Methods for sequencing and synthesizing l-polynucleotides

Abstract

The present disclosure provides new methods of amplifying, sequencing, or synthesizing L-form polynucleotides. Further provided are L-form nucleotides and D-form enzymes that can be used in the methods. Methods of making the L-form nucleotides and D-form enzymes are also provided. The nucleotides have structure I, II, or III:

Description

[0001] 1. Sequence List

[0002] This application contains a sequence list.

[0003] 2. Background

[0004] Chirality is a geometric property of molecules. A molecule is chiral if it cannot be superimposed on its mirror image by a combination of rotations, translations, and some conformational changes.

[0005] Many natural biomolecules, such as proteins, DNA, and RNA, are chiral. Amino acids, except for glycine, have a chiral carbon atom adjacent to a carboxyl group. This chiral center allows amino acids to exist as stereoisomers (also called enantiomers) that can be characterized as L or D based on optical activity. However, most amino acids found in nature, except for the achiral glycine, are L-type. Nucleic acids have their chiral centers on their backbones. Unlike amino acids, nucleic acids are predominantly D-type in nature. Due to chiral specificity, biomolecules can interact and utilize other molecules or substrates only when they possess a certain degree of chirality. For example, L-type polymerases can synthesize polynucleotides using D-type nucleotides instead of L-type nucleotides.

[0006] Recently, researchers have generated mirror images of several biomolecules, attempting to create mirror-image artificial systems based on D-amino acids, L-nucleic acids, and D-proteins. Furthermore, mirror-image therapeutic proteins or polynucleotides have been developed, for example, exhibiting a more favorable, slower biodegradation in vivo. However, the development of mirror-image biomolecules and systems faces significant obstacles due to the lack of efficient and reliable technologies for sequencing or synthesizing molecules.

[0007] 3. Overview

[0008] This disclosure provides a novel method for sequencing or synthesizing L-polynucleotides. Compositions for the sequencing or synthesis method, such as novel L-nucleotides and D-enzymes, are also provided. This method is expected to enable high-throughput sequencing and synthesis of L-polynucleotides.

[0009] Therefore, in a first aspect, this disclosure provides mirror-image nucleotides comprising:

[0010] a. A pentose sugar selected from (3R,4S)-3,4,5-trihydroxypentanal and (4R)-4,5-dihydroxypentanal; wherein the H of the 5' hydroxyl group is substituted with one or more phosphate groups; and wherein the H of the 3' hydroxyl group (if present) is optionally substituted with a cleavable protecting group;

[0011] b. Nitrogenous bases, and

[0012] c. Optional cleavable marker comprising a cleavable linker and a marker;

[0013] It contains at least one of a cleavable protecting group and a cleavable label.

[0014] In some embodiments, this disclosure provides nucleotides comprising:

[0015] a. Pentose sugars in cyclic form containing (3R,4S)-3,4,5-trihydroxypentanal or (4R)-4,5-dihydroxypentanal;

[0016] In this pentose, the H of the 5-hydroxy group is replaced by one or more phosphate groups; and

[0017] In (3R,4S)-3,4,5-trihydroxypentanal, the H of the 3-hydroxy group is optionally replaced by a cleavable protecting group.

[0018] b. The nitrogenous base attached to the 2-position of the pentose sugar, and

[0019] c. Optional cleavable marker comprising a cleavable linker and a marker;

[0020] It contains at least one of a cleavable protecting group and a cleavable label.

[0021] In some embodiments, a cleavable marker is present. In some embodiments, the cleavable marker is attached to a nitrogenous base, a 3'O, or a 5' phosphate group.

[0022] In some embodiments, the mirror nucleotide has a structure according to Formula I:

[0023] Formula I

[0024] The bases are nitrogen-containing bases, and R' is a cleavable protecting group.

[0025] In some embodiments, the mirror nucleotide has a structure according to Formula II.

[0026] Formula II

[0027] Where the base is a nitrogen-containing base; R' is a cleavable protecting group or H; R2-mark is a cleavable mark containing a cleavable linker R2 and the mark.

[0028] In some embodiments, the mirror nucleotide has a structure according to Formula III:

[0029] Formula III

[0030] The bases are nitrogen-containing bases; the R2-label is a cleavable label containing a cleavable linker R2 and a label.

