cyano-ethyl group, linked by a phosphate triester, wherein R⁴ comprises 3-15 carbon atoms.

In some embodiments, LG¹ is a chloride, LG² is 2,4,6-trifluorophenolate, and R⁴ comprises or consists of a hydrocarbon, and G² represents an alkyne group, wherein R⁴ comprises 1-20 carbon atoms, such as 3-15 carbon atoms, 3-10 carbon atoms or 3-6 carbon atoms. Most preferably, the chiral compound has the following structure:

In preferred embodiments, the chiral compound is enantiopure (with regard to the phosphorus stereocenter). This allows diastereoselective synthesis of nucleoside monophosphoramidates or triphosphates. In a preferred example, the chiral compound has the structure:

This configuration (for TRIPP) can be identified as eluting first in a chiral chromatography separation as described in the Examples. This configuration can be further identified, for example, by separating the two enantiomers via chiral chromatography and testing whether expandable nucleoside triphosphates synthesized with a given enantiomer are suitable for Xpandomer synthesis (see e.g. Example 10 of WO 2020/172479).

In a preferred example, the chiral compound has the structure: wherein LG¹ is a conjugate base of a strong acid; R⁴ comprises or consists of a hydrocarbon; and G², when present, represents a terminal conjugable group, preferably a clickable group; and LG² has the following structure: wherein R, when present, represents one or more substituents, preferably electron-withdrawing substituents.

More preferably, the chiral chiral compound has the following structure: wherein R⁴ comprises or consists of a hydrocarbon; and G², when present, represents a terminal conjugable group, preferably a clickable group; and LG² has the following structure: wherein R, when present, represents one or more substituents, preferably electron-withdrawing substituents.

As a preferred example thereof, the chiral compound can have the following structure:

The disclosure also provides a composition that comprises the chiral compound disclosed herein. In some embodiments, the composition comprises both enantiomers of the chiral compound disclosed herein. For example, the composition can comprise or consists of a racemic mixture of both enantiomers. In preferred examples, at least 80%, preferably at least 90%, such as at least 95%, at least 99%, or 100% of the chiral compound comprised in the composition has the following structure:

The composition can further comprise an organic solvent, such as 2-methyltetrahydrofuran or EtOAc/w-hexane (10:90) or DCM. The composition is preferably water-free.

The disclosure also provides a method for producing a chiral compound as disclosed herein. An exemplary reaction scheme is given in Fig. 5. In some embodiments, the method for producing the chiral compound comprises reacting a phosphoryl halide with H-LG² and with HO- R⁴-G², wherein R⁴, G² and LG² are as defined in the context of the chiral compound. Preferably, the phosphoryl halide is phosphoryl chloride (POCI3).

In some embodiments, the method for producing the chiral compound comprises:

1) reacting a phosphoryl halide with H-LG²; and

2) reacting the product of step 1) with HO-R⁴-G².

In some embodiments, the method for producing the chiral compound comprises:

1) reacting a phosphoryl halide with HO-R⁴-G²; and

2) reacting the product of step 1) with H-LG².

As a preferred example, HO-R⁴-G² can be hex-5-yn-l-ol.

As preferred examples, H-LG² can be 2,4,6-trifluorophenol, 2,6-difluorophenol, 4- nitrophenol, 2-methyl-4-nitrophenol, or 3,5-bis(methoxycarbonyl)phenol, preferably 2,4,6- trifluorophenol.

In embodiments in which LG¹ and LG² are linked, the phosphoryl halide can be reacted with a single compound comprising both LG¹ and LG², such as substituted or unsubstituted salicylic acid.

For example, the reactions can be carried out in an organic solvent, such as DCM or 2- methyltetrahydrofuran. The reactions are preferably carried out under water-free conditions.

The reaction temperature is preferably lower than 30 °C, such as -20 °C to 25 °C, preferably 0 °C. It is also possible to start the reaction at 0 °C and gradually increase the temperature over time up to 20-25 °C.

The reaction mixture can further comprise an organic base, such as trimethylamine or pyridine, preferably pyridine.

The method may further comprise purifying the chiral compound, for example by normal phase chromatography. The method may further comprise separating the two enantiomers of the chiral compound, for example by chiral chromatography. Chiral chromatography can be high-performance liquid chromatography (HPLC), for example. Suitable examples of chiral selector used in chiral chromatography includes immobilized cellulose tri s-(3, 5 -dimethylphenylcarbamate). This allows the isolation of an enantiomer of choice, allowing diastereoselective synthesis of nucleoside monophosphoramidates or triphosphates.

The disclosure also provides a use of the chiral compound or a composition comprising the chiral compound as disclosed herein for producing a nucleoside monophosphoramidate as disclosed herein.

The disclosure also provides a use of a chiral compound or a composition comprising a chiral compound as disclosed herein for producing a nucleoside triphosphate as disclosed herein.

III. Nucleoside monophosphoramidates

The disclosure also provides a nucleoside monophosphoramidate with a 5’ phosphoramidate. Such nucleoside monophosphoramidate is obtainable by reacting the chiral compound disclosed herein with a 5 ’-amino-5’ -deoxynucleoside. The nucleoside monophosphoramidate has the following structure: wherein LG² is a conjugate base of a weak acid; NB is a nucleobase; R¹, when present, comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 -ribose modification; R³ is H or any protecting group; R⁴ comprises or consists of a hydrocarbon; and G¹ and G², when present, independently represent terminal conjugable groups, preferably clickable groups.

Also provided is a nucleoside monophosphoramidate having the following structure: wherein LG² is a substituted or unsubstituted cyclic or heterocyclic compound that is linked to the phosphorus via an exocyclic or an endocyclic group; NB is a nucleobase; R¹, when present, comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 '-ribose modification; R³ is H or any protecting group; R⁴ comprises or consists of a hydrocarbon; and G¹ and G², when present, independently represent terminal conjugable groups, preferably clickable groups.

NB is a nucleobase, and generally will be a pyrimidine nucleobase or a purine nucleobase. This includes naturally occurring nucleobases, like adenine, guanine, cytosine, uracil or thymine, and nucleobases with modifications that do not interfere with base pairing to a complementary nucleobase. For instance, pyrimidine nucleobases can be modified at the position 5, and purine nucleobases can be modified at the position 7. Non-limiting examples of nucleobases are adenine, guanine, thymine, cytosine, uracil, xanthine, hypoxanthine, 8-azapurine, purines substituted at the 8 position with methyl or bromine, 9-oxo-N6-methyladenine, 2-aminoadenine, 7-deazaxanthine, 7-deazaguanine, 7-deazaadenine, N4-ethanocytosine, 2,6-diaminopurine, N6-ethano-2,6- diaminopurine, 5-methylcytosine, 5-(C3-C10)-alkynylcytosine, 5 -fluorouracil, 5 -bromouracil, thiouracil, pseudoisocytosine, 2-hydroxy-5-methyl-4-triazolopyridine, isocytosine, isoguanine, inosine, 7,8-dimethylalloxazine, 6-dihydrothymine, 5,6-dihydrouracil, 4-methyl-indole, ethenoadenine and the non-naturally occurring nucleobases described in U.S. Pat. Nos. 5,432,272 and 6,150,510 and published PCT applications WO 92/002258, WO 93/10820, WO 94/22892 and WO 94/24144, and Fasman ("Practical Handbook of Biochemistry and Molecular Biology", pp. 385-394, 1989, CRC Press, Boca Raton, La.), all herein incorporated by reference in their entireties. In one embodiment, the nucleobase is selected from adenine, guanine, uracil, and cytosine, and modified versions of these nucleobases, such as those disclosed herein (e.g. 7- deazaadenine or 7-deazaguanine). NB is preferably selected from cytosine, thymine, 7- deazaadenine and 7-deazaguanine. In preferred embodiments, NB has one of the following structures: wherein R⁵ = H or a protective group, preferably H.

When the nucleobase is based on guanine, R⁵ can be, for example, N2-p-isopropyl- phenoxyacetyl, N2-phenoxyacetyl, N2-acetyl, N2-dimethylformamidine or N2-isobutyryl. When the nucleobase is based on adenine, R⁵ can be, for example, N6-phenoxyacetyl, N6-acetyl or N6- benzoyl. When the nucleobase is based on cytosine, R⁵ can be, for example, N4-acetyl or N4- benzyol.

R¹ can be present or absent, but is preferably present. When present, R¹ is typically such that it does not interfere with base-pairing with a complementary nucleobase. For example, R¹ is attached to position 5 of the nucleobase when the nucleobase is a pyrimidine nucleobase, and to position 7 of the nucleobase when the nucleobase is a purine nucleobase (wherein a naturally occurring nitrogen at position 7 can be replaced by a carbon, for example, as e.g. in 7-deazaadenine or 7-deazaguanine). Nucleobases with modifications, for example at position 5 (pyrimidine bases) or 7 (purine bases), and their synthesis are commonly known, see e.g. Kozak et al., 2020 (Russ. Chem. Rev., 2020, 89 (3) 281-310) and Matyugina et al., 2021 (Russ. Chem. Rev., 2021, 90 (11) 1454-1491). For concrete synthesis methods for nucleosides with bases as shown above, see also WO 2016/081871.

R¹ comprises or consists of a hydrocarbon. R¹ preferably consists of a hydrocarbon. The hydrocarbon can be substituted or unsubstituted, preferably unsubstituted. The hydrocarbon can be saturated or unsaturated, preferably unsaturated. For example, R¹ can comprise or consist of an alkyl, alkenyl, or alkynyl.

Preferably, R¹ comprises 1-100 carbon atoms, preferably 1-30 carbon atoms or 1-20 carbon atoms, such as 3-20 carbon atoms, 3-10 carbon atoms or 5-10 carbon atoms, such as 6 or 8 carbon atoms. Typically, R¹ will be acyclic. Preferably, R¹ is linear. In some embodiments, the molecular weight of R1 is 1500 g/mol or less, 1000 g/mol or less, 500 g/mol or less, 200 g/mol or less, or 100 g/mol or less.

In some embodiments, R¹ comprises or consists of a branched, linear, cyclic or heterocyclic, substituted or unsubstituted, saturated or unsaturated hydrocarbon, optionally including one or more heteroatoms, optionally selected from nitrogen, oxygen, phosphorus and sulfur. A cyclic or heterocyclic hydrocarbon can be 5-membered or 6-membered, for example. A cyclic or heterocyclic hydrocarbon can be aromatic, for example.

In some embodiments, R¹ is a substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group comprising 1-100 carbon atoms, which optionally includes one or more oxygen, nitrogen, phosphorus or sulfur heteroatoms (e.g. to include an ether, a thioether, a phosphordiester or phosphortriester, or PEG, a heterocycle, such as a triazole or imidazole).

In some embodiments, R¹ is -R^w-Z, wherein R^w is a substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group having between 1 and 100 carbon atoms, which optionally includes one or more oxygen, nitrogen, phosphorus or sulfur heteroatoms, and where Z is alkyl, alkenyl, alkynyl, acyl, -Het, or -CH2-Het, where "Het" is a substituted or unsubstituted 5- or 6-membered heterocyclic moiety.

In some embodiments, R¹ does not comprise an amino group, a hydroxyl group, or a thiol group. In some embodiments, R¹ can comprise a protected amino group, a protected hydroxyl group, or a protected thiol group or a protected or unprotected functional group that is compatible with the synthesis and usage of the phosphorylation reagent. Deprotection can then be done e.g. at the monophosphoramidate or triphosphate stage.

In preferred examples, R¹ is a hexa-l-ynyl or a octa-l-ynyl group. In most preferred examples, R¹ with G¹ is an octa-l,7-diynyl or a deca-l,9-diynyl group.

R² is independently H, OH or any 2 -ribose modification. In some embodiments, both R² are H or one R² is H and the other R² is OH. Preferably, both R² are H. 2’-ribose modifications are known in the art and include, for example, tert-butyldimethylsilyl and tri-iso-propylsilyloxymethyl ether groups as well as a 2’-O-methyl group or a 2’-fluoro group.

R³ is H or any protecting group, preferably H. Protecting groups are known in the art. Examples of a protecting group include acetyl, benzoyl, benzyl, methoxyethoxymethyl ether, dimethoxytrityl, ethoxymethyl ether, methoxytrityl, p-methoxybenzyl ether, p-methoxyphenyl ether, methylthiomethyl ether, pivaloyl, tert-butyl ethers, tetrahydropyranyl, tetrahydro furan, trityl, silyl ether (e.g. trimethylsilyl, triethylsilyl, tert-butyldimethylsilyl, tri-iso- propylsilyloxymethyl, or triisopropylsilyl ethers), methyl ethers, and ethoxyethyl ethers.

G¹ can be present or absent, and is preferably present. When present, G¹ represents a terminal conjugable group, preferably a clickable group.

The description of LG², R⁴ and G² in the context of the phosphorylation reagent above applies also to the nucleoside monophosphoramidate. In preferred embodiments, G¹ and G² represent terminal conjugable groups, preferably clickable groups, of the same type. Thus, for example, G¹ and G² can both be terminal alkyne groups or terminal azide groups, and preferably both are terminal alkyne groups.

In preferred embodiments, LG² is 2,4,6-trifluorophenolate, and R⁴ with G² has one of the following structures: preferably the following structure: Thus, for example, the nucleoside monophosphoramidate can have one of the following structures:

In particular, the nucleoside monophosphorami date can have one of the following structures:

Preferably, the nucleoside monop hosphoramidate has one of the following structures:

More preferably, the nucleoside monop hosphoramidate has one of the following structures:

In preferred embodiments, the nucleoside monophosphoramidate is diastereomerically

5 pure with regard to the phosphorus stereocenter. As a preferred example, the nucleoside monophosphoramidate has the following structure: wherein LG² is a conjugate base of a weak acid; NB is a nucleobase; R¹, when present, comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 -ribose modification; R³ is H or any protecting group; R⁴ comprises or consists of a hydrocarbon; and G¹ and G², when present, independently represent terminal conjugable groups, preferably clickable groups.

As another preferred example, the nucleoside monophosphoramidate has the following structure: wherein LG² is a substituted or unsubstituted cyclic or heterocyclic compound that is linked to the phosphorus via an exocyclic or an endocyclic group; NB is a nucleobase; R¹, when present, comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 '-ribose modification; R³ is H or any protecting group; R⁴ comprises or consists of a hydrocarbon; and G¹ and G², when present, independently represent terminal conjugable groups, preferably clickable groups.

Thus, for example, the nucleoside monophosphoramidate can have one of the following structures:

More particularly, the nucleoside monophosphoramidate can have one of the following structures:

In preferred embodiments, the nucleoside monophosphoramidate has one of the following structures:

In more preferred embodiments, the nucleoside monophosphoramidate has one of the

The disclosure also provides a composition comprising the nucleoside monophosphoramidate disclosed herein. In preferred embodiments, at least 80%, preferably at least 90%, such as at least 95%, at least 99%, or 100% of the nucleoside monophosphoramidate comprised in the composition has the following structure:

The disclosure also provides a method for producing a nucleoside monophosphoramidate as disclosed herein, comprising reacting the chiral compound as disclosed herein with a 5 ’-aminos’ -deoxynucleoside. The method can further comprise purifying the product of this reaction.

Preferably, the method comprises the following steps: wherein LG¹, LG², R¹, R², R³, R⁴, G¹ and G² are as described herein.

The method is preferably diastereoselective (with regard to the phosphorus stereocenter). To this end, the step is preferably carried out with an enantiopure chiral compound or a composition comprising an excess of one enantiomer over the other as disclosed herein. This allows the diastereoselective synthesis of the nucleoside monophosphoramidate. The chiral compound used for diastereoselective synthesis preferably has the structure:

Thus, more preferably, the method comprises the following steps:

The reaction of the chiral compound and the 5 ’-amino-5’ -deoxynucleoside can be carried out in an organic solvent, such as MeCN/MeTHF. Typically, the reaction is carried out under water-free conditions.

The temperature of the reaction is not particularly limited, and can be, for example, 0°C to 30°C, such as 15°C to 25°C.

The reaction of the chiral compound and the 5 ’-amino-5 ’-deoxynucleoside is typically carried out in the presence of a non-nucleophilic base, such as Triethylamine (TEA), N,N- Diisopropylethylamine (DIPEA), 1,8-Bis(dimethylamino)naphthalene, Tripropylamine, Triisopropylamine, 2,6-Lutidine, l,8-Diazabicyclo(5.4.0)undec-7-ene (DBU), preferably TEA, DIPEA or 1,8-Bis(dimethylamino)naphthalene, most preferably TEA.