[0031] In some embodiments of the mirror nucleotides of Formula II or III, the label is selected from the group consisting of:

[0032]

[0033] In some embodiments of the mirror nucleotide of Formula I or II, the cleavable protecting group is selected from allyl, dimethyl disulfide, nitrobenzyl, and azide protecting groups.

[0034] In some embodiments of the mirror nucleotide of Formula I or II, the cleavable protecting group is selected from:

[0035] .

[0036] In some embodiments of the mirror nucleotide of formula II or III, the cleavable linker is photocleavable, cleavable by contact with a water-soluble phosphine, or cleavable by a water-soluble catalyst containing a transition metal.

[0037] In some embodiments of the mirror nucleotide of formula II or III, the cleavable linker comprises an allyl, disulfide, or azide group.

[0038] In certain embodiments of mirror nucleotides of Formula II or III, the cleavable linker comprises:

[0039] ,or

[0040] .

[0041] In some embodiments of the mirror nucleotide of Formula I, the compound has a structure selected from the following:

[0042] ;and .

[0043] In some embodiments, R' comprises a methyl disulfide, allyl, azide, or nitrobenzyl moiety. In some embodiments, R' is selected from:

[0044] .

[0045] In some embodiments of the mirror nucleotide of Formula II, the compound has a structure selected from the following:

[0046]

[0047] and .

[0048] In some embodiments, R' comprises a methyl disulfide, allyl, azide, or nitrobenzyl moiety. In some embodiments, R' is selected from:

[0049] H, ;and

[0050] In some implementations, R2 includes:

[0051] ,or

[0052] .

[0053] In some embodiments of the mirror nucleotide of Formula III, the compound has a structure selected from the following:

[0054]

[0055] and

[0056] .

[0057] In some implementations, R2 includes:

[0058] ,or .

[0059] In some embodiments of the mirror nucleotide of Formula II, the compound has a structure selected from the following:

[0060]

[0061] and

[0062] .

[0063] R' is selected from:

[0064] H, .

[0065] In some embodiments of the mirror nucleotide of Formula III, the compound has a structure selected from the following:

[0066]

[0067]

[0068] and

[0069] .

[0070] In some embodiments, R' comprises a methyl disulfide, allyl, azide, or nitrobenzyl moiety. In some embodiments, R' is selected from:

[0071] H, .

[0072] In a second aspect, this disclosure provides a mirror-image nucleic acid polymerase comprising a sequence having at least 90% sequence identity with SEQ ID NO: 1, wherein the polymerase comprises D-type amino acids.

[0073] In some implementations, the polymerase is composed of D-amino acids.

[0074] In some embodiments, the polymerase comprises a sequence having at least 95% sequence identity with SEQ ID NO: 1. In other embodiments, the polymerase comprises a sequence having at least 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 1.

[0075] In some embodiments, compared to SEQ ID NO: 1, the polymerase includes one or more modifications at one or more amino acid sites selected from E276, K317, N424 and S651.

[0076] In some embodiments, compared to SEQ ID NO: 1, the polymerase comprises one or more substitutions selected from E276A, K317G, N424A and S651A.

[0077] In some embodiments, the polymerase comprises substitutions of E276A, K317G, N424A, and S651A compared to SEQ ID NO: 1.

[0078] In some embodiments, compared to SEQ ID NO: 1, the polymerase includes one or more modifications at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744.

[0079] In some embodiments, compared to SEQ ID NO: 1, the polymerase comprises one or more substitutions from Ile to Ala, Val, Leu, or Tyr at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744.

[0080] In some embodiments, compared to SEQ ID NO: 1, the polymerase comprises one or more substitutions selected from I80V, I127V, I171A, I176V, I191V, I228V, I256V, I264A, I268L, I400V, I597V, I610V, I618A, I630L, I642V, I715Y, I733V, and I744V.

[0081] In some embodiments, the polymerase comprises substitutions of I80V, I127V, I171A, I176V, I191V, I228V, I256V, I264A, I268L, I400V, I597V, I610V, I618A, I630L, I642V, I715Y, I733V, and I744V compared to SEQ ID NO: 1.

[0082] In some embodiments, compared to SEQ ID NO: 1, the polymerase includes one or more modifications at one or more amino acid sites selected from M129, I130, G131, D141, E143, L408, Y409, P410, A485, T514, and I521.

[0083] In some embodiments, compared to SEQ ID NO: 1, the polymerase includes one or more modifications at one or more amino acid sites selected from D141, E143, Y409, and A485.

[0084] In some embodiments, the polymerase comprises one or more substitutions selected from D141A, E143A, Y409V and A485L compared to SEQ ID NO: 1.

[0085] In some embodiments, the polymerase comprises substitutions of D141A, E143A, Y409V, and A485L compared to SEQ ID NO: 1.

[0086] In some embodiments, compared to SEQ ID NO: 1, the polymerase includes one or more modifications at one or more amino acid sites selected from D141, E143, L408, Y409, P410, A485, T514, and I521.

[0087] In some embodiments, compared to SEQ ID NO: 1, the polymerase comprises one or more substitutions selected from D141A, E143A, L408A, Y409A, P410I, A485V, T514S and I521L.

[0088] In some embodiments, the polymerase comprises substitutions of D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L compared to SEQ ID NO: 1.

[0089] In some embodiments, compared to SEQ ID NO: 1, the polymerase comprises one or more substitutions selected from M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, I521L, or a modification of adding D between I130 and G131.

[0090] In some embodiments, compared to SEQ ID NO: 1, the polymerase comprises substitutions of M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L, and the addition of D between I130 and G131.

[0091] In some embodiments, the polymerase comprises a sequence selected from SEQ ID NOs: 2-7.

[0092] In some embodiments, the polymerase consists of sequences selected from SEQ ID NOs: 2-7.

[0093] In a third aspect of this disclosure, a method for replicating L-polynucleotides is provided, the method comprising the steps of: incubating a mixture comprising (i) L-polynucleotide, (ii) L-primer, (iii) L-dNTP, (iv) polymerase (e.g., as described herein) and (v) buffer, thereby inducing replication of L-polymerase.

[0094] In some implementations, the L-polynucleotide is DNA or RNA.

[0095] In some embodiments, the mixture comprises L-dATP, L-dGTP, L-dCTP, and L-dTTP.

[0096] In some implementations, the buffer solution contains 50 mM Tris-HCl, 20 mM MgCl2, 1 mM MTT, and 50 mM KCl at pH 7.5.

[0097] In some implementations, the incubation step includes PCR.

[0098] In a fourth aspect of this disclosure, a method for sequencing L-polynucleotides is provided, the method comprising the following cycle:

[0099] a. Incubate a mixture containing (i) L-polynucleotide, (ii) L-primer, (iii) L-3'-O-R'-dNTP-R2-label, (iv) polymerase (e.g., as described herein) and (v) buffer to obtain the replication product;

[0100] b. Detect the signal from the L-3'-O-R'-dNTP-R2- tag incorporated into the replication product; and

[0101] c. Inducing the cleavage of the R' and R2 groups of the L-3'-O-R'-dNTP-R2-labeled group incorporated into the replication product.

[0102] In some implementations, the cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times.

[0103] In some embodiments, the L-3'-O-R'-dNTP-R2-label comprises L-3'-O-R'-dATP-R2-label, L-3'-O-R'-dTTP-R2-label, L-3'-O-R'-dGTP-R2-label, and L-3'-O-R'-dCTP-R2-label, wherein each label is distinct. In some embodiments, the L-3'-O-R'-dNTP-R2-label has a structure according to Formula II as defined in section 5.2.2.1.2. In some embodiments, the signal is a fluorescent signal.

[0104] In a fifth aspect of this disclosure, a method for sequencing L-polynucleotides is provided, the method comprising the following steps:

[0105] a. Incubate a mixture containing (i) L-polynucleotide, (ii) L-primer, (iii) L-dNTP, (iv) L-ddNTP-R2-label or L-3'-O-R'-dNTP-R2-label, (v) polymerase (e.g., as described herein) and (vi) buffer to obtain the replication product;

[0106] b. Separate the replication products; and

[0107] c. Detect signals from L-ddNTP-R2- tags or L-3'-O-R'-dNTP-R2- tags incorporated into the replication product.