Diastereoselective synthesis of the nucleoside monophosphoramidate is preferably performed without pyridine. For example, A-biityl-2-pyrrolidonc may be used instead. Moreover, the synthesis is preferably performed with low amounts of TEA or more bulky non-nucleophilic bases (e.g. 1-10 equivalents, such as 5 equivalents with respect to the 5’ -amino-5 ’- deoxynucleoside). Examples of bulky non-nucleophilic bases include DIPEA, DBU, 1,5- Diazabicyclo(4.3.0)non-5-ene (DBN), 1,8-Bis(dimethylamino)naphthalene and 2,6-Di-tert- butylpyridine.

The method can further comprise purifying the nucleoside monophosphoramidate. The purification can comprise separating the two diastereomers (with regard to the phosphorus stereocenter), e.g. by (preparative) HPLC. This may be relevant in embodiments, in which racemic phosphorylation reagent is used, which yields in a crude mixture of both active and inactive diastereomers of the nucleoside monophosphorami date.

When R³ is a protecting group, the method can further comprise removing the protecting group. This will typically result in replacement of the protecting group by a hydrogen. When R⁵ is a protective group, the method can further comprise removing the protective group. This will typically result in replacement of the protective group by a hydrogen. When G¹ and/or G² consist of a terminal alkyne group protected by a silyl group, the method can further comprise removing the silyl group to yield a terminal alkyne group. Deprotection can take place at any time during the method as disclosed herein. However, removal of a protective group R⁵ is typically done already on the 5’azido-5’deoxynucleoside.

The 5 ’-amino-5’ -deoxynucleoside can be obtained, for example, by selective reduction of a 5 ’-azido-5’ -deoxynucleoside. Thus, in some embodiments, the method further comprises a step of selectively reducing a 5’-azido-5’-deoxynucleoside to a 5 ’-amino-5 ’-deoxynucleoside. In some embodiments, the method further comprises the following step:

Undesired side-products (e.g. by water addition to alkyne groups) should be avoided during the reduction step. The preferred reducing agent is DTT. If protecting groups are used in the synthesis of the 5 ’-azido-5 ’-deoxynucleoside, deprotection and purification is preferably performed ahead of reduction to the 5 ’-amino-5 ’-deoxynucleoside in order to avoid inseparable impurities with 5’-OH-nucleosides. The 5 ’-azido-5 ’-deoxynucleoside is preferably free from Pd and Cu traces to reduce side products such as thiol adducts of DTT.

The reduction step can be carried out over a range of temperature, such as at 15° C to 30° C.

The disclosure also provides a nucleoside monophosphoramidate produced by the method disclosed herein.

The disclosure also provides a 5 ’-amino-5 ’-deoxynucleoside having the following structure: wherein NB is a nucleobase; R¹ comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 -ribose modification; R³ is H or any protecting group; G¹ represents terminal conjugable group, preferably clickable group. The further description of NB, R¹, R², R³ and G¹ from the context of the nucleoside monophosphoramidate equally applies.

The disclosure also provides a nucleoside monophosphate with a 5 ’-organophosphate. Such nucleoside monophosphate is obtainable by reacting the chiral compound disclosed herein with a 5 ’-hydroxynucleoside. The nucleoside monophosphate has the following structure: wherein LG², R^x-R⁴, G¹ and G² are as described herein; and a version thereof that is diastereomerically pure with regard to the phosphorus stereocenter, preferably:

The disclosure also provides a method for producing such nucleoside monophosphate comprising reacting the phosphorylation reagent described herein with a 5 ’-hydroxynucleoside. The disclosure on the method for producing a nucleoside monophosphoramidate applies mutatis mutandis. The disclosure also provides a use of a nucleoside monophosphoramidate or a composition comprising a nucleoside monophosphoramidate as disclosed herein for producing a nucleoside triphosphate as disclosed herein.

IV. Nucleoside triphosphates Also disclosed is a nucleoside triphosphate with a 5’ phosphoramidate. Such nucleoside triphosphate is obtainable by reacting the nucleoside monophosphoramidate disclosed herein with a pyrophosphate. The nucleoside triphosphate has the following structure: wherein NB is a nucleobase; R¹, when present, comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 -ribose modification; R³ is H or any protecting group; R⁴ comprises or consists of a hydrocarbon; and G¹ and G², when present, independently represent terminal conjugable groups, preferably clickable groups. Preferably, the nucleoside triphosphate is a modified 2 ’-deoxynucleoside triphosphate (dNTP). NB, R¹, R², R³, R⁴, G¹ and G² can further be as described in the context of the phosphorylation reagent and the nucleoside monophosphoramidate.

For example, the nucleoside triphosphate can have one of the following structures:

In preferred embodiments, the nucleoside triphosphate has one of the following structures: The nucleoside triphosphate is preferably diastereomerically pure with regard to the phosphorus stereocenter. Thus, in a preferred example, the nucleoside triphosphate has the following structure: wherein NB is a nucleobase; R¹, when present, comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 -ribose modification; R³ is H or any protecting group; R⁴ comprises or consists of a hydrocarbon; and G¹ and G², when present, independently represent terminal conjugable groups, preferably clickable groups.

Preferably, the nucleoside triphosphate has one of the following structures:

Most preferably, the nucleoside triphosphate has one of the following structures:

The nucleoside triphosphates can be used, for example, for sequencing by expansion. Thus, in such embodiments, the a-phosphoramidate is linked to the nucleobase, e.g. via a tether molecule (to form an XNTP). For example, R¹ can be linked to R⁴, e.g. via a tether molecule.

The tether molecule is not particularly limited, but will typically comprise a reporter (to allow specific identification of the attached nucleobase, e.g. via nanopore-based sequencing). A tether molecule can be, for example, a symmetrically synthesized reporter tether (SSRT) as disclosed in WO 2020/236526 Al. Such tether molecules typically have the following structure: Linker A - reporter - Linker B. Linker A can be attached to the a-phosphoramidate and linker B to the nucleobase, or vice versa. For example, Linker A and Linker B can be polymers comprising two or more repeat units selected from: spermine (Q), hexaethylene glycol (D), 2-((4-((3- (benzoyloxy)-2-((( 1 -(3 -(benzoyloxy)-2-((benzoyloxy)methyl)-2-((phosphodiester- oxy)methyl)propyl)- 1 H- 1 ,2, 3 -triazol-4-yl)methoxy)methyl)-2- ((benzoyloxy)methyl)propoxy)methyl)-lH-l,2,3-triazol-l-yl)methyl)-2-O-phosphodiester- propane- 1 ,3 -diyl dibenzoate, 1 ,3 -O-bis(phosphodiester-2,2-bis( 1 -Me-4-(Me-O-PEG2-O-Bz)- l,2,3-triazole)-propane, l,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-l-(Et-O-Ac)-l,2,3- triazole)-propane, l,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG7)-l-(Et-OBz)-l,2,3-triazole)- propane, l,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-l-(Et-2,2,2-Tris-(Me-O-Bz))-l,2,3- triazole)-propane, l,3-O-bis(phosphodiester-2-(4-(Me-O-PEG5)-l-(Et-2,2,2-Tris-(Me-O-Ac))- l,2,3-triazole)-propane, l,2-O-bis(phosphodiester)-3-(4-(Me-O-PEG3-O-Bz)-l-(l,2,3-triazole))- propane, l,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG2-O-Me)-l-(Et-O-Bz)-l,2,3-triazole)- propane, l,3-O-bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-l-(Et-2,2,2-Tris-(Me-O-Bz))- l,2,3-triazole)-propane, l,2-O-bis(phosphodiester)-3-(4-methylpiperazine-l-yl)-propane, 1,3-0- bis(phosphodiester-2,2-bis(4-(Me-O-PEG3-O-Me)-l-(Et-O-Bz)-l,2,3-triazole)-propane, and l,l’-O-bis(phosphodiester)-N(p-tolyl)-diethanolamine, preferably spermine. In some embodiments, linker A and B are inverted copies of each other. For example, a reporter can be a polymer comprising two or more repeat units selected from: hexaethylene glycol (D), ethane (L), triaethylene glycol (X), l,3-O-bis(phosphodiester)-2S- O-mPEG4-propane, l,3-O-bis(phosphodiester)-2-(4-Me-O-PEG3)-l-(Et-O-Ac)-l,2,3-triazole)- propane, l,3-O-bis(phosphodiester-2,2-bis(Me-O-mPEG2)-propane, l,3-O-bis(phosphodiester- 2S-O-(PEG4-O-Bz)-propane, l,3-O-bis(phosphodiester)-2s-O-mPEG6-propane, 1,3-0- bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-l-(Et-2,2,2-Tris-(Me-O-Bz))-l,2,3-triazole)-propane, l,3-O-bis(phosphodiester-2s-O-(4-(Me-O-PEG3)-l-(Me-acetate)-l,2,3-triazole)-propane, 1,3-0- bis(phosphodiester)-2s-O-(4-(Me-O-PEG2)-l-(Et-OBz)-l,2,3-triazole)-propane, 1,3-0- bis(phosphodiester)-2-(4-Et-l-(Et-O-mPEGl)-l,2,3-triazole)-propane, 2,3-0- bis(phosphodiester)-l-(l dimethoxyquinazolinedione)- propane , 2,3-O-bis(phosphodiester)-l- (N9-(3,6-dimethoxycarbazole)-propane, l,r-O-bis(phosphodiester)-2,2’-(sulfonylbis(benz-4- yl))- diethanol, l,r-O-bis(phosphodiester)-2,2’-bipyridin-4,4’-yl)-dimethanol, 2,3-0- bis(phosphodiester)-l-(Nl-(4,6-dimethoxy-3-Me-indole)-propane, 3-(l,2-O-bis(phosphodiester)- propyl)-8,8-dimethylhexahydro-3H-3a,6-methanobenzo[c]isothiazole 2,2-dioxide, 2,3-0- bis(phosphodiester)-l-(Nl-(6-Azathymine))-propane, l,5-O-bis(phosphodiester)- hexahydrofuro[2,6]furan, l,l’-O-bis(phosphodiester)-octahydro-2,6-dimethyl-3, 8:4,7- dimethano-2,6-naphthyridin-4,8-diyl)-dimethanol, 2,3-O-bis(phosphodiester)-l-(Nl-(2-Me-5- nitroindole)-propane, 2,3-O-bis(phosphodiester)-l-(Nl-(2-Me-5-nitroindole)-propane, 2,3-0- bis(phosphodiester)-l-(5-benzofuran)-propane, l,2-O-bis(phosphodiester)-3-O-mPEG2-propane, 1 ,3 -O-bis(phosphodiester)-2-(4-Et- 1 -(Et-0-mPEG3)- 1 ,2,3 -triazole)-propane, and 1,3-0- bis(phosphodiester)-3-O-mPEG4-propane (see WO 2020/236526 Al). In some embodiments, the reporter comprises or consists of two inverted copies of the same polymer comprising two or more repeat units selected from the above. The two inverted copies of the same polymer in the reporter can be linked via a branching element that is further linked to a translocation control element (TCE), as described in WO 2020/236526 Al.

For example, the nucleoside triphosphate can have the following structure: wherein T is a tether molecule; NB is a nucleobase; R¹ comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 -ribose modification; R³ is H or any protecting group; R⁴ comprises or consists of a hydrocarbon; and L¹ and L² independently represent a linking group.

Preferably, the tether molecule is attached via click chemistry reactions. In other words, when terminal conjugable groups, preferably clickable groups, G¹ and G² are present, they can be used to link a tether molecule via a click reaction. These G¹ and G² are typically conjugable groups, preferably clickable groups, of the same type. When the tether is attached via click reactions, L¹ and L² will be a products of a click reaction, such as a 1,2, 3 -triazole. For example, when both G¹ and G² are terminal alkyne groups, a tether molecule attached to terminal azide groups on two ends can be reacted with G¹ and G² (thereby yielding two 1,2, 3 -triazoles). Thus, in preferred embodiments, L¹ and L² each represents a 1,2,3-triazole. In more preferred embodiments, the nucleoside triphosphate has one of the following structures:

These structures are obtainable by reacting a nucleoside triphosphate (dNTP-2c) as disclosed herein with R¹ with G¹ = octa-1, 7-diynyl or deca-l,9-diynyl group, and R⁴ with G² = hex-5-ynyl group with a tether attached to terminal azide groups.

In preferred embodiments, the nucleoside triphosphate has one of the following structures:

As mentioned before, the nucleoside triphosphate is preferably diastereomerically pure with regard to the phosphorus stereocenter. When the a-phosphoramidate is linked to the nucleobase, the nucleoside triphosphate therefore preferably has the following structure:

When click chemistry is used for attaching the tether molecule, the diastereomerically pure nucleoside triphosphate preferably has one of the following structures:

In most preferred embodiments, the diastereomerically pure nucleoside triphosphate has one of the following structures:

The disclosure also provides a composition comprising the nucleoside triphosphate disclosed herein. In preferred embodiments, at least 80%, preferably at least 90%, such as at least 95%, at least 99%, or 100% of the nucleoside triphosphate comprised in the composition has the following structure: wherein NB is a nucleobase; R¹ comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 -ribose modification; R³ is H or any protecting group; R⁴ comprises or consist of a hydrocarbon; G¹ and G², when present, independently represent terminal conjugable groups, preferably clickable groups; and L¹ and L² independently represent a linking group. The disclosure on the nucleoside triphosphate as such applies mutatis mutandis.

The disclosure also provides a method for producing a nucleoside triphosphate or composition comprising the same as disclosed herein. The method comprises reacting the nucleoside monophosphoramidate as disclosed herein with a pyrophosphate (preferably a pyrophosphate salt, such as tris(tetrabutylammonium) hydrogen pyrophosphate). In preferred examples, the method can comprise the following step: wherein LG² is a conjugate base of a weak acid; NB is a nucleobase; R¹, when present, comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 -ribose modification; R³ is H or any protecting group; R⁴ comprises or consists of a hydrocarbon; and G¹ and G², when present, independently represent terminal conjugable groups, preferably clickable groups; R⁶ is H or any organic or inorganic cation; and Z⁺ is H⁺ or any (monovalent or bivalent) cation.

The nucleoside monophosphoramidate can be obtained by the method for producing a nucleoside monophosphoramidate described herein.

Thus, in some embodiments, the method for producing a nucleoside triphosphate comprises:

(a) reacting the chiral compound as disclosed herein with a 5 ’-amino-5’ -deoxynucleoside, and (b) reacting the product of step (a) with a pyrophosphate.

In a preferred example, the method can further comprise the following steps: wherein NB, LG¹, LG², R¹, R², R³, R⁴, G¹ and G² are as defined above.

Typically, the reactions are substitution reactions proceeding with inversion of configuration, such as an Sx2-type reaction. For instance, each of the above steps is a Sx2-type reaction. When both reactions are SN2-type reactions (that inverse the configuration at the phosphorus stereocenter) the nucleoside triphosphate will have the same configuration as the phosphorylation reagent used as input. Conversely, when starting from a nucleoside monophosphoramidate and the reaction with pyrophosphate is a SN2-type reaction, the configuration of the nucleoside triphosphate will be inverted compared to the nucleoside monophosphoramidate used as input. This allows for the diastereoselective production of the nucleoside triphosphate.

When R⁴ comprises a hydrocarbon with a beta-cyano-ethyl group linked to a phosphate, said hydrocarbon is typically removed after the reaction with the pyrophosphate.

The method is preferably a method for diastereoselectively producing a nucleoside triphosphate or composition comprising the same as disclosed herein. To this end, the nucleoside monophosphoramidate used is preferably diastereomerically pure with regard to the phosphorus stereocenter. The nucleoside monophosphoramidate used for diastereo selective production preferably has the structure:

Thus, in preferred embodiments of diastereoselective production, the method comprises the following step: wherein NB, LG², R¹, R², R³, R⁴, G¹, G², R⁶, and Z⁺ are as defined above.

When starting from a 5 ’-amino-5’ -deoxynucleoside, the diastereoselective production can further comprise the following step: wherein NB, LG¹, LG², R¹, R², R³, R⁴, G¹ and G² are as defined above.

The reaction of the nucleoside monophosphoramidate with a pyrophosphate can be carried out in an organic solvent, such as MeCN. Typically, the reaction mixture will also comprise triethylamine. The reactions are typically conducted under water-free conditions.