[0108] In some embodiments, L-dNTPs include L-dATP, L-dTTP, L-dGTP, and L-dCTP. In some embodiments, L-ddNTP-R2-labeling includes L-ddATP-R2-labeling, L-ddTTP-R2-labeling, L-ddGTP-R2-labeling, and L-ddCTP-R2-labeling, wherein each labeling is different.

[0109] In some embodiments, the L-ddNTP-R2-label has Formula III as defined in section 5.2.2.2.1. In some embodiments, the L-3'-O-R'-dNTP-R2-label comprises L-3'-O-R'-dATP-R2-label, L-3'-O-R'-dTTP-R2-label, L-3'-O-R'-dGTP-R2-label, and L-3'-O-R'-dCTP-R2-label, wherein each label is distinct. In some embodiments, the L-3'-O-R'-dNTP-R2-label has Formula II as defined in section 5.2.2.1.2. In some of these embodiments, the signal is a fluorescent signal.

[0110] In some of these embodiments, the incubation step includes PCR. In some of these embodiments, the separation step includes separating the replication products by size.

[0111] In a sixth aspect of this disclosure, a method for sequencing L-polynucleotides is provided, the method comprising the following cycle:

[0112] a. Incubate a mixture containing (i) L-polynucleotide, (ii) L-primer, (iii) L-3'-O-R'-dNTP, (iv) L-ddNTPs-R2-labeled or L-dNTPs-R2-labeled, (v) polymerase (e.g., as described herein) and (vi) buffer to obtain the replication product;

[0113] b. Detecting signals from L-ddNTP-R2-labels or L-3'-O-R'-dNTP-R2-labels incorporated into the replication product; and

[0114] c. Inducing the cleavage of the following: i) the R' group of L-3'-O-R'-dNTP and the R2 group of L-ddNTPs-R2-labeled in the replication product; or ii) the R' group of L-3'-O-R'-dNTP, and the R' and R2 groups of L-3'-O-R'-dNTP-R2-labeled in the replication product.

[0115] In some implementations, the cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times.

[0116] In some implementations, L-3'-O-R'-dNTP includes L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, and L-3'-O-R'-dCTP.

[0117] In some implementations, L-3'-O-R'-dNTP has a structure according to Formula I as defined in section 5.2.2.1.1.

[0118] In some implementations, the L-ddNTP-R2-label includes L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label, and L-ddCTP-R2-label, where each label is different.

[0119] In some embodiments, the L-ddNTP-R2-label has a structure according to Formula III as defined in section 5.2.2.2.1. In some embodiments, the L-3'-O-R'-dNTP-R2-label comprises L-3'-O-R'-dATP-R2-label, L-3'-O-R'-dTTP-R2-label, L-3'-O-R'-dGTP-R2-label, and L-3'-O-R'-dCTP-R2-label, wherein each label is distinct. In some embodiments, the L-3'-O-R'-dNTP-R2-label has a structure according to Formula II as defined in section 5.2.2.1.2. In some embodiments, the signal is a fluorescent signal.

[0120] The seventh aspect of this disclosure provides a mirror-terminal deoxynucleotidyl transferase comprising a sequence having at least 90% sequence identity with SEQ ID NO: 36, wherein the polymerase comprises D-type amino acids.

[0121] In some embodiments, the transferase is composed of D-type amino acids. In some embodiments, the transferase comprises a sequence having at least 95% sequence identity with SEQ ID NO: 36. In some embodiments, the transferase comprises a sequence having at least 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO: 36. In some embodiments, the transferase comprises one or more modifications at one or more amino acid sites of SEQ ID NO: 36.

[0122] In a seventh aspect, this disclosure provides a method for synthesizing L-polynucleotides, comprising the steps of incubating a mixture containing (i) an L-primer, (ii) an L-dNTP or L-ddNTP, (iii) a transferase disclosed herein, and (iv) a buffer solution to induce the synthesis of L-polynucleotides.

[0123] In some embodiments, the L-polynucleotide is DNA or RNA. In some embodiments, the mixture comprises L-dATP, L-dGTP, L-dCTP, or L-dTTP. In some embodiments, the L-dNTP comprises (i) L-dATP or L-dTTP and (ii) L-dGTP or L-dCTP. In some embodiments, the L-dNTP comprises L-dATP, L-dGTP, L-dCTP, and L-dTTP.

[0124] In some embodiments, the mixture contains radiolabeled L-ddNTPs. In some embodiments, the L-ddNTPs are radiolabeled L-ddATP, L-ddGTP, L-ddCTP, or L-ddTTP. In some embodiments, the L-ddNTPs contain L-ddNTP-R2-labeling or L-3'-O-R'-dNTP-R2-labeling.

[0125] In some embodiments, the method further includes a step of stopping the reaction by heating or by adding a chelating agent. In some embodiments, the chelating agent is EDTA.

[0126] In an eighth aspect of this disclosure, a kit for replicating or synthesizing L-polynucleotides is provided, the kit comprising (i) the polymerase or transferase described in any of the preceding embodiments and (ii) optional buffer.

[0127] In some embodiments, the kit contains L-dNTPs. In some embodiments, the L-dNTPs include L-dATP, L-dGTP, L-dCTP, L-dTTP, or L-UTP.

[0128] In some embodiments, the kit contains an L-3'-O-R'-dNTP-R2- label. In some embodiments, the kit contains an L-3'-O-R'-dNTP-R2- label, which includes an L-3'-O-R'-dATP-R2- label, an L-3'-O-R'-dTTP-R2- label, an L-3'-O-R'-dGTP-R2- label, an L-3'-O-R'-dCTP-R2- label, or an L-3'-O-R'-dUTP-R2- label. In some embodiments, the kit contains an L-3'-O-R'-dNTP-R2- label having Formula II as defined in section 5.2.2.1.2.

[0129] In some embodiments, the kit contains an L-ddNTP-R2-label. In some embodiments, the kit contains an L-ddNTP-R2-label, which includes an L-ddATP-R2-label, an L-ddTTP-R2-label, an L-ddGTP-R2-label, an L-ddCTP-R2-label, or an L-ddUTP-R2-label. In some embodiments, the L-ddNTP-R2-label has Formula III as defined in section 5.2.2.2.1.

[0130] In some embodiments, the kit comprises L-3'-O-R'-dNTPs. In some embodiments, the L-3'-O-R'-dNTPs comprise L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, and L-3'-O-R'-dCTP. In some embodiments, the L-3'-O-R'-dNTPs have Formula I as defined in section 5.2.2.1.1. 4. Brief description of the attached diagram

[0132] These and other features, aspects, and advantages of the invention will become more readily understood with reference to the following description and accompanying drawings, wherein:

[0133] Figure 1 The amino acid sequence (SEQ ID NO: 8) of the (D)-type mutant 9°N DNA polymerase (candidate #1-1) is provided. All amino acids are D-type amino acids. Rhombus = mutation introduced for NCL; circle = NCL site (start of the "second" strand); square = potential substitution site for isoleucine; triangle = mutation introduced for modifying nucleotide.

[0134] Figure 2A The sequence of the D-type 9°N DNA polymerase mutant 1-I, 9°NN fragment is shown (SEQ ID NO: 9), in which the sequences of nine synthetic peptides are presented in separate lines. Figure 2BThe synthetic pathway for the D-type 9°N DNA polymerase mutant 1-I, 9°NN fragment is shown.

[0135] Figure 3A The sequence of the D-type 9°N DNA polymerase mutant 1-I, 9°NC fragment is shown (SEQ ID NO: 10), in which the sequences of the six synthetic peptides are presented in separate lines. Figure 3B The synthetic pathway for the D-type 9°N DNA polymerase mutant 1-I, 9°NC fragment is shown.

[0136] Figure 4A Tfa-(D)-Thz-OH (i.e., ( S TLC staining of 3-(2,2,2-trifluoroacetyl)thiazolidin-4-carboxylic acid. Figure 4B Tfa-(D)-Thz-OH (i.e., ( S )-3-(2,2,2-trifluoroacetyl)thiazolidin-4-carboxylic acid) 1 H NMR (500 MHz, CDCl3) results.

[0137] Figure 5 The synthesis of 2-Cl-(Trt)-NHNH2 is shown as detailed in Example 13.