The reaction can be carried out at a wide range of temperatures, e.g. -20 °C to 30 °C, preferably at 0 °C to 20 °C, such as 0 °C to 8 °C, preferably 0 °C.

As in the context of the production of a nucleoside monophosphoramidate, the 5 ’-aminos’ -deoxynucleoside can be obtained, for example, by reduction of a 5 ’-azido-5’ -deoxynucleoside. Thus, in some embodiments, the method further comprises a step of reducing a 5’ -azido-5 ’- deoxynucleoside to a 5 ’-amino-5 ’-deoxynucleoside.

In some embodiments, e.g. when LG² is 2,4,6-trifluorophenolate, the reaction with a pyrophosphate is preferably to be performed at lower temperatures (e.g. 0 °C) for highest yields. Thus, in some embodiments, the reaction with a pyrophosphate is performed at -10 °C to 10 °C, preferably -5°C to 5°C, most preferably 0°C.

Preferably, the pyrophosphate is water-free for optimal nucleophilicity. Dry tris(tetrabutylammonium) hydrogen pyrophosphate is preferred. Moreover, the amount of residual monophosphoramidate in the pyrophosphate starting material is typically low and controlled, and the content of tetrabutylammonium ions is typically controlled. The (diastereoselective) synthesis of the nucleoside monophosphoramidate and the corresponding nucleoside triphosphate works in liquid phase with conventional laboratory equipment as used in any chemical laboratory or small scale production, and can be readily scaled up. Thus, in preferred embodiments, the methods disclosed herein are performed in liquid phase.

In addition, the (diastereoselective) synthesis of the nucleoside monophosphoramidate and the corresponding nucleoside triphosphate can be performed in one pot without isolation or workup of the intermediate compound.

The nucleoside triphosphates can be used, for example, for sequencing by expansion. To this end, in some embodiments, the method further comprises a step of linking the a- phosphoramidate to the nucleobase of the nucleoside triphosphate produced by the reaction with pyrophosphate. The a-phosphoramidate can be linked to the nucleobase via a tether molecule, for example. In preferred embodiments, R¹ is linked to R⁴, e.g. via a tether molecule, as described for the nucleoside triphosphate as such. The tether molecule to be used is not particularly limited, and can be, for example, a symmetrically synthesized reporter tether (SSRT) as disclosed in WO 2020/236526 Al. Preferably, the tether molecule is attached via click chemistry reactions. Typically, the product of the step of linking the a-phosphoramidate to the nucleobase is an XNTP. Thus, for production of XNTPs, the method preferably further comprises the following step: wherein NB is a nucleobase; R¹ comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 -ribose modification; R³ is H or any protecting group; R⁴ comprises or consists of a hydrocarbon; G¹ and G² independently represent terminal conjugable groups, preferably clickable groups; G^la and G^2a independently represent terminal conjugable groups, preferably clickable groups; L¹ and L² independently represent linking groups formed by reacting G¹ with G^la and G² with G^2a; and T is a tether molecule; and Z⁺ is H⁺ or any (monovalent or bivalent) cation.

During diastereoselective production (of the active diastereomer), the above step preferably looks as follows:

In preferred embodiments, L¹ and L² each represent a 1,2,3-triazole. 1,2,3-triazoles can be formed, for example, by reacting terminal alkyne groups with terminal azide groups. Thus, preferably, G¹ and G² represent terminal alkyne groups and G^la and G^2a represent terminal azide groups or G¹ and G² represent terminal azide groups and G^la and G^2a represent terminal alkyne groups. More preferably, G¹ and G² represent terminal alkyne groups and G^la and G^2a represent terminal azide groups.

The method can further comprise purifying the nucleoside triphosphate. Purification can take place e.g. between the reaction with a pyrophosphate and linking the a-phosphoramidate to the nucleobase and/or after linking the a-phosphoramidate to the nucleobase. The purification can comprise separating the two diastereomers (with regard to the phosphorus stereocenter), e.g. by (preparative) HPLC. This may be relevant in embodiments, in which racemic phosphorylation reagent is used, which yields in a crude mixture of both active and inactive diastereomer of the nucleoside triphosphate. If necessary, separation of the two diastereomers is typically done between the reaction with a pyrophosphate and linking the a-phosphoramidate to the nucleobase. Purification may also include anion exchange chromatography.

When R³ is a protecting group, the method can further comprise removing the protecting group. This will typically result in replacement of the protecting group by a hydrogen. When R⁵ is a protective group, the method can further comprise removing the protective group. This will typically result in replacement of the protective group by a hydrogen. When G¹ and/or G² consist of a terminal alkyne group protected by a silyl group, the method can further comprise removing the silyl group to yield a terminal alkyne group. Deprotection can take place at any time during the method as disclosed herein. However, removal of a protective group R⁵ is typically done already on the 5’azido-5’deoxynucleoside.

The disclosure also provides a nucleoside triphosphate obtained by the methods disclosed herein.

The methods disclosed herein are not limited to the production of nucleoside triphosphates, but nucleosides with two or more than three phosphates can also be produced. The disclosure thus also provides a method for producing a nucleoside diphosphate or nucleoside oligophosphate, comprising reacting the nucleoside monophosphoramidate or the composition comprising the same as disclosed herein with a monophosphate or oligophosphate, preferably with a monophosphate or oligophosphate salt. An oligophosphate can comprise 3-6 phosphate units, for example, and is preferably a triphosphate or tetraphosphate. The disclosure on the method for producing a nucleoside triphosphate applies mutatis mutandis.

The disclosure further provides a nucleoside triphosphate with a 5’ a-organophosphate. Such nucleoside triphosphate is obtainable by reacting the modified nucleoside monophosphate with a 5’ organophosphate disclosed herein with a pyrophosphate. The nucleoside triphosphate has the following structure: wherein NB, R¹, R², R³, R⁴, G¹ and G² are as described herein; and a version thereof that is diastereomerically pure with regard to the phosphorus stereocenter, preferably:

The disclosure also provides a method for producing such nucleoside triphosphate comprising reacting the nucleoside monophosphate with a 5’ organophosphate disclosed herein with a pyrophosphate. In some embodiments, the method comprises reacting the phosphorylation reagent described herein with a 5 ’-hydroxynucleoside and a pyrophosphate (salt). The disclosure on the method for producing a nucleoside triphosphate with a 5’ phosphoramidate applies mutatis mutandis. V. Examples

The following non-limiting examples show preferred methods for the production of preferred types of the phosphorylation reagent, of nucleoside monophosphoramidates and triphosphates.

Example 1: Preparation of racemic TRIPP

In a Schlenk-flask under Argon, a solution of POCI3 (4.93 mL, 54.0 mmol, 2.00 eq.) in 2-methyltetrahydrofuran (2-MeTHF, 125 mL, dry) was cooled to 0 °C. In a separate Schlenk-flask 2,4,6-Trifluorophenol (FPhOH, 4.00 g, 27.0 mmol, 1.00 eq.) and triethylamine (TEA, 4.52 mL, 32.4 mmol, 1.2 eq.) were dissolved in dry 2-MeTHF (125 mL) under Argon. The solution of 3FPhOH and TEA was added dropwise to the POCI3 solution at 0 °C over 2 h using a syringe pump. The reaction mixture was stirred for 20 min at 0 °C. A sample was analyzed by LC-MS (1 pL of reaction mixture was quenched with 100 pL MeOH).

TEA (4.90 mL, 35.1 mmol, 1.3 eq.) and Hexynol (3.98 g, 40.5 mmol, 1.5 eq.) were added at 0 °C and the reaction was stirred at 0 °C for 30 min. A sample was analyzed by LC-MS (1 pL of reaction mixture was quenched with 100 pL MeOH).

The reaction was filtered using a fritted glass. Subsequently, the filter was washed with EtOAc and the filtrate was concentrated in vacuo to remove most of 2-MeTHF and EtOAc.

The crude product was purified via flash chromatography (Puriflash 5250, column: PF- 30SIHP-F0220). The start point was at 100 % n-hexane and the end point was at 100% EtOAc.

Yield (racemate): 3.96 g, 12.1 mmol, 45% (with respect to the input amount of 3FPhOH).

In another protocol, 2,4,6-trifluorophenol (5.00 g, 33.8 mmol, 1.00 eq.) was dissolved in 102 mL MeTHF (dry). At 0 °C POCI3 (5.55 mL, 60.8 mmol, 1.80 eq.) was added in one portion. Subsequently pyridine (dry, 4.72 mL, 58.4 mmol, 1.73 eq.) was added via syringe pump (flow: 14.2 mL/h). 30 min after completion of the addition, a sample was analyzed by LC-MS (quench 1 pL of reaction mixture in 200 pL MeOH) indicating full conversion of the phenol. After 45 min total reaction time, hex-5-yn-l-ol (6.7 mL, 60.8 mmol, 1.80 eq.) and fast dropwise pyridine (dry, 5.46 mL, 67.5 mmol, 2.00 eq.) was added at 0 °C. After 30 min a sample was analyzed by LC-MS (quench 1 pL of reaction mixture in 300 pL MeOH) indicating full conversion of the intermediate. After 45 min the reaction mixture was filtered through a schlenk frit under argon. The filter cake was washed with EtOAc (2*5 mL). The filtrate was concentrated in vacuo (30 °C). The crude product was purified via column chromatography. For enantioselective separation CHIRALPAK IB-N (5 pm, 50*250 mm) and acetone :n-heptane (3:97) with a flow of 185 mL/min was used. 31 P (Acetonitril-d3) 1.48 (1 P,s). 19F (Acetonitril-d3) -110.99 (2 F, m), -123.95 (1 F, m) For LC-MS analysis the phosphorylation reagent was reacted with MeOH. LC-MS (YMC Triart Cl 8, Buffer A: H2O + 0.1 % formic acid, Buffer B: MeCN + 0.1 % formic acid, gradient elution: 100% Buffer A for 0.5 min, then 0% to 100% Buffer B in Buffer A over 6 min, flow rate 0.45 mL/min, 50 °C): RT = 6.56 min, calc, for C13H15F3O4P+: 323.1, found: 323.1.

Example 2: Enantioselective separation of TRIPP

2 cm x 25 cm column:

-Interchim Puriflash 4250

-Chiral column: Daicel Chiral Technologies, CHIRALPAK® IB N-5 20x250mm, particle size: 5 pm, compatible with HPLC (Normal Phase and Reversed-Phase)

-equilibration of the system: eluent: acetone/w-hexane (3:97), isocratic, eluent was premixed

Flow: 25 mL/min

Total run time: 20 min

22 °C

-method: eluent: acetone/w-hexane (3:97), isocratic, eluent was premixed

Flow: 30 mL/min

Total run time: 15 min

22 °C

Wavelength: 254 nm

First enantiomer (El, 10:00 min) was collected (see Fig. 7A)

-200 pL of a TRIPP solution (200 mg/mL of racemic TRIPP in EtOAc/w-hexane (10:90)) -criteria of collection:

From the starting point of the elution of the first enantiomer to max. 30% intensity after maximum.

Enantioselective separation was executed on an Interchim Puriflash 4250 system using a CHIRALPAK® IB N-5 column (20x250mm, particle size: 5 pm, compatible with HPLC).

Before separation, the column was equilibrated with manually premixed acetone/w-hexane (3:97) and a flow of 25 mL/min for 20 min.

For enantioselective separation, a 200 pL loop was charged manually with a 200 pL solution of racemic TRIPP reagent (200 mg/mL in EtOAc/w-hexane (10:90)). The enantioselective separation was carried out with manually premixed acetone/w-hexane (3:97) and a flow of 30 mL/min at 22 °C. The first enantiomer (10:00 min) was collected from the starting point of the elution of the first enantiomer to max. 30% intensity after maximum. The method was run for 15 min. The fraction collector was washed for 1 min within the method. Subsequently further runs for enantioselective separation can be carried out. The resolution as well as the peak shape are improved by successively executed runs. Also the elution time is shortened.

The isolated TRIPP enantiomer was quantified after evaporation (1 mbar, 30 °C for 1 h) and drying under high vacuum for 1 h. The identity and purity was analyzed by NMR spectroscopy. In addition to NMR, the identity and purity as well as reactivity of the TRIPP reagent was verified by quenching 1 pL of reagent solution (50 mg/mL in 2-MeTHF) in 100 pL MeOH and analyzing the MeOH-adduct by LC-MS. TRIPP reagent was obtained as colorless oil with a yield of 12.3 mg per run (45% of theoretical yield). The reagent was stored under argon at -20 °C.

Upscale to a 5 cm x 25 cm column

-Interchim Puriflash 4250

-Chiral column: Daicel Chiral Technologies, CHIRALPAK® IB N-5 50x250mm, particle size: 5 pm, compatible with HPLC (Normal Phase and Reversed-Phase)

-equilibration of the system: eluent: acetone/zz-hexane (3:97), isocratic, eluent was premixed

Flow: 120 mL/min

Total run time: 20 min

22 °C

-method: eluent: acetone/zz-hexane (3:97), isocratic, eluent was premixed

Flow: 185 mL/min

Total run time: 15 min

22 °C

Wavelength: 254 nm

First enantiomer (El, 9: 19 min) was collected (see Fig. 7B)

-1.5 mL of a TRIPP solution (200 mg/mL of racemic TRIPP in EtOAc/zz-hexane (10:90)) -criteria of collection:

From the starting point of the elution of the first enantiomer to max. 30% intensity after maximum.

Enantioselective separation was executed on an Interchim Puriflash 4250 system using a CHIRALPAK® IB N-5 column (50x250mm, particle size: 5 pm, compatible with HPLC). Before separation the column was equilibrated with manually premixed acetone/w-hexane (3:97) and a flow of 120 mL/min for 20 min.

For enantio selective separation, a 2 mL loop was charged manually with a 1.5 mL solution of racemic TRIPP reagent (200 mg/mL in EtOAc/w-hexane (10:90)). The enantioselective separation was carried out with manually premixed acetone/w-hexane (3:97) and a flow of 185 mL/min at 22 °C. The first enantiomer (9:19 min) was collected from the starting point of the elution of the first enantiomer to max. 30% intensity after the peak maximum. The method was run for 15 min. The fraction collector was washed for 1 min within the method. Subsequently further runs for enantioselective separation can be carried out. The resolution as well as the peak shape are improved by successively executed runs. Also the elution time is shortened.

The isolated TRIPP enantiomer was quantified after evaporation (1 mbar, 30 °C for 1 h) and drying under high vacuum for 1 h. The identity and purity was analyzed by NMR spectroscopy (Fig. 8A). In addition to NMR, the identity and purity as well as reactivity of the TRIPP reagent was verified by quenching 1 pL of reagent solution (50 mg/mL in 2-MeTHF) in 100 pL MeOH and analyzing the MeOH-adduct by LC-MS (Fig. 8B). Enantiopure TRIPP reagent was obtained as a colorless oil with a yield of 583 mg in ten runs with a total injection amount of 2.0 g racemic TRIPP reagent using an injection amount ranging from of 100 mg to 300 mg racemic TRIPP per run (58% of theoretical yield). The reagent was stored under argon at -20 °C.

Example 3: Diastereoselective synthesis

1. C10-dTMP-2c 3FP phosphoramidate

To a suspension of 5’-NH2-C10-dT (43.1 mg, 120 pmol, 1.0 eq) in dry MeCN (2.2 mL) and dry 2-MeTHF (2.2 mL) was added tri ethylamine (21.7 pL, 156 pmol, 1.3 eq) and a solution of TRIPP reagent (50 mg/mL in dry MeTHF, single enantiomer El, 1.0 mL, 156 pmol, 1.3 eq) was added dropwise. The reaction mixture was stirred at room temperature for 15 minutes. The clear reaction solution was analyzed by LC-MS indicating quantitative conversion of the starting material. The reaction was quenched by addition of MeOH and the solution was concentrated in vacuo to dryness, dried under high vacuum overnight and used without further purification.

LC-MS (YMC Triart C18, Buffer A: H₂O + 0.1% FA, Buffer B: MeCN + 0.1% FA, gradient elution: 100% Buffer A for 1 min, then 0% to 100% Buffer B in Buffer A over 10 min, flow rate 0.3 mL/min, 30 °C): RT = 10.6 min, calc, for CsiHseFsNsOyP : 650.2, found: 650.3.