[0138] Figure 6A It is the peptide sequence of D-9°NN-6@5-mer (SEQ ID NO: 11). Figure 6B An analytical HPLC chromatogram (λ=214 nm) of crude D-9°NN-6@5-mer is provided. Column: Welch C4. Gradient: 0–20 min 100%–80% A in B, 20–40 min 80%–50% A in B, 40–60 min 50%–20% A in B, and 60–70 min 20%–0% A in B. Wherein A = H₂O (0.1% TFA) and B = CH₃CN (0.1% TFA). Figure 6C Provided pure D-9 oAnalytical HPLC chromatogram of NN-6@5-mer. Gradient: 95%-5% CH3CN (0.1% TFA) in H2O (0.1% TFA) for 30 min. Purification conditions: Column: Welch C4 semi-preparative column. Gradient: 1-20 min in B 100%-80% A, 20-40 min in B 80%-50% A, 40-50 min in B 50%-20% A, and 50-55 min in B 20%-0% A. Wherein A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA).

[0139] Figure 7A It is the peptide sequence of D-9°NN-6@11-mer (SEQ ID NO: 12); Figure 7B An analytical HPLC chromatogram (λ=214 nm) of crude D-9°NN-6@11-mer is provided. Column: Welch C4. Gradient: 95%-5% CH3CN (0.1% TFA) in H2O (0.1% TFA) for 30 min. Figure 7C An analytical HPLC chromatogram of pure D-Pfu-9°oN-N-6@11-mer is provided. Gradient: 95%-5% CH3CN (0.1% TFA) in H2O (0.1% TFA) for 30 min. Purification conditions: Column: Welch C4 semi-preparative column. Gradient: 1-20 min in B 100%-80% A, 20-40 min in B 80%-50% A, 40-50 min in B 50%-20% A, and 50-55 min in B 20%-0% A. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA); Figure 7D MALDI-TOF mass spectra of D-9°oN-N-6@11-mer are provided.

[0140] Figure 8A It is the peptide sequence of D-9°NN-6@24-mer (SEQ ID NO: 13); Figure 8B An analytical HPLC chromatogram (λ=214 nm) of crude D-9°NN-6@24-mer is provided. Column: Welch C4. Gradient: 0-20 min 100%-80% A in B, 20-40 min 80%-50% A in B, 40-60 min 50%-20% A in B, and 60-70 min 20%-0% A in B. Wherein A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA); Figure 8C An analytical HPLC chromatogram of pure D-Pfu-9°NN-6@5-mer is provided. Gradient: 95%-5% CH3CN (0.1% TFA) in H2O (0.1% TFA) for 30 min. Purification conditions: Column: Welch C4 semi-preparative column. Gradient: 1-20 min in B 100%-80% A, 20-40 min in B 80%-50% A, 40-50 min in B 50%-20% A, and 50-55 min in B 20%-0% A. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA).

[0141] Figure 9A It is the peptide sequence of D-9°NC-3@9-mer (SEQ ID NO: 14); Figure 9B An analytical HPLC chromatogram (λ=214 nm) of crude D-9°NC-3@9-mer is provided. Column: Welch C4. Gradient: 0-20 min 100%-80% A in B, 20-40 min 80%-50% A in B, 40-60 min 50%-20% A in B, and 60-70 min 20%-0% A in B. Wherein A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semi-preparative column. Gradient: 1-20 min 100%-80% A in B, 20-40 min 80%-50% A in B, 40-60 min 50%-20% A in B, and 60-70 min 20%-0% A in B. Where A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA); Figure 9C HRMS (ESI) spectra of D-9°NC-3@9-mer are provided: for C 36 H 59 N 13 O 15 The calculated value of S is [M+H]. + 946.4053; measured value 946.4064.

[0142] Figure 10AAn analytical HPLC chromatogram (λ=214 nm) of purified D-9°NC-1@33-mer is provided. Column: Welch C4. Gradient: 0–20 min 75%–35% A in B; 20–23 min 35%–0% A in B. Wherein A = H₂O (0.1% TFA) and B = CH₃CN (0.1% TFA). b) D-9 o ESI-MS spectrum of NC-1@33-mer: observed value 3985.5, calculated value 3985.6. Figure 10B D-9 was provided o ESI-MS spectrum of NC-1@33-mer.