Tris(tetrabutylammonium) hydrogen pyrophosphate (PPi) was dried under high vacuum overnight before usage. The crude C10-dTMP-2c 3FP phosphoramidate (78.0 mg, 120 pmol, 1.0 eq) was dissolved in dry MeCN (8.5 mL). The solution was cooled to 0 °C and a solution of PPi (487 mg, 540 pmol, 4.5 eq) in dry MeCN (8.5 mL) was added quickly and stirred at 0 °C. The reaction progress was monitored by LC-MS, indicating full consumption of phosphoramidate within 20-40 minutes. The reaction mixture was diluted with triethylammonium acetate (TEAA) buffer (100 mM, pH 7, 15 mL) and concentrated in vacuo (30 °C, 50 mbar) to remove MeCN. The resulting suspension was filtered (syringe filter, 0.45 pm). The filter was washed with additional TEAA buffer (100 mM, pH 7, 2 mL) and subjected to preparative HPLC (isocratic elution using 100 mM TEAA in 48.5% MeOH for 100 min, flow rate 75 mL/min). Collected fractions of desired product were pooled on ice, diluted with H2O (1 :1) and desalted via Oasis HLB 20cc 1g column. After eluting with H2O/MeCN (1 : 1, 15 mL), the eluate was lyophilized to yield C10-dTTP-2c (49 pmol, 41%) which was dissolved in 20 mM TrisCi buffer at pH 7.5-8.5, analyzed by HPLC (min. 98.0 area% purity) and stored at -80 °C.

Analytical HPLC (Atlantis T3 150 x 4.6 mm, 3 pm; Buffer A: l0 mM TEAA in H2O, Buffer B: 10 mM TEAA in 95% MeOH, isocratic elution: 50% buffer B for 58 min, flow rate 0.7 mL/min): RT = 24.0 min (kmax = 292 nm). Example 4: Synthesis of nucleoside triphosphates and intermediates

General Methods

Unless otherwise noted, reactions were carried out under argon. Reactions were monitored by LC-MS using YMC Triart C8 or C18 columns (50 x 2.0mm, I.D. S-l,9pm ,12nm). Unless otherwise noted, crude reaction mixtures and isolated products were analyzed using a gradient of MeCN (+0.1% FA) in H2O (+0.1% FA) from 0% to 100% over 10 minutes after 1 minute of isocratic 0% MeCN (+0.1% FA). Triphosphate Synthesis was monitored using a C18 column (YMC Triart C18, 50 x 2.0mm, 3.0 pm,12nm) using a gradient MeCN in 10 mM TEAA (pH = 7) from 0% to 100% over 12 minutes.

1. TRIPP reagent a. C6pC6-TRIPP reagent

A solution of POCI3 (123 pL, 1.35 mmol, 3.1 eq) in dry DCM (5 mL) was cooled to 0 °C under argon atmosphere. A solution of 2,4,6-trifluorophenol (100 mg, 675 pmol, 1.5 eq) and triethylamine (113 pL, 0.81 mmol, 1.8 eq) in dry DCM (5 mL) was added dropwise over 15 minutes. The reaction mixture was stirred for 3 hours at 0 °C and monitored by LC-MS (monitoring MeOH adducts). Triethylamine (113 pL, 0.81 mmol, 1.8 eq) and 2-cyanoethyl hex-5-yn-l-yl (6- hydroxyhexyl) phosphate as a solution in dry DCM (50 mg/mL, 2.9 mL, 438 pmol, 1.0 eq) were added at 0 °C and the reaction mixture was stirred for 16 hours, allowing the reaction mixture to warm to room temperature. The reaction mixture was directly subjected to column chromatography (PuriFlash, NP, 40g, nHex/EtOAc: 0CV - 0%, 2CV - 0%, 12CV - 10%, 15CV - 20% EtOAc, UV-VIS: 254 nm, RT = 10 CV) to yield the product (58.0 mg, 104 pmol, 24%) as colorless oil.

The C6pC6-TRIPP reagent was stored as a solution in dry DCM (25 mg/mL) at -20 °C. For LC-MS analysis, a sample (2 pL) was quenched with MeOH (50 pL) to generate the corresponding methyl ester of the C6pC6-TRIPP reagent. 'H NMR (400 MHz, ACETONITRILE-d3) 5 ppm 1.42 - 1.50 (m, 2 H) 1.56 - 1.65 (m, 1 H) 1.68 - 1.83 (m, 3 H) 2.19 (q, J=2.51 Hz, 1 H) 2.21 - 2.27 (m, 1 H) 2.75 - 2.86 (m, 1 H) 4.02 - 4.25 (m, 4 H) 6.93 - 7.02 (m, 1 H).

³¹P NMR (162 MHz, ACETONITRILE-d3) 5 ppm -5.67 (s, 1 P) -2.42 (s, 1 P) -2.28 (s, 1 P) -1.69 (s, 1 P) 2.45 (s, 1 P).

¹⁹F NMR (376 MHz, ACETONITRILE-d3) 5 ppm -125.05 (br d, J=7.15 Hz, 1 F) -123.57 (br s, I F) -113.61 - -113.49 (m, 1 F) -112.58 (br s, I F).

LC-MS (YMC Triart C18, Buffer A: H₂O + 0.1% FA, Buffer B: MeCN + 0.1% FA, gradient elution: 100% Buffer A for 1 min, then 0% to 100% Buffer B in Buffer A over 10 min, flow rate 0.3 mL/min, 30 °C): RT = 10.6 min, calc, for methyl ester C22H3iF3NOsP2⁺: 556.1, found: 556.2.

2. 5’-amino-5’-deoxynucleosides

5’-Azido-dG (156 mg, 395 pmol, 1.0 eq) was dissolved in pyridine (1 mL) and H2O (1 mL). NaHCCh (113 mg, 1.34 mmol, 3.4 eq) and DTT (122 mg, 789 pmol, 2.0 eq) were added in one portion and stirred at rt for 2 hours. LC-MS indicated quantitative conversion. The solvent was removed in vacuo and the residue was dissolved in 5% aqueous MeCN (2 mL) and directly subjected to reverse phase column chromatography (PuriFlash, RP25g, H2O/MeCN: OCV - 5%, 2CV - 5%, 13CV - 40%, 15CV - 100% MeCN). The collected fractions were concentrated in vacuo to remove MeCN and lyophilized to yield 5’-Amino-dG (80 mg, 218 pmol, 55%) as a colorless solid.

'H NMR (400 MHz, METHANOL-d4 ) 5 ppm 1.65 - 1.75 (m, 4 H) 2.21 (br d, J=12.67 Hz, 4 H) 2.43 (s, 2 H) 2.49 - 2.59 (m, 1 H) 2.77 - 2.97 (m, 2 H) 3.85 (br d, J=3.76 Hz, 1 H) 4.30 - 4.42 (m, 1 H) 6.35 (t, J=6.84 Hz, 1 H) 6.98 (s, 1 H). LC-MS (YMC Triart C8, Buffer A: H₂O + 0.1% FA, Buffer B: MeCN + 0.1% FA, gradient elution: 100% Buffer A for 1 min, then 0% to 100% Buffer B in Buffer A over 10 min, flow rate 0.3 mL/min, 30 °C): RT = 7.6 min, calc, for C_I9H₂4N₅O₃ : 370.2, found:370.2

5’-Azido-dC (100 mg, 251 pmol, 1.0 eq) was dissolved in pyridine (1.39 mL) and H₂O (1.39 mL). NaHCCh (72.1 mg, 858 pmol, 3.4 eq) and DTT (116 mg, 753 pmol, 3.0 eq) were added in one portion and stirred at rt for 2 hours. LC-MS indicated quantitative conversion. The solvent was removed in vacuo and the residue was dissolved in 10% aqueous MeCN (2 mL) and directly subjected to column chromatography (PuriFlash, RP25g, H₂O/MeCN: 0CV - 5%, 2CV - 5%, 13CV - 40%, 15CV - 100% MeCN). The collected fractions were concentrated in vacuo to remove MeCN and lyophilized to yield 5’-Amino-dC (37 mg, 112 pmol, 45%) as a colorless solid.

'H NMR (400 MHz, METHANOL-d4 ) 5 ppm 1.61 - 1.77 (m, 4 H) 2.18 - 2.28 (m, 4 H) 2.30 - 2.40 (m, 1 H) 2.48 (s, 2 H) 2.85 - 3.06 (m, 2 H) 3.88 (dt, J=7.37, 4.47 Hz, 1 H) 4.23 (dt, J=6.90, 4.33 Hz, 1 H) 6.14 (s, 1 H) 7.89 (s, 1 H).

LC-MS (YMC Triart C18, Buffer A: H₂O + 0.1% FA, Buffer B: MeCN + 0.1% FA, gradient elution: 100% Buffer A for 1 min, then 0% to 100% Buffer B in Buffer A over 10 min, flow rate 0.3 mL/min, 30 °C): RT = 7.0 min, calc, for C17H23N4O3 : 331.2, found: 331.2 5’-Azido-dA (634 mg, 1.67 mmol, 1.0 eq) was dissolved in pyridine (4.18 mL) and H2O (4.18 rnL). NaHCCh (480 mg, 5.71 mmol, 3.4 eq) and DTT (515 mg, 3.34 mmol, 2.0 eq) were added in one portion and stirred at rt for 2 hours. LC-MS indicated quantitative conversion. The solvent was removed in vacuo and the residue was dissolved in 20% aqueous MeCN and directly subjected to column chromatography (PuriFlash, RP25g, H2O/MeCN). The collected fractions were concentrated in vacuo to remove MeCN and lyophilized to yield 5’-Amino-dA (490 mg, 1.39 mmol, 83%) as a colorless solid.

'H NMR (400 MHz, METHANOL-d4) 5 ppm 1.63 - 1.80 (m, 4 H) 2.23 - 2.28 (m, 3 H) 2.30 - 2.38 (m, 1 H) 2.49 - 2.55 (m, 2 H) 2.58 - 2.70 (m, 1 H) 2.78 - 2.96 (m, 2 H) 3.88 (s, 1 H) 4.38 (br d, J=6.53 Hz, 1 H) 6.52 (s, 1 H) 7.44 (s, 1 H) 8.10 (s, 1 H).

LC-MS (YMC Triart C18, Buffer A: H₂O + 0.1% FA, Buffer B: MeCN + 0.1% FA, gradient elution: 100% Buffer A for 1 min, then 0% to 100% Buffer B in Buffer A over 10 min, flow rate 0.3 mL/min, 30 °C): RT = 7.11 min, calc, for C19H24N5O2 : 354.2, found: 354.2.

5’-Azido-C8-dT (500 mg, 1.40 mmol, 1.0 eq) was dissolved in pyridine (7.0 mL) and H2O (7.0 mL). NaHCCh (402 mg, 4.79 mmol, 3.4 eq) and DTT (432 mg, 2.80 mmol, 2.0 eq) were added in one portion and stirred at rt for 2 hours. LC-MS indicated quantitative conversion. The solvent was removed in vacuo and the residue was dissolved in 20% aqueous MeCN and directly subjected to column chromatography (PuriFlash, RP25g, H2O/MeCN). The collected fractions were concentrated in vacuo to remove MeCN and lyophilized to yield 5’-Amino-C8-dT (300 mg, 905 pmol, 65%) as a colorless solid.

'H NMR (400 MHz, METHANOL-d4) 5 ppm 1.60 - 1.76 (m, 4 H) 2.18 - 2.25 (m, 2 H) 2.26 - 2.36 (m, 2 H) 2.39 - 2.46 (m, 2 H) 2.83 - 2.97 (m, 2 H) 3.78 - 3.89 (m, 1 H) 4.21 - 4.30 (m, 1 H) 6.16 (t, J=6.78 Hz, 1 H) 7.83 (s, 1 H). LC-MS (YMC Triart C8, Buffer A: H₂0 + 0.1% FA, Buffer B: MeCN + 0.1% FA, gradient elution: 100% Buffer A for 1 min, then 0% to 100% Buffer B in Buffer A over 10 min, flow rate 0.3 mL/min, 30 °C): RT = 7.0 min, calc, for C17H22N3O/: 332.2, found: 332.2

5’-Azido-C10-dT (250 mg, 649 pmol, 1.0 eq) was dissolved in pyridine (3.50 mL) and H2O (1.75 mL). NaHCCh (185 mg, 2.21 mmol, 1.5 eq) and DTT (200 mg, 1.30 mmol, 2.0 eq) were added in one portion and stirred at rt for 2 hours. LC-MS indicated quantitative conversion. The solvent was removed in vacuo and the residue was dissolved in 10% aqueous MeCN and directly subjected to column chromatography (PuriFlash, RP25g, H₂O/MeCN). The collected fractions were concentrated in vacuo to remove MeCN and lyophilized to yield 5’-Amino-C10-dT (160 mg, 445 pmol, 69%) as a colorless solid.

'H NMR (400 MHz, DMSO-d6 ) 5 ppm 1.32 - 1.54 (m, 6 H) 2.03 - 2.10 (m, 1 H) 2.12 - 2.24 (m, 2 H) 2.31 - 2.39 (m, 2 H) 2.64 - 2.70 (m, 1 H) 2.74 (s, 2 H) 3.64 - 3.75 (m, 1 H) 4.14 -

4.22 (m, 1 H) 5.14 - 5.23 (m, 1 H) 6.03 - 6.14 (m, 1 H) 8.10 (s, 1 H).

LC-MS (YMC Triart C18, Buffer A: H₂O + 0.1% FA, Buffer B: MeCN + 0.1% FA, gradient elution: 100% Buffer A for 1 min, then 0% to 100% Buffer B in Buffer A over 10 min, flow rate 0.3 mL/min, 30 °C): RT = 8.02 min, calc, for C19H26N3OC: 360.2, found: 360.2. 3. Nucleoside monophosphoramidates a. dGMP-2c 3FP phosphoramidate

To a solution of 5’-NH2-dG (70 mg, 189 pmol, 1.0 eq) in dry pyridine (16 mL) was added triethylamine (2.20 mL, 16.0 mmol, 85 eq) and a solution of TRIPP reagent (50 mg/mL in dry DCM, 1.9 mL, 284 pmol, 1.5 eq) was added dropwise. The reaction mixture was stirred at room temperature for 16 hours and analyzed by LC-MS indicating quantitative conversion of the starting material. The solution was concentrated in vacuo to dryness, dried under high vacuum overnight and used without further purification. LC-MS (YMC Triart C8, Buffer A: H₂O + 0.1% FA, Buffer B: MeCN + 0.1% FA, gradient elution: 100% Buffer A for 1 min, then 0% to 100% Buffer B in Buffer A over 10 min, flow rate 0.3 mL/min, 30 °C): RT = 10.6 min, calc, for C^I I MF^NSCLP : 660.2, found: 660.4 b. C6-dCMP-2c 3FP phosphoramidate

To a solution of 5’-NH2-dC (49.2 mg, 149 pmol, 1.0 eq) in dry pyridine (9.3 mL) was added triethylamine (1.76 mL, 12.7 mmol, 85 eq) and a solution of TRIPP reagent (50 mg/mL in dry DCM, 1.5 mL, 223 pmol, 1.5 eq) was added dropwise. The reaction mixture was stirred at room temperature for 16 hours and analyzed by LC-MS indicating quantitative conversion of the starting material. The solution was concentrated in vacuo to dryness, dried under high vacuum overnight and used without further purification.

LC-MS (YMC Triart C18, Buffer A: H₂O + 0.1% FA, Buffer B: MeCN + 0.1% FA, gradient elution: 100% Buffer A for 1 min, then 0% to 100% Buffer B in Buffer A over 10 min, flow rate 0.3 mL/min, 30 °C): RT = 9.96 min, calc, for C29H33F3N4OeP⁺: 621.2, found: 621.3 c. C6pC6-dCMP-2c 3FP phosphoramidate

To a solution of 5’-NH2-dC (15.0 mg, 45.4 pmol, 1.0 eq) in dry pyridine (9.1 mL) was added triethylamine (1.27 mL, 9.08 mmol, 200 eq) and a solution of C6pC6-TRIPP reagent (25 mg/mL in dry DCM, 1.5 mL, 68.1 pmol, 1.5 eq) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour and analyzed by LC-MS indicating quantitative conversion of the starting material. The solution was concentrated in vacuo to dryness, dried under high vacuum overnight and used without further purification. LC-MS (YMC Triart C18, Buffer A: H₂O + 0.1% FA, Buffer B: MeCN + 0.1% FA, gradient elution: 100% Buffer A for 1 min, then 0% to 100% Buffer B in Buffer A over 10 min, flow rate 0.3 mL/min, 30 °C): RT = 10.3 min, calc, for CssFL^FsNsOioP? : 854.3, found: 854.4. d. dAMP-2c 3FP phosphoramidate

To a solution of 5’-NH2-dA (106 mg, 300 pmol, 1.0 eq) in dry pyridine (25 mL) was added triethylamine (3.55 mL, 25.5 mmol, 85 eq) and a solution of TRIPP reagent (50 mg/mL in dry DCM, 2.9 mL, 450 pmol, 1.5 eq) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour and analyzed by LC-MS indicating quantitative conversion of the starting material. The solution was concentrated in vacuo to dryness, dried under high vacuum overnight and used without further purification.