[0143] Figure 11A An analytical HPLC chromatogram (λ=214 nm) of purified D-9°NC-2@39-mer is provided. Column: Welch C4. Gradient: 0–20 min 75%–35% A in B; 20–23 min 35%–0% A in B. Wherein A = H₂O (0.1% TFA) and B = CH₃CN (0.1% TFA). Figure 11B D-9 was provided o ESI-MS spectrum of NC-2@39-mer: observed value 4921.5, calculated value 4921.6.

[0144] Figure 12A The peptide sequence of D-9°NC-3@21-mer is provided (SEQ ID NO: 15); Figure 12B An analytical HPLC chromatogram (λ=214 nm) of crude D-9°NC-3@21-mer is provided. Column: Welch C4. Gradient: 0-30 min 90%-50% A in B, 30-40 min 50%-5% A in B. Wherein A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semi-preparative column. Gradient: 0-30 min 90%-50% A in B, 30-40 min 50%-5% A in B. Wherein A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Figure 12C The ESI-MS spectrum of D-9°NC-3@21-mer is provided. Figure 12D MS spectra of D-9°NC-3@21-mer without TFA protection and observation quality 2196.1 are provided.

[0145] Figure 13AIt is the peptide sequence of D-9°NC-3@Cys35-mer (SEQ ID NO: 16); Figure 13B Provided crude D-9 o Analytical HPLC chromatogram of NC-3@Cys35-mer (λ=214 nm). Column: Welch C4. Gradient: 0-30 min 70%-20% A in B, 30-40 min 20%-5% A in B. Wherein A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semi-preparative column. Gradient: 0-30 min 70%-20% A in B, 30-40 min 20%-5% A in B. Wherein A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Figure 13C ESI-MS spectrum of D-9°NC-3@21-mer is provided; Figure 13D The observation quality of D-9°NC-3@Cys35-mer is 4276.

[0146] Figure 14A It is the peptide sequence of D-9°NC-3@56-mer (SEQ ID NO: 17); Figure 14B Provided crude D-9 o Analytical HPLC chromatogram of NC-3@56-mer (λ=214 nm). Column: Welch C4. Gradient: 0-30 min 80%-30% A in B, 30-40 min 30%-5% A in B. Wherein A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semi-preparative column. Gradient: 0-30 min 80%-30% A in B, 30-40 min 30%-5% A in B. Wherein A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA).

[0147] Figure 15A It is the peptide sequence of D-Pfu-9°NC-3@35-mer (SEQ ID NO: 18); Figure 15BAn analytical HPLC chromatogram (λ=214 nm) of crude D-Pfu-9°NC-3@35-mer is provided. Column: Welch C4. Gradient: 0-30 min 80%-30% A in B, 30-40 min 30%-5% A in B. Wherein A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA). Purification conditions: Column: Polaris C18-A semi-preparative column. Gradient: 0-30 min 80%-30% A in B, 30-40 min 30%-5% A in B. Wherein A = H2O (0.1% TFA) and B = CH3CN (0.1% TFA).

[0148] Figure 16A These are the peptide sequences of D-9°NN-6@5-mer and D-9°NN-6@11-mer (SEQ ID NO: 19 and SEQ ID NO: 20); Figure 16B A synthetic route for preparing D-9°NN-6@16-mer is provided. D-9°NN-6@5-mer (4 mg, 1 equivalent) was dissolved in 0.4 mL of acidified ligation buffer (aqueous solution of 6 M Gn·HCl and 0.1 M NaH2PO4, pH 3.0). The mixture was cooled in an ice-salt bath (-15 °C), and 40 μl of 0.5 M NaNO2 (in acidified ligation buffer) was added. The reaction was maintained in an ice-salt bath with stirring for 15 min, followed by the addition of 0.4 mL of 0.2 M MPAA (in 6 M Gn·HCl and 0.1 M Na2HPO4, pH 5.7). After the addition of D-Pfu-9°NN-6@11-mer (7.5 mg), the pH of the reaction mixture was adjusted to 6.6–6.8 at room temperature using NaOH solution. After 12 h, the reaction mixture was reduced by TCEP and purified by HPLC (purification conditions: 5%–95% CH3CN (0.1% TFA) gradient in H2O (with 0.1% TFA) over