LC-MS (YMC Triart C18, Buffer A: H₂O + 0.1% FA, Buffer B: MeCN + 0.1% FA, gradient elution: 100% Buffer A for 1 min, then 0% to 100% Buffer B in Buffer A over 10 min, flow rate 0.3 mL/min, 30 °C): RT = 10.2 min, calc, for C31H34F3N5O5P : 644.2, found: 644.3. e. C8-dTMP-2c 3FP phosphoramidate

To a solution of 5’-NH2-C8-dT (100 mg, 302 pmol, 1.0 eq) in dry pyridine (17 mL) was added triethylamine (3.57 mL, 25.6 mmol, 85 eq) and a solution of TRIPP reagent (50 mg/mL in dry DCM, 3.0 mL, 453 pmol, 1.5 eq) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour and analyzed by LC-MS indicating quantitative conversion of the starting material. The solution was concentrated in vacuo to dryness, dried under high vacuum overnight and used without further purification.

LC-MS (YMC Triart C18, Buffer A: H₂O + 0.1% FA, Buffer B: MeCN + 0.1% FA, gradient elution: 100% Buffer A for 1 min, then 0% to 100% Buffer B in Buffer A over 10 min, flow rate 0.3 mL/min, 30 °C): RT = 9.62 min, calc, for C29H32F3N3O?P⁺: 622.2, found: 622.3. f. C10-dTMP-2c 3FP phosphoramidate

To a solution of 5’-NH2-C10-dT (50 mg, 139 pmol, 1.0 eq) in dry pyridine (8.7 mL) was added triethylamine (1.65 mL, 11.8 mmol, 85 eq) and a solution of TRIPP reagent (50 mg/mL in dry DCM, 1.4 mL, 209 pmol, 1.5 eq) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour and analyzed by LC-MS indicating quantitative conversion of the starting material. The solution was concentrated in vacuo to dryness, dried under high vacuum overnight and used without further purification.

LC-MS (YMC Triart C18, Buffer A: H₂O + 0.1% FA, Buffer B: MeCN + 0.1% FA, gradient elution: 100% Buffer A for 1 min, then 0% to 100% Buffer B in Buffer A over 10 min, flow rate 0.3 mL/min, 30 °C): RT = 10.6 min, calc, for CsiHseFsNsOyP : 650.2, found: 650.3.

4. Nucleoside triphosphates Tris(tetrabutylammonium) hydrogen pyrophosphate (PPi) was dried under high vacuum overnight before use. The crude dGMP-2c 3FP phosphoramidate (46 mg, 70 pmol, 1.0 eq) was dissolved in dry MeCN (5 mL) and dry pyridine (0.11 mL, 1.40 mmol, 20 eq) was added. The solution was cooled to 0 °C and a solution of PPi (280 mg, 310 pmol, 4.5 eq) in dry MeCN (5 mL) was added quickly and stirred at 0 °C. The reaction progress was monitored by LC-MS, indicating full consumption of phosphoramidate within 20-40 minutes. The reaction mixture was diluted with TEAA buffer (100 mM, pH 7, 11 mL) and concentrated in vacuo (30 °C, 50 mbar) to remove MeCN. The resulting suspension was filtered (syringe filter, 0.45 pm). The filter was washed with additional TEAA buffer (100 mM, pH 7, 2 mL) and subjected to preparative HPLC (isocratic 100 mM TEAA in 46% MeOH for 90 minutes, flow rate 63 mL/min). Collected fractions of leading isomer were pooled, diluted with H2O (1 : 1) and desalted via Oasis HLB 20cc 1g column. After eluting with H2O/MeCN (1 : 1, 15 mL), the eluate was lyophilized to yield dGTP-2c (24.3 pmol, 35%) which was dissolved in 20 mM Tris-(hydroxymethyl)-aminomethane-hydrochloride (TrisCi) buffer, analyzed by HPLC (min. 98.0 area% purity) and stored at -80 °C.

Analytical HPLC (Atlantis T3 150 x 4.6 mm, 3 pm; Buffer A: l0 mM TEAA in H2O, Buffer B: 10 mM TEAA in 95% MeOH, gradient elution: 45% buffer B for 1 min, 45% to 60% buffer B over 27 min, flow rate 0.7 mL/min): RT = 16.2 min.

Xmax 294 nm

HRMS ((-)-ESI): calculated for C2₅H33N₅Oi2P3’: 688.1344, found: 688.1359 b. C6-dCTP-2c

Tris(tetrabutylammonium) hydrogen pyrophosphate (PPi) was dried under high vacuum overnight before use. The crude C6-dCMP-2c 3FP phosphoramidate (31.0 mg, 50.0 pmol, 1.0 eq) was dissolved in dry MeCN (3.5 mL) and dry pyridine (80.9 pL, 1.00 mmol, 20 eq) was added. The solution was cooled to 0 °C and a solution of PPi (203 mg, 225 pmol, 4.5 eq) in dry MeCN (3.5 mL) was added quickly and stirred at 0 °C. The reaction progress was monitored by LC-MS, indicating full consumption of phosphoramidate within 20-40 minutes. The reaction mixture was diluted with TEAA buffer (100 mM, pH 7, 15 mL) and concentrated in vacuo (30 °C, 50 mbar) to remove MeCN. The resulting suspension was filtered (syringe filter, 0.45 pm). The filter was washed with additional TEAA buffer (100 mM, pH 7, 2 mL), split in two equal portions and subjected to preparative HPLC (isocratic 100 mM TEAA in 39% MeOH for 110 minutes, flow rate 75 mL/min) separately. Collected fractions of leading isomer were pooled from both HPLC runs, diluted with H2O (1 : 1) and desalted via Oasis HLB 20cc 1g column. After eluting with H2O/MeCN (1 : 1, 15 mL), the eluate was lyophilized to yield C6-dCTP-2c (7.89 pmol, 32%) which was dissolved in 20 mM TrisCi buffer, analyzed by HPLC (min. 98.0 area% purity) and stored at -80 °C.

Analytical HPLC (Atlantis T3 150 x 4.6 mm, 3 pm; Buffer A: l0 mM TEAA in H2O, Buffer B: 10 mM TEAA in 95% MeOH, gradient elution: 35% buffer B for 1 min, 35% to 58% buffer B over 30 min, flow rate 0.7 mL/min): RT = 19.1 min.

Xmax = 299 nm

HRMS ((-)-ESI): calculated for C23H32N₄Oi2P3’: 649.1235, found: 649.1238 c. C6pC6-dCTP-2c

The crude C6pC6-dCMP-2c 3FP phosphoramidate was treated with diethylamine (20% solution in MeCN) for 2 hours. The reaction was monitored by LC-MS and after complete deprotection, the reaction mixture was concentrated in vacuo, dried under high vacuum overnight and used without further purification.

Tris(tetrabutylammonium) hydrogen pyrophosphate (PPi) was dried under high vacuum overnight before use. The crude C6pC6-dCMP-2c 3FP phosphoramidate (12.0 mg, 15.0 pmol, 1.0 eq) was dissolved in dry MeCN (1.1 mL) and dry pyridine (24.3 pL, 300 pmol, 20 eq) was added. The solution was cooled to 0 °C and a solution of PPi (67.7 mg, 75 pmol, 5.0 eq) in dry MeCN (1.1 mL) was added quickly and stirred at 0 °C. The reaction progress was monitored by LC-MS, indicating full consumption of phosphoramidate within 20-40 minutes. The reaction mixture was diluted with TEAA buffer (100 mM, pH 7, 11 mL) and concentrated in vacuo (30 °C, 50 mbar) to remove MeCN. The resulting suspension was filtered (syringe filter, 0.45 pm). The filter was washed with additional TEAA buffer (100 m , pH 7, 2 mL) and subjected to preparative HPLC (buffer A: 100 mM TEAA in 40% MeOH, buffer B: 100 mM TEAA in 47% MeOH, gradient elution: 100% Buffer A for 3 min, then 0% to 100% Buffer B in Buffer A over 67 min, flow rate 63 mL/min). Collected fractions of leading isomer were pooled, diluted with H2O (1 : 1) and desalted via Oasis HLB 20cc 1g column. After eluting with H2O/MeCN (1 :1, 15 mL), the eluate was lyophilized to yield C6pC6-dCTP-2c (0.96 pmol, 6.4%) which was dissolved in 20 mM TrisCi buffer, analyzed by HPLC (min. 98.0 area% purity) and stored at -80 °C.

Analytical HPLC (Atlantis T3 150 x 4.6 mm, 3 pm; Buffer A: l0 mM TEAA in H2O, Buffer B: 10 mM TEAA in 95% MeOH, gradient elution: 35% buffer B for 1 min, 35% to 58% buffer B over 30 min, flow rate 0.7 mL/min): RT = 21.9 min. kmax = 299 nm

HRMS ((+)-ESI): calculated for C29H₄6LiN₄Oi6P4⁺: 837.2014, found: 837.1443 d. dATP-2c

Tris(tetrabutylammonium) hydrogen pyrophosphate (PPi) was dried under high vacuum overnight before use. The crude dAMP-2c 3FP phosphoramidate (90.0 mg, 140.0 pmol, 1.0 eq) was dissolved in dry MeCN (9.0 mL) and dry pyridine (80.9 pL, 1.00 mmol, 20 eq) was added. The solution was cooled to 0 °C and a solution of PPi (380 mg, 420 pmol, 4.5 eq) in dry MeCN (9.0 mL) was added quickly and stirred at 0 °C. The reaction progress was monitored by LC-MS, indicating full consumption of phosphoramidate within 20-40 minutes. The reaction mixture was diluted with TEAA buffer (100 mM, pH 7, 10 mL) and concentrated in vacuo (30 °C, 50 mbar) to remove MeCN. The resulting suspension was filtered (syringe filter, 0.45 pm). The filter was washed with additional TEAA buffer (100 mM, pH 7, 2 mL), split in two portions and subjected to preparative HPLC (isocratic 100 mM TEAA in 47% MeOH for 100 minutes, flow rate 75 mL/min) separately. Collected fractions of leading isomer were pooled from both HPLC runs, diluted with H2O (1 :1) and desalted via Oasis HLB 20cc 1g column. After eluting with H2O/MeCN (1 : 1, 15 mL), the eluate was lyophilized to yield dATP-2c (32.2 pmol, 23%) which was dissolved in 20 mM TrisCi buffer, analyzed by HPLC (min. 98.0 area% purity) and stored at -80 °C.

Analytical HPLC (Atlantis T3 150 x 4.6 mm, 3 pm; Buffer A: l0 mM TEAA in H2O, Buffer B: 10 mM TEAA in 95% MeOH, gradient elution: 45% buffer B for 1 min, 45% to 65% buffer B over 27 min, flow rate 0.7 mL/min): RT = 18.2 min.

Xmax 281 nm

HRMS ((-)-ESI): calculated for C25H33N₅OnP3’: 672.1395, found: 672.1409 e. C8-dTTP-2c

Tris(tetrabutylammonium) hydrogen pyrophosphate (PPi) was dried under high vacuum overnight before use. The crude C8-dTMP-2c 3FP phosphoramidate (24.0 mg, 39.0 pmol, 1.0 eq) was dissolved in dry MeCN (4.5 mL) and dry pyridine (63.0 pL, 0.78 mmol, 20 eq) was added. The solution was cooled to 0 °C and a solution of PPi (110 mg, 0.12 mmol, 4.5 eq) in dry MeCN (4.5 mL) was added quickly and stirred at 0 °C. The reaction progress was monitored by LC-MS, indicating full consumption of phosphoramidate within 20-40 minutes. The reaction mixture was diluted with TEAA buffer (100 mM, pH 7, 11 mL) and concentrated in vacuo (30 °C, 50 mbar) to remove MeCN. The resulting suspension was filtered (syringe filter, 0.45 pm). The filter was washed with additional TEAA buffer (100 mM, pH 7, 2 mL) and subjected to preparative HPLC (isocratic 100 mM TEAA in 39% MeOH for 110 minutes, flow rate 75 mL/mins). Collected fractions of leading isomer were pooled, diluted with H2O (1 : 1) and desalted via Oasis HLB 20cc 1g column. After eluting with H2O/MeCN (1 :1, 15 mL), the eluate was lyophilized to yield C8- dTTP-2c (6.92 pmol, 18%) which was dissolved in 20 mM TrisCi buffer, analyzed by HPLC (min. 98.0 area% purity) and stored at -80 °C. Analytical HPLC (Atlantis T3 150 x 4.6 mm, 3 pm; Buffer A: l0 mM TEAA in H2O, Buffer B: 10 mM TEAA in 95% MeOH, gradient elution: 35% buffer B for 1 min, 35% to 55% buffer B over 30 min, flow rate 0.7 mL/min): RT = 21.8 min.

Xmax ^— 292 nm

HRMS ((+)-ESI): calculated for C₂3H32LiN3Oi₃P3⁺: 658.1303, found: 658.0750 f. C10-dTTP-2c

Tris(tetrabutylammonium) hydrogen pyrophosphate (PPi) was dried under high vacuum overnight before use. The crude C10-dTMP-2c 3FP phosphoramidate (22.7 mg, 35.0 pmol, 1.0 eq) was dissolved in dry MeCN (2.5 mL) and dry pyridine (56.6 pL, 700 pmol, 20 eq) was added. The solution was cooled to 0 °C and a solution of PPi (142 mg, 157 pmol, 4.5 eq) in dry MeCN (2.5 mL) was added quickly and stirred at 0 °C. The reaction progress was monitored by LC-MS, indicating full consumption of phosphoramidate within 20-40 minutes. The reaction mixture was diluted with TEAA buffer (100 mM, pH 7, 11 mL) and concentrated in vacuo (30 °C, 50 mbar) to remove MeCN. The resulting suspension was filtered (syringe filter, 0.45 pm). The filter was washed with additional TEAA buffer (100 mM, pH 7, 2 mL), split in two equal portions and subjected to preparative HPLC (buffer A: 100 mM TEAA in 47% MeOH, buffer B: 100 mM TEAA in 50% MeOH, gradient elution: 100% Buffer A for 3 min, then 0% to 100% Buffer B in Buffer A over 100 min, flow rate 75 mL/min) separately. Collected fractions of leading isomer were pooled from both HPLC runs, diluted with H2O (1 :1) and desalted via Oasis HLB 20cc 1g column. After eluting with H2O/MeCN (1 : 1, 15 mL), the eluate was lyophilized to yield C10- dTTP-2c (3.65 pmol, 10%) which was dissolved in 20 mM TrisCi buffer, analyzed by HPLC (min. 98.0 area% purity) and stored at -80 °C.

Analytical HPLC (Atlantis T3 150 x 4.6 mm, 3 pm; Buffer A: l0 mM TEAA in H2O, Buffer B: 10 mM TEAA in 95% MeOH, gradient elution: 50% buffer B for 1 min, 50% to 65% buffer B over 27 min, flow rate 0.7 mL/min): RT = 15.3 min. Xmax ^— 292 nm HRMS ((-)-ESI): calculated for C2₅H3₅N3Oi3P3’: 678.1388, found: 678.1364

Example 5: Phosphorylation reagents with LG² other than 3FP

1. Phenolate as LG2:

Phosphorylation reagent synthesis:

In a Schlenk flask under argon, phenol (659 mg, 7.00 mmol, 1.00 eq.) and then POCh (1.15 mL, 12.6 mmol, 1.80 eq.) was dissolved in 21 mL anhydrous MeTHF at 4°C. Anhydrous pyridine (979 pL, 12.1 mmol, 1.73 eq.) was added using syringe pump (flow: 3.2 mL/h) at 0 °C and was stirred additional 1.5 h. A sample was analyzed by LC-MS (quench 1 pL of reaction mixture in 300 pL MeOH).

At 0 °C anhydrous pyridine (1.08 mL, 13.3 mmol, 1.90 eq.) and hex-5-yn-l-ol (1.3 mL, 11.9 mmol, 1.70 eq.) was added in one portion. The reaction was stirred for 100 min. Then hex-5- yn-l-ol (150 pL, 1.40 mmol, 0.20 eq) was added and stirred for an additional 30 min. The reaction mixture was filtered using a syringe filter (5 pm, 25 mm) and concentrated in vacuo. The crude mixture was purified by column chromatography. The racemic product was obtained as colorless oil (yield: 760.6 mg, 2.79 mmol, 39.9%).

³¹P NMR (162 MHz, ACETONITRILE-d3) 5 ppm -1.08 (s, 1 P).For LC-MS analysis the phosphorylation reagent was reacted with MeOH. LC-MS (YMC Triart Cl 8, Buffer A: H2O + 0.1% formic acid, Buffer B: MeCN + 0.1% formic acid, gradient elution: 100% Buffer A for 0.5 min, then 0% to 100% Buffer B in Buffer A over 6 min, flow rate 0.45 mL/min, 50 °C): RT = 5.79 min, calc, for Ci3Hi₈O₄P+: 269.1, found: 269.1.

Triphosphate synthesis:

In a Schlenk flask under argon, amino-dG (3.7 mg, 10 pmol, 1.00 eq.) was suspended in anhydrous MeCN (1.1 mL) and TEA (2.1 pL, 15 pmol, 1.50 eq.) was added. A solution of the phosphorylation reagent in anhydrous MeTHF (99 pL, c = 50 mg/mL, 20 pmol, 2.00 eq.) was added and stirred at rt. The reaction was analyzed by LC-MS. After 2 h, the clear solution was cooled to 4 °C and a solution of PPi (110 mg, 120 pmol, 12.00 eq.) in anhydrous MeCN (880 pL) was added quickly. After 24 h, no conversion was observed at 4 °C. The reaction was then stirred at rt for 23-138 h, which led to the consumption of the intermediate, but only a small amount of desired dGTP-2c was observed (0.70%).

2. 2,6-Difluorophenolate as LG2

Phosphorylation reagent synthesis:

In a Schlenk flask under argon, POCI3 (1.40 mL, 15.4 mmol, 2.00 eq.) was added to anhydrous DCM (30 mL) and cooled to 4°C. In a separate Schlenk-flask 2,6-difluorophenol (1.00 g, 7.69 mmol, 1.00 eq.) was placed under argon and dissolved in anhydrous DCM (30 mL). TEA (1.23 mL, 8.84 mmol, 1.15 eq) was added and the solution of 2,6-difluorophenol and TEA was added dropwise to the POC13 solution at 4 °C over 30 min using a syringe pump. The reaction mixture was stirred for 15 min at 4 °C. A sample was analyzed by LC-MS (quench 1 pL of reaction mixture in 100 pL MeOH).

TEA (2.14 mL, 15.4 mmol, 2.00 eq) and hex-5-yn-l-ol (1.7 mL, 15.4 mmol, 2.00 eq.) were added at 0 °C and the reaction was stirred at 4 °C for 2 h. Because the reaction was not completed Hexynol (0.42 mL, 3.84 mmol, 0.5 eq) and TEA (536 pL, 3.84 mmol, 0.50 eq.) was added and stirred overnight at rt. A sample was analyzed by LC-MS (quench 1 pL of reaction mixture in 100 pL MeOH).

The reaction was concentrated in vacuo. The resulting suspension was filtered. The filter was washed two times with DCM/hexane 1 : 1 (2 x 3 mL). The crude product was purified via flash chromatography. The racemic product was obtained as colorless oil (yield: 675.9 mg, 2.19 mmol, 28.5%). The enantiomers were separated using chiral chromatography (Chiralpak IB-N5 2*25 cm, n-hexane: acetone 97:3, Flow 30 mL/min). The results from chiral chromatography is shown in Fig. 9A.

For LC-MS analysis the phosphorylation reagent was reacted with MeOH. LC-MS (YMC Triart C18, Buffer A: H2O + 0.1% formic acid, Buffer B: MeCN + 0. 1% formic acid, gradient elution: 100% Buffer A for 0.5 min, then 0% to 100% Buffer B in Buffer A over 6 min, flow rate 0.45 mL/min, 50 °C): RT = 5.75 min, calc, for C13H16F2O4P+: 305.1, found: 305.1.

Triphosphate synthesis: In a Schlenk flask under argon, amino-dG (3.7 mg, 10 pmol, 1.00 eq.) was suspended in anhydrous MeCN (1.1 mL) and TEA (2.1 pL, 15 pmol, 1.50 eq.) was added. A solution of the racemic phosphorylation reagent in anhydrous MeTHF (93 pL, c = 50 mg/mL, 15 pmol, 1.50 eq.) was added and stirred at rt. The reaction was analyzed by LC-MS. After 45 minutes, the clear solution was cooled to 4 °C and a solution of PPi (54 mg, 60 pmol, 6.00 eq.) in anhydrous MeCN (440 pL) was added quickly. The reaction was stirred at 4 °C for 38 h (full conversion of the intermediate was observed). Then the reaction was quenched with 100 mM TEAA pH 7 solution (1 mL), concentrated in vacuo (30 °C, 50 mbar) to remove excess of MeCN. The resulting suspension was filtered (10 mm syringe filter, 0.25 pm) in a vial. The crude product was analyzed by HPLC. Total area% of both diastereomers (Peak 7 and 8): 83.0%. The results are shown in Fig. 9B.

3. 4-Nitrophenolate as LG2

Phosphorylation reagent synthesis:

In a Schlenk flask under argon, 4-nitrophenol (1.00 g, 7.19 mmol, 1.00 eq.) and then POCI3 (1.18 mL, 12.9 mmol, 1.80 eq.) was dissolved in 21 mL anhydrous MeTHF at 4°C. Anhydrous pyridine (1.01 mL, 12.4 mmol, 1.73 eq.) was added using syringe pump (flow: 3.2 mL/h) at 4 °C and was stirred additional 2 h. A sample was analyzed by LC-MS (quench 1 pL of reaction mixture in 300 pL MeOH).

At 4 °C anhydrous pyridine (1.10 mL, 13.7 mmol, 1.90 eq.) and hex-5-yn-l-ol (1.3 mL, 12.2 mmol, 1.70 eq.) was added in one portion. The reaction was stirred overnight. The reaction mixture was filtered using a Schlenk frit and concentrated in vacuo. The crude mixture was purified by column chromatography. The racemic product was obtained as colorless oil (791 mg, 2.49 mmol, 34.6%).

The enantiomers were separated using chiral chromatography (Chiralpak IB-N5 2*25 cm, n-hexane: acetone 97:3, Flow 30 mL/min). The results from chiral chromatography is shown in Fig. 10 A. For LC-MS analysis the phosphorylation reagent was reacted with MeOH. LC-MS (YMC Triart C18, Buffer A: H2O + 0.1% formic acid, Buffer B: MeCN + 0.1% formic acid, gradient elution: 100% Buffer A for 0.5 min, then 0% to 100% Buffer B in Buffer A over 6 min, flow rate 0.45 mL/min, 50 °C): RT = 5.75 min, calc, for Ci3Hi₇NO₆P+: 314.1, found: 314.1.

Triphosphate synthesis:

In a Schlenk flask under argon, amino-dG (3.7 mg, 10 pmol, 1.00 eq.) was suspended in anhydrous MeCN (1.1 mL) and TEA (2.1 pL, 15 pmol, 1.50 eq.) was added. A solution of the racemic phosphorylation reagent in anhydrous MeTHF (48 pL, c = 100 mg/mL, 15 pmol, 1.50 eq.) was added and stirred at rt. The reaction was analyzed by LC-MS. After 4h, the clear solution was cooled to 4 °C and a solution of PPi (54 mg, 60 pmol, 6.00 eq.) in anhydrous MeCN (440 pL) was added quickly. The reaction was stirred at 0 °C for 24 h (full conversion of the intermediate was observed). Then the reaction was quenched with 100 mM TEAA pH 7 solution (1 mL), concentrated in vacuo (30 °C, 50 mbar) to remove excess of MeCN. The resulting suspension was filtered (10 mm syringe filter, 0.25 pm) in a vial. The crude product was analyzed by HPLC. Total area% of both diastereomers (Peak 5 and 6): 95.4%. The results are shown in Fig. 10B.

4. 2-methyl-4-nitrophenolate as LG2

Phosphorylation reagent synthesis:

In a schlenk flask under argon, 2-methyl-4-nitrophenol (1.07 g, 7.00 mmol, 1.00 eq.) and then POCI3 (1.15 mL, 12.6 mmol, 1.80 eq.) was dissolved in 21 mL anhydrous MeTHF at 4°C. Anhydrous pyridine (979 pL, 12.1 mmol, 1.73 eq.) was added using syringe pump (flow: 3.2 mL/h) at 4 °C and was stirred additional 30 min. A sample was analyzed by LC-MS (quench 1 pL of reaction mixture in 300 pL MeOH).

At 4 °C anhydrous pyridine (1.08 mL, 13.3 mmol, 1.90 eq.) and hex-5-yn-l-ol (1.3 mL, 11.9 mmol, 1.70 eq.) was added in one portion. The reaction was stirred for 1.5 h at 0 °C. The reaction mixture was filtered using a Schlenk frit and concentrated in vacuo. The crude mixture was purified by column chromatography. The racemic product was obtained as colorless oil (1.04 g, 3.14 mmol, 44.8%).

For LC-MS analysis the phosphorylation reagent was reacted with MeOH. LC-MS (YMC Triart C18, Buffer A: H2O + 0.1% formic acid, Buffer B: MeCN + 0.1% formic acid, gradient elution: 100% Buffer A for 0.5 min, then 0% to 100% Buffer B in Buffer A over 6 min, flow rate 0.45 mL/min, 50 °C): RT = 6.06 min, calc, for Ci4Hi₉NO₆P+: 328.1, found: 328.1.

In a Schlenk flask under argon, amino-dG (3.7 mg, 10 pmol, 1.00 eq.) was suspended in anhydrous MeCN (1.1 mL) and TEA (2.1 pL, 15 pmol, 1.50 eq.) was added. A solution of the racemic phosphorylation reagent in anhydrous MeTHF (100 pL, c = 50 mg/mL, 15 pmol, 1.50 eq.) was added and stirred at rt. The reaction was analyzed by LC-MS. After 45 minutes, the clear solution was cooled to 4 °C and a solution of PPi (54 mg, 60 pmol, 6.00 eq.) in anhydrous MeCN (440 pL) was added quickly. The reaction was stirred at 0 °C for 1 h (full conversion of the intermediate was observed). Total area% of both diastereomers (Peak 3 and 4): 89.6%. The results are shown in Fig. 11.

5. 3,5-bis(methoxycarbonyl)phenolate as LG2

Phosphorylation reagent synthesis:

In a Schlenk flask under argon, Dimethyl 5-hydroxyisophthalate (1.50 g, 7. 14 mmol, 1.00 eq.) and then POCI3 (1.17 mL, 12.8 mmol, 1.80 eq.) was dissolved in 21 mL anhydrous MeTHF at 4°C. Anhydrous pyridine (999 pL, 12.3 mmol, 1.73 eq.) was added using syringe pump (flow: 3.2 mL/h) at 4 °C and was stirred additional 2 h. A sample was analyzed by LC-MS (quench 1 pL of reaction mixture in 300 pL MeOH).

At 0 °C anhydrous pyridine (1.15 mL, 14.3 mmol, 2.00 eq.) and hex-5-yn-l-ol (1.4 mL, 12.8 mmol, 1.80 eq.) was added in one portion. The reaction was stirred for 2 h at 4 °C. The reaction mixture was filtered using a Schlenk frit and concentrated in vacuo. The crude mixture was purified by column chromatography. The racemic product was obtained as colorless oil (1.24 g, 3.19 mmol, 44.7%).

The enantiomers were separated using chiral chromatography (Chiralpak IB-N5 2*25 cm, n-hexane: acetone 97:3, Flow 30 mL/min). ³¹P NMR (162 MHz, ACETONITRILE-d3) 5 ppm - 1.26 (s, 1 P). The results from chiral chromatography is shown in Fig. 12A.

For LC-MS analysis the phosphorylation reagent was reacted with MeOH. LC-MS (YMC Triart C18, Buffer A: H2O + 0.1% formic acid, Buffer B: MeCN + 0.1% formic acid, gradient elution: 100% Buffer A for 0.5 min, then 0% to 100% Buffer B in Buffer A over 6 min, flow rate 0.45 mL/min, 50 °C): RT = 5.77 min, calc, for C17H22O8P+: 385.1, found: 385.1.

Triphosphate synthesis:

In a Schlenk flask under argon, amino-dG (3.7 mg, 10 pmol, 1.00 eq.) was suspended in anhydrous MeCN (1.1 mL) and TEA (2.1 pL, 15 pmol, 1.50 eq.) was added. A solution of the racemic phosphorylation reagent in anhydrous MeTHF (39 pL, c = 150 mg/mL, 15 pmol, 1.50 eq.) was added and stirred at rt. The reaction was analyzed by LC-MS. After 90 min, the clear solution was cooled to 4 °C and a solution of PPi (54 mg, 60 pmol, 6.00 eq.) in anhydrous MeCN (440 pL) was added quickly. After 80 h additional solution of PPi (54 mg, 60 pmol, 6.00 eq.) in anhydrous MeCN (440 pL) was added. The reaction was stirred at 4 °C for additional 70 h (full conversion of the intermediate was observed). Then the reaction was quenched with 100 mM TEAA pH 7 solution (1 mL), concentrated in vacuo (30 °C, 50 mbar) to remove excess of MeCN. The resulting suspension was filtered (10 mm syringe filter, 0.25 pm) in a vial. The crude product was analyzed by HPLC. Total area% of both diastereomers (Peak 6 and 7): 72.2%. The results are shown in Fig. 12B. 6. 2,6-Dimethylphenolate as LG2

Phosphorylation reagent synthesis:

In a Schlenk flask under argon, 2,6-dimethylphenol (855 mg, 7.00 mmol, 1.00 eq.) and then POCh (1.15 mL, 12.6 mmol, 1.80 eq.) was dissolved in 21 mL anhydrous MeTHF at 4°C. Anhydrous pyridine (979 pL, 12.1 mmol, 1.73 eq.) was added using syringe pump (flow: 3.2 mL/h) at 4 °C and was stirred additional 45 min. A sample was analyzed by LC-MS (quench 1 pL of reaction mixture in 300 pL MeOH).

At 0 °C anhydrous pyridine (1.08 mL, 13.3 mmol, 1.90 eq.) and hex-5-yn-l-ol (1.3 mL, 11.9 mmol, 1.70 eq.) was added in one portion. The reaction was stirred for 2 h at 4 °C. The reaction mixture was filtered using a Schlenk frit and concentrated in vacuo. The crude mixture was purified by column chromatography. The racemic product was obtained as colorless oil (569 mg, 1.89 mmol, 27.0%).

For LC-MS analysis the phosphorylation reagent was reacted with MeOH. LC-MS (YMC Triart C18, Buffer A: H2O + 0.1% formic acid, Buffer B: MeCN + 0.1% formic acid, gradient elution: 100% Buffer A for 0.5 min, then 0% to 100% Buffer B in Buffer A over 6 min, flow rate 0.45 mL/min, 50 °C): RT = 6.19 min, calc, for C15H22O4P+: 279.1, found: 279.1.

Triphosphate synthesis:

In a Schlenk flask under argon, amino-dG (3.7 mg, 10 pmol, 1.00 eq.) was suspended in anhydrous MeCN (1.1 mL) and TEA (2.1 pL, 15 pmol, 1.50 eq.) was added. A solution of the racemic phosphorylation reagent in anhydrous MeTHF (90 pL, c = 50 mg/mL, 15 pmol, 1.50 eq.) was added and stirred at rt. The reaction was analyzed by LC-MS. After 45 min, the clear solution was cooled to 4 °C and a solution of PPi (54 mg, 60 pmol, 6.00 eq.) in anhydrous MeCN (440 pL) was added quickly. After 6 d at 4 °C the reaction mixture was analyzed by analytical HPLC (quench 10 pL of reaction mixture in 40 pL 100 mM TEAA buffer). Total area% of both diastereomers (Peak 8 and 9): 2.04%. The results are shown in Fig. 13 7. 2-Ethylphenolate as LG2

Phosphorylation reagent synthesis:

In a Schlenk flask under argon, 2-ethylphenol (855 mg, 7.00 mmol, 1.00 eq.) and then POCI3 (1.15 mL, 12.6 mmol, 1.80 eq.) was dissolved in 21 mL anhydrous MeTHF at 4°C. Anhydrous pyridine (979 pL, 12.1 mmol, 1.73 eq.) was added using syringe pump (flow: 3.2 mL/h) at 4 °C and was stirred additional 45 min. A sample was analyzed by LC-MS (quench 1 pL of reaction mixture in 300 pL MeOH).

At 0 °C anhydrous pyridine (1.08 mL, 13.3 mmol, 1.90 eq.) and hex-5-yn-l-ol (1.3 rnL, 11.9 mmol, 1.70 eq.) was added in one portion. The reaction was stirred for 2 h at 4 °C. The reaction mixture was filtered using a Schlenk frit and concentrated in vacuo. The crude mixture was purified by column chromatography. The racemic product was obtained as colorless oil (757 mg, 2.52 mmol, 36.0%).

For LC-MS analysis the phosphorylation reagent was reacted with MeOH. LC-MS (YMC Triart C18, Buffer A: H2O + 0.1% formic acid, Buffer B: MeCN + 0.1% formic acid, gradient elution: 100% Buffer A for 0.5 min, then 0% to 100% Buffer B in Buffer A over 6 min, flow rate

0.45 mL/min, 50 °C): RT = 6.19 min, calc, for C15H22O4P+: 279.1, found: 279.1.

Triphosphate synthesis:

In a Schlenk flask under argon, amino-dG (3.7 mg, 10 pmol, 1.00 eq.) was suspended in anhydrous MeCN (1.1 mL) and TEA (2.1 pL, 15 pmol, 1.50 eq.) was added. A solution of the racemic phosphorylation reagent in anhydrous MeTHF (90 pL, c = 50 mg/mL, 15 pmol, 1.50 eq.) was added and stirred at rt. The reaction was analyzed by LC-MS. After 45 min, the clear solution was cooled to 4 °C and a solution of PPi (54 mg, 60 pmol, 6.00 eq.) in anhydrous MeCN (440 pL) was added quickly. After 6 d at 4 °C the reaction mixture was analyzed by analytical HPLC (quench 10 pL of reaction mixture in 40 pL 100 mM TEAA buffer). Total area% of both diastereomers (Peak 8 and 10): 10.65%. The results are shown in Fig. 14. 8. 2-Isopropylphenolate as LG2

Phosphorylation reagent synthesis:

In a Schlenk flask under argon, 2-isopropylphenol (953 mg, 7.00 mmol, 1.00 eq.) and then POCI3 (1.15 mL, 12.6 mmol, 1.80 eq.) was dissolved in 21 mL anhydrous MeTHF at 4°C. Anhydrous pyridine (979 pL, 12.1 mmol, 1.73 eq.) was added using syringe pump (flow: 3.2 mL/h) at 4 °C and was stirred additional 45 min. A sample was analyzed by LC-MS (quench 1 pL of reaction mixture in 300 pL MeOH).

At 0 °C anhydrous pyridine (1.08 mL, 13.3 mmol, 1.90 eq.) and hex-5-yn-l-ol (1.3 mL, 11.9 mmol, 1.70 eq.) was added in one portion. The reaction was stirred for 2 h at 4 °C. The reaction mixture was filtered using a Schlenk frit and concentrated in vacuo. The crude mixture was purified by column chromatography. The racemic product was obtained as colorless oil (757 mg, 2.52 mmol, 36.0%).

For LC-MS analysis the phosphorylation reagent was reacted with MeOH. LC-MS (YMC Triart C18, Buffer A: H2O + 0.1% formic acid, Buffer B: MeCN + 0.1% formic acid, gradient elution: 100% Buffer A for 0.5 min, then 0% to 100% Buffer B in Buffer A over 6 min, flow rate

0.45 mL/min, 50 °C): RT = 6.35 min, calc, for C16H24O4P+: 311.1, found: 311.2.

Triphosphate synthesis:

In a Schlenk flask under argon, amino-dG (3.7 mg, 10 pmol, 1.00 eq.) was suspended in anhydrous MeCN (1.1 mL) and TEA (2.1 pL, 15 pmol, 1.50 eq.) was added. A solution of the racemic phosphorylation reagent in anhydrous MeTHF (90 pL, c = 50 mg/mL, 15 pmol, 1.50 eq.) was added and stirred at rt. The reaction was analyzed by LC-MS. After 45 min, the clear solution was cooled to 4 °C and a solution of PPi (54 mg, 60 pmol, 6.00 eq.) in anhydrous MeCN (440 pL) was added quickly. After 6 d at 4 °C the reaction mixture was analyzed by analytical HPLC (quench 10 pL of reaction mixture in 40 pL 100 mM TEAA buffer). Total area% of both diastereomers (Peak 7 and 8): 9.00%. The results are shown in Fig. 15. 9. Summary

The following Table 3 and 4 show results obtained with different phosphorylation reagents. The phenols used for synthesizing the phosphorylation reagents cover a broad pKa area (5.4 to -10.5), steric influence (see bigger substituents e.g. -(COOMe)?, methyl, ethyl or isopropyl) and several electron withdrawing substituents attached to the phenol. Hereby, a preferred pKa area could be determined. The derivatives of phenol were converted to the phosphorylation reagent with POCI3 and hex-5-yn-l-ol. Chiral separation of suitable phosphorylation reagents was tested where applicable. For the sake of simplicity, only racemic phosphorylation reagents were tested in these examples, except for 2,4,6-trifluorphenolate. These racemic phosphorylation reagents were reacted with NH2-dG-c to give the nucleoside monophosphoramidate and subsequently the nucleoside triphosphate (dGTP-2c in this case). As shown in Table 3 and Table 4, a higher pKa value (e.g. phenol, pKa = 10.0) leads to more stable phosphorylation reagents which can be converted to the nucleoside monophosphoramidate. However, a conversion of the nucleoside monophosphoramidate to the corresponding triphosphate is extremely slow at 0 °C due to bad LG² leaving group properties. When the reaction is performed at room temperature (rt), decomposition of the triphosphate is predominantly observed (see Table 3; unsubstituted phenol). Phosphorylation reagents containing the conjugate bases of 2,6-difluorophenol, 2,4,6- trifluorophenol, 4-nitrophenol or dimethyl 5-hydroxyisophthalate are stable during storage, their enantiomers can be separated by chiral chromatography, converted to the nucleoside monophosphoramidate and the desired nucleoside triphosphate (dGTP-2c in this case). Phosphoramidate containing 2-methyl-4-nitrophenol reacts drastically faster than the 4- nitrophenol. The reaction is completed within 1 h. The phosphorylation reagent of 2,4,6- trifluorophenol shows optimal properties (e.g. short reaction times at 0 °C). LG² derived from dimethyl 5-hydroxyisophthalate exhibited longer reaction times. Extremely electron poor phenols such as 2,3,5,6-tetrafluorphenol and 2,3,4,5,6-pentafluorphenol with a low pKa value lead to phosphorylation reagents, which are more reactive and decompose rather quickly, for example during normal phase chromatography. Therefore, separation of the enantiomers by chiral chromatography was not tested. Furthermore, the resulting monophosphoramidates are less stable. Main product of the reaction with pyrophosphate is the nucleoside monophosphoramidate with the loss of the phenolate. In conclusion, most preferred phosphorylation reagents have a phenolate with electron- withdrawing substituents, ideally with a pKa of 6 to 9, as LG², but synthesis of the nucleoside monophosphoramidate and the nucleoside triphosphate also works with LG² having a lower or higher pKa than that (more than 3.5 to 10.5). Table 3

Furthermore, the influence of sterically demanding groups at the ortho-position were analyzed. The results are summarized in Table 4. Therefore, phenols substituted with one or two alkane groups such as methyl, ethyl or isopropyl were used to synthesize the corresponding phosphorylation reagents. All four tested phenols can first be converted to the desired phosphorylation reagent and second subsequently in the corresponding phosphoramidate. The conversion of monophosphoramidates to the respective nucleoside triphosphate without an electronwithdrawing group in the phenolate in LG² is very slow at 4 °C. However, after 6 days at 4 °C up to 10.7 area% of nucleoside triphosphate could be detected. Remarkably, a more sterically demanding group than a proton at the ortho position of the 4-nitrophenol, for example a methyl group (see 2-methyl-4-nitrophenolate), leads to a better phenol leaving group.

Table 4

A further derivatization of the meta- and/or para-positions of phenols with different functional groups (e.g. carboxylic esters) allows to increase the separation efficiency during chiral chromatography in order to separate the enantiomers of the racemic phosphorylation reagents. Example 6: dCTP-2c synthesis

C6-dCTP-2c

To a solution of 5’-NH2-dC (32.9 mg, 100 pmol, 1.00 eq.) in anhydrous MeCN (6.5 mL) and /V-butyl-2- pyrrolidone (1.4 mL) was added TEA (18.1 pL, 130 pmol. 1.30 eq.) and a solution of enantiopure TRIPP reagent (50 mg/mL in dry MeTHF, 0.85 mL, 130 pmol. 1.30 eq.) was added dropwise. The reaction mixture was stirred at room temperature for 10 minutes. LC-MS indicated quantitative conversion of the starting material.

The clear solution was cooled to 0 °C, a solution of Tris(tetrabutylammonium) hydrogen pyrophosphate (PPi, 609 mg, 675 pmol, 6.75 eq.) in anhydrous MeCN (6.5 mL) was added quickly and further stirred at 0 °C. The reaction progress was monitored by LC-MS, indicating full consumption of phosphoramidate within 20-40 minutes. The reaction mixture was diluted with TEAA buffer (100 mM, pH 7, 8 mL) and concentrated in vacuo (30 °C, 50 mbar) to remove MeCN. The resulting suspension was filtered (syringe filter, 0.45 pm) and the filter was washed with additional TEAA buffer (100 mM, pH 7, 2 mL) and subjected to preparative HPLC (isocratic elution, 100 mM TEAA in 20% MeCN, flow rate 75 mL/min). Collected fractions were pooled, diluted with H2O (1 : 1) and desalted via anion exchange chromatography. After eluting with H2O/MeCN (1: 1, 15 mL), the eluates were lyophilized to yield C6-dCTP-2c (64.5 pmol, 65%) which were dissolved in 20 mM TrisCi buffer, analyzed by HPLC and stored at -80 °C.

Analytical HPLC (Atlantis T3 150 x 4.6 mm, 3 pm; Buffer A: 100 mM TEAA in H2O, Buffer B: 100 mM TEAA in 95% MeOH, gradient elution: 35% buffer B for 1 min, 35% to 58% buffer B over 30 min, flow rate 0.7 mL/min): RT = 21.2 min.

Xmax = 299 nm

MS ((-)-ESI): calculated for C23H33N4O12P3 : 649.1235, found: 649.1320

Example 7: Azido TRIPP

Azido-TRIPP

In a Schlenk flask under argon, 2,4,6-trifluorphenol (7.50 g, 50.6 mmol, 1.00 eq.) was dissolved in 153 mL MeTHF (dry). At 0 °C, POCI3 (23.1 mL, 253 mmol, 5.00 eq.) was added in one portion. Subsequently, anhydrous pyridine (dry, 8.19 mL, 101 mmol, 2.00 eq.) was added over a period of 20 min using a syringe pump (flow: 0.410 mL/min). The reaction mixture was additionally stirred for 1.5 h. All volatiles were removed and the mixture was coevaporated with ACN/toluene (2*5 mL:25 mL) and MeTHF (1*25 mL) to removed the excess of POCI3. After drying in vacuo the reaction mixture was suspended in 150 mL MeTHF (dry) and cooled to 0°C. Then pyridine (dry, 3.64 mL, 45.0 mmol, 1.50 eq) in one portion and 6-azidohexan- l-ol (4.51 g, 31.5 mmol, 1.05 eq.) over a period of 5 min were added. The reaction was stirred at 0 °C for 2.5 h (LC-MS after 2 h indicated full conversion). The reaction mixture was allowed to warm up to rt, filtered under Schlenk conditions and concentrated in vacuo. The crude product was purified by column chromatography (silica, n-hexane:EtOAc 100:0 to 80:20).

’H NMR (400 MHz, ACETONITRILE-d3) 5 ppm 1.34 - 1.49 (m, 4 H); 1.54 - 1.64 (m, 2 H); 1.74 - 1.84 (m, 2 H); 3.29 (t, J=6.84 Hz, 2 H); 4.39 (dt, J=8.72, 6.31 Hz, 2 H); 6.99 - 7.08 (m, 2 H).

¹⁹F NMR (376 MHz, ACETONITRILE-d3) 5 ppm -124.05 to -123.96 (m, 1 F); -111.09 (dtt, J=8.94, 6.56, 6.56, 3.88, 3.88 Hz, 1 F).

³¹P NMR (162 MHz, ACETONITRILE-d3) 5 ppm 0.82 (m, 1 P).

Example 8: Side chains with protecting groups

Base-protected b(TES)-5’-NH2-dG b(TES)-5’-Azido-dG (50.0 mg, 98.1 pmol, 1.0 eq) was dissolved in pyridine (0.25 mL) and H2O (0.25 mL). NaHCOs (28.2 mg, 335 pmol, 3.4 eq) and DTT (30.3 mg, 196 pmol, 2.0 eq) were added in one portion and stirred at rt for 3.5 hours. LC-MS indicated quantitative conversion. The solvent was removed in vacuo and the residue was dissolved in DCM (2 mL) and directly subjected to column chromatography (PuriFlash, NP12g, DCM/MeOH: 0CV - 0%, 2CV - 0%, 20CV - 100% MeOH). The collected fractions were concentrated in vacuo to yield b(TES)-5’-Amino-dG (38 mg, 79 pmol, 80%) as a colorless solid. Xmax = 294 nm

LC-MS (YMC Triart C18, Buffer A: H₂O + 0.1% FA, Buffer B: MeCN + 0.1% FA, gradient elution: 100% Buffer A for 0.5 min, then 0% to 100% Buffer B in Buffer A over 6 min, flow rate 0.45 mL/min, 50 °C): RT = 5.88 min, calc, for C25H38N5O3SC: 484.3, found: 484.3

Base-protected b(TES) dGTP-2c

To a suspension of b(TES)-5’-NH2-dG (38.0 mg, 78.6 pmol, 1.00 eq.) in anhydrous MeCN (8.6 mL) was added TEA (14.2 pL, 102 pmol. 1.30 eq.) and a solution of enantiopure TRIPP reagent (50 mg/mL in dry MeTHF, 0.67 mL, 102 pmol, 1.30 eq.) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour. LC-MS indicated quantitative conversion of the starting material.

The clear solution was cooled to 0 °C, a solution of Tris(tetrabutylammonium) hydrogen pyrophosphate (PPi, 479 mg, 530 pmol, 6.75 eq.) in anhydrous MeCN (2.6 mL) was added quickly and further stirred at 0 °C. The reaction progress was monitored by LC-MS, indicating full consumption of phosphoramidate within 20-40 minutes. The reaction mixture was diluted with TEAA buffer (100 mM, pH 7, 15 mL) and concentrated in vacuo (30 °C, 50 mbar) to remove MeCN. The resulting suspension was filtered (syringe filter, 0.45 pm) and the filter was washed with additional TEAA buffer (100 mM, pH 7, 2 mL) and subjected to preparative HPLC (isocratic elution, 100 mM TEAA in 48% MeCN, flow rate 63 mL/min). Collected fractions were pooled, diluted with H2O (1:3) and desalted via Oasis HLB 20cc 6g column. After eluting with H2O/MeCN (1: 1, 15 mL), the eluate was lyophilized to yield b(TES)-dGTP-2c (26.4 pmol, 34%) which was dissolved in 20 mM TrisCi buffer, analyzed by HPLC and stored at -80 °C. Analytical HPLC (Atlantis T3 150 x 4.6 mm, 3 pm; Buffer A: 100 mM TEAA in H2O, Buffer B: 100 mM TEAA in 90% MeCN, gradient elution: 45% buffer B for 1 min, 45% to 54% buffer B over 18 min, 54% to 90% buffer B over 3 min, 90% to 100% buffer B over 24 min, flow rate 0.7 mL/min): RT = 26.5 min. Xmax = 294 nm

MS ((-)-ESI): calculated for C₃iH4₇N₅Oi₂P₃Si : 802.22, found: 802.6

6-(TES)-hex-5-yn-l-ol

1 . n-BuLi

2. CISiEt₃

In a flame dried Schlenk flask under argon, 5-hexyn-l-ol (4.4 mL, 3.93 g, 40.0 mmol) was dissolved in anhydrous MeTHF. At -78 °C n-BuLi (1.6 M, in hexane, 52.5 mL, 84.0 mmol, 2.1 eq.) was added dropwise. The reaction was stirred for 60 min at -78 °C. Then chlorotriethylsilane (12.1 g, 13.4 mL, 80.0 mmol, 2.00 eq.) was added at the same temperature and stirred for 2 h. Then HC1 solution (aq., 6 M, 50 mL) was added and vigorously stirred for 10 min. The aq. layer was extracted with EtOAc (3*100 mL), the combined organic layers were dried over Na2SC>4 and all volatiles were removed under reduced pressure. The crude product was purified via column chromatography (silica, n-hexane: EtOAc (8:2)). The product was obtained as a colorless oil (2.72 g, 12.8 mmol, 32%).

’H NMR (400 MHz, ACETONITRILE-d3) 5 ppm 0.57 (q, J=7.91 Hz, 6 H, -Si-CH₂-CH₃); 0.98 (t, J=7.91 Hz, 9 H, -Si-CH₂-CH₃); 1.48 - 1.63 (m, 4 H, alkyl); 2.21 - 2.28 (m, 2 H, alkyl); 2.53 (t, J=5.33 Hz, 1 H, - OH); 3.50 (q, J=6.11 Hz, 2 H, HO-CH2-).

¹³C NMR (101 MHz, ACETONITRILE-d3) 5 ppm 5.26 (s, 3 C, -Si-C H₂-C H0: 7.87 (s, 3 C, -Si-CH?-CH₃); 20.13 (s, 1 C, alkvne-CH2-); 26.13 (s, 1 C, alkyl); 32.66 (s, 1 C, alkyl); 62.07 (s, 1 C, HO-CH2-); 82.23 (s, 1 C, alkyne); 109.94 (s, 1 C, alkyne).

TES-TRIPP

In a Schlenk flask under argon, 2,4,6-trifluorphenol (2.00 g, 13.5 mmol, 1.00 eq.) was dissolved in 41 mL MeTHF (dry). At 0 °C, POC1₃ (6.16 mL, 67.5 mmol, 5.00 eq.) was added in one portion. Subsequently, anhydrous pyridine (2.18 mL, 27.0 mmol, 2.00 eq.) was added over a period of 20 min using a syringe pump (flow: 6.5 mL/h). The reaction mixture was additionally stirred for 130 min. Then 6- (triethylsilyl)hex-5-yn-l-ol (1.89 g, 8.91 mmol, 0.66 eq.) and subsequently anhydrous pyridine (1.64 mL, 20.3 mmol, 1.50 eq.) was added and stirred at 0 °C for additional 2.5 h. The reaction mixture was fdtered through fritted glass under argon. The filter cake was extracted with anhydrous MeTHF (5*10 mL). The combined organic layers were evaporated under reduced pressure. The crude product was purified via column chromatography (n-hexane:EtOAc 100:0 to 85: 15). The product was obtained as a colorless oil (3.07 g, 6.96 mmol, 52%).

’H NMR (400 MHz, ACETONITRILE-d3) 5 ppm 0.56 (q, J=7.70 Hz, 6 H, -Si-CHg-Me); 0.97 (t, J=7.91 Hz, 9 H, Si-CH2-CHs); 1.56 - 1.65 (m, 2 H, alkyl); 1.85 - 1.93 (m, 2 H, alkyl); 2.29 (t, J=6.96 Hz, 2 H, alkyl); 4.42 (dt, J=8.78, 6.34 Hz, 2 H, -O-CHg-alkyl); 6.99 - 7.08 (m, 2 H, ar).

¹⁹F NMR (376 MHz, ACETONITRILE-d3) 5 ppm -124.09 - -123.98 (m, 2 F); -111.05 (tq, J=8.79, 4.07 Hz, I F).

³¹P NMR (162 MHz, ACETONITRILE-d3) 5 ppm 0.83 (q, J=3.01 Hz, 1 P).

Phosphate-protected p(TES) dGTP-2c

To a suspension of 5’-NHg-dG (20.0 mg, 54.1 pmol, 1.00 eq.) in anhydrous MeCN (3.9 mL) was added TEA (9.81 pL, 70.4 pmol, 1.30 eq.) and a solution of racemic TES-TRIPP reagent (50 mg/mL in dry MeTHF, 0.62 mL, 70.4 pmol, 1.30 eq.) was added dropwise. The reaction mixture was stirred at room temperature for 1 hour. LC-MS indicated quantitative conversion of the starting material.

The clear solution was cooled to 0 °C, a solution of Tris(tetrabutylammonium) hydrogen pyrophosphate (PPi, 330 mg, 365 pmol, 6.75 eq.) in anhydrous MeCN (3.9 mL) was added quickly and further stirred at 0 °C. The reaction progress was monitored by LC-MS, indicating full consumption of phosphoramidate within 20-40 minutes. The reaction mixture was diluted with TEAA buffer (100 mM, pH 7, 8 mL) and concentrated in vacuo (30 °C, 50 mbar) to remove MeCN. The resulting suspension was filtered (syringe filter, 0.45 pm) and the filter was washed with additional TEAA buffer (100 mM, pH 7, 2 mL) and subjected to preparative HPLC (gradient elution, Buffer A: 100 mM TEAA in 35% MeCN, Buffer B: 100 mM TEAA in 50% MeCN, 0% to 100% buffer B in 40 minutes, flow rate 63 mL/min). Collected fractions of leading and lagging diastereomers were pooled separately, diluted with H2O (1 : 1) and desalted via Oasis HLB 20cc 1g column. After eluting with H2O/MeCN (1: 1, 15 mL), the eluates were lyophilized to yield two diastereomers of p(TES)-dGTP-2c (leading diastereomer: 12.6 pmol, 23%, lagging diastereomer: 15.6 pmol, 29%) which were dissolved in 20 mM TrisCi buffer, analyzed by HPLC and stored at -80 °C.

Analytical HPLC (Atlantis T3 150 x 4.6 mm, 3 pm; Buffer A: 100 mM TEAA in H2O, Buffer B: 100 mM TEAA in 90% MeCN, gradient elution: 45% buffer B for 1 min, 45% to 54% buffer B over 18 min, 54% to 90% buffer B over 3 min, 90% to 100% buffer B over 24 min, flow rate 0.7 mL/min): RT (leading diastereomer) = 26.0 min, RT (lagging diastereomer) = 26.2 min.

Xmax = 294 nm

MS ((-)-ESI): calculated for C’SI HTYN OOPSSO: 802.22, found: 802.7

Example 9: dNTP-2c with 5’ organophosphate

5’-O-dGMP-2c TFP ester

In a Schlenk tube under argon, 5'-OH-dG was dissolved in 4.15 mL anhydrous MeTHF and anhydrous 1- methylimidazole (NMI, 161 pL, 2.02 mmol, 7.50 eq.) was added. The reaction was cooled to 0 °C. After 10 min racemic TRIPP (50 mg/mL in anhydrous MeTHF) was added dropwise. The reaction was stirred overnight and was allowed to warm up to room temperature. Reaction progress was monitored via LC-MS: 5 pL reaction mixture was quenched in 50 pL MeOH. All volatiles were removed under reduced pressure and the crude product was purified by column chromatography (silica, DCM:MeOH 100:0 to 90: 10). The product was obtained as pale yellow film (16.6 mg, 25.1 pmol, 9.3%). NMR analysis shows only one formed diastereomer.

¹⁹F NMR (376 MHz, ACETONITRILE-d3) 5 ppm -125.16 to -124.93 (m, 1 F); -112.88 to -112.66 (m, 2 F).

³¹P NMR (162 MHz, ACETONITRILE-d3) 5 ppm -5.46 (m, 1 P).

LC-MS (YMC Triart C18, Buffer A: H2O + 0.1% FA, Buffer B: MeCN + 0.1% FA, gradient elution: 100% Buffer A for 0.5 min, then 0% to 100% Buffer B in Buffer A over 6 min, flow rate 0.45 mL/min, 50 °C): RT = 5.89 min, calc, for C31H33F3N4O7P : 661.2, found: 661.3.

5’-O-dGTP-2c

In a Schlenk tube under argon, TFP-5’-O-dGMP (16.6 mg, 25.1 pmol, 1.00 eq.) is dissolved in anhydrous MeCN (1.8 mL) and cooled to 0 °C. A solution of Tris(tetrabutylammonium) hydrogen pyrophosphate (PPi, 136 mg, 151 pmol, 6.75 eq.) in anhydrous MeCN (1.8 mL) is added quickly and further stirred at 0 °C. The reaction progress is monitored by LC-MS. The reaction mixture is diluted with TEAA buffer (100 mM, pH 7, 4 mL) and concentrated in vacuo (30 °C, 50 mbar) to remove MeCN. The resulting suspension is filtered (syringe filter, 0.45 pm) and the filter is washed with additional TEAA buffer (100 mM, pH 7, 1 mL) and subjected to preparative HPLC (gradient elution, Buffer A: 100 mM TEAA in water, Buffer B: 100 mM TEAA in MeCN). Collected fractions are diluted with H2O (1: 1) and desalted via Oasis HLB 20cc 1g column. After eluting with H2O/MeCN (1: 1, 15 mL), the eluates are lyophilized to yield 5 ’ -O-dGTP-2c which is dissolved in 20 mM TrisCi buffer, analyzed by HPLC and stored at -80 °C.

Claims

PATENT CLAIMS

1. A chiral compound having the following structure: wherein LG¹ is a conjugate base of a strong acid (H-LG¹); R⁴ comprises or consists of a hydrocarbon; G², when present, represents a terminal conjugable group, preferably a clickable group; and LG² is a substituted or unsubstituted cyclic or heterocyclic compound that is linked to the phosphorus via an exocyclic or an endocyclic group or/and wherein LG² is a conjugate base of a weak acid (H-LG²).

2. A nucleoside monophosphoramidate having the following structure: wherein NB is a nucleobase; R¹, when present, comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 -ribose modification; R³ is H or any protecting group; R⁴ comprises or consists of a hydrocarbon; G¹ and G², when present, independently represent terminal conjugable groups, preferably clickable groups; and LG² is a conjugate base of a weak acid or a substituted or unsubstituted cyclic or heterocyclic compound that is linked to the phosphorus via an exocyclic or an endocyclic group.

3. The chiral compound of claim 1, wherein the conjugate acid of LG² has a Ka that is at least

10 times lower than the Ka of the conjugate acid of LG¹.

4. The chiral compound of claim 1 or 3, wherein LG¹ is a halide, preferably chloride, and optionally the conjugate acid of LG² has a pKa of 5 to 10.5, a pKa of 5 to 10, a pKa of 6 to 9, or a pKa of 7 to 8.

5. The chiral compound of claim 1, 3 or 4 or the nucleoside monophosphoramidate of claim 2, wherein LG² is a phenolate, an imidazole, a hydroxypyridine, such as 4-hydroxypyridine, a thiophenolate, a naphtholate, such as 2-naphtholate, a benzimidazole, a hydroxy-quinoline or hydroxy-isoquinoline, or an imidazopyridines, optionally with one or more substituents; and wherein LG² is preferably a phenolate with one or more substituents selected from a halide, a nitro, a nitroso group, a sulfonyl group, sulfonamide group, cyano group, a halogenated alkyl group, a carboxyester, and a substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group or cyclic or heterocyclic (aromatic or non-aromatic) group comprising 1-10, such as 1-6 or 1-3, carbon atoms (such as a methyl, ethyl or isopropyl group), which substituted or unsubstituted, branched or unbranched, saturated or unsaturated alkyl group or cyclic or heterocyclic group optionally includes one or more oxygen, nitrogen, phosphorus or sulfur heteroatoms.

6. The chiral compound of any one of claims 1 or 3-5 or the nucleoside monophosphoramidate of claim 2 or 5, wherein LG² is 2,4,6-trifluorophenolate, 2,6- difluorophenolate, 4-nitrophenolate, 2-methyl-4-nitrophenolate, or 3,5- bis(methoxycarbonyl)phenolate, preferably 2,4,6-trifluorophenolate.

7. The chiral compound of any one of claims 1 or 3-6 or the nucleoside monophosphoramidate of any one of claims 2 or 5-6, wherein R¹ and/or R⁴ comprises or consists of a branched, linear, cyclic or heterocyclic, substituted or unsubstituted, saturated or unsaturated hydrocarbon, optionally including one or more heteroatoms, optionally selected from nitrogen, oxygen, phosphorus and sulfur.

8. The chiral compound of any one of claims 1 or 3-7 or the nucleoside monophosphoramidate of any one of claims 2 or 5-7, wherein G¹ and G² independently represent terminal conjugable groups, preferably clickable groups, wherein optionally G¹ and/or G² is an alkyne group or an azide group, preferably an alkyne group.

9. The chiral compound of any one of claims 1 or 3-8, having the following structure:

10. A composition comprising the chiral compound of any one of claims 1 or 3-9, wherein at least 80%, preferably at least 90%, such as at least 95%, at least 99% or 100% of the chiral compound has the structure:

11. A composition comprising the nucleoside monophosphoramidate of any one of claims 2 or 5-8, wherein at least 80%, preferably at least 90%, such as at least 95%, at least 99%, or 100% of the nucleoside monophosphoramidate has the structure:

12. A method for producing a chiral compound of any one of claims 1 or 3-9 or a composition of claim 10, comprising reacting a phosphoryl halide with H-LG² and with HO-R⁴-G², wherein optionally the phosphoryl halide is phosphoryl chloride.

13. A method for producing a nucleoside monophosphoramidate according to any one of claims 2, 5-8 or the composition of claim 11, comprising reacting the compound of any one of 1 or 3-9 or the composition of claim 10 with a 5 ’-amino-5’ -deoxynucleoside, and optionally purifying the product of this reaction.

14. The method of claim 13, that is for diastereoselectively producing a nucleoside monophosphoramidate, comprising the following step:

15. A method for producing a nucleoside triphosphate, comprising reacting the nucleoside monophosphoramidate of any one of claims 2, 5-8 or the composition of claim 11 with a pyrophosphate, preferably with a pyrophosphate salt, wherein the method is optionally for diastereoselectively (with regard to the phosphorus stereocenter), and wherein the method optionally comprises deprotecting and/or purifying the nucleoside triphosphate.

16. The method of claim 15, that is for diastereoselectively producing a nucleoside triphosphate, comprising the following step: wherein R⁶ is H or any organic or inorganic cation; and Z⁺ is H⁺ or any (monovalent or bivalent) cation.

17. The method of claim 15 or 16, comprising:

(a) reacting the compound of any one of claims 1 or 3-9 or a composition of claim 10 with a 5’- amino-5’ -deoxynucleoside, and

(b) reacting the product of step (a) with a pyrophosphate.

18. The method of any one of claims 15-17, further comprising linking the a-phosphoramidate to the nucleobase.

19. The method of claim 18, wherein linking the a-phosphoramidate to the nucleobase comprises the following step: wherein NB is a nucleobase; R¹ comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 -ribose modification; R³ is H or any protecting group; R⁴ comprises or consists of a hydrocarbon; G¹ and G² independently represent terminal conjugable groups, preferably clickable groups; G^la and G^2a independently represent terminal conjugable groups, preferably clickable groups; L¹ and L² independently represent linking groups formed by reacting G¹ with G^la and G² with G^2a, preferably 1,2, 3 -triazoles; and T is a tether molecule; and Z⁺ is H⁺ or any (monovalent or bivalent) cation.

20. A method for producing a nucleoside diphosphate or oligophosphate, such as a tetraphosphate, comprising reacting the nucleoside monophosphoramidate of any one of claims 2 or 5-8 or the composition of claim 11 with a monophosphate or oligophosphate, preferably with a monophosphate or oligophosphate salt, wherein the oligophosphate optionally comprises 4-6 phosphate units, such as 4 phosphate units.

21. A 5 ’-amino-5’ -deoxynucleoside having the following structure: - I l l - wherein NB is a nucleobase; R¹ comprises or consists of a hydrocarbon; R² is independently H, OH or any 2 -ribose modification; R³ is H or any protecting group; G¹ represents terminal conjugable group, preferably clickable group.

22. A method for producing a 5 ’-amino-5’ deoxynucleoside of claim 21, comprising selective reduction of a 5 ’-azido-5’ -deoxynucleoside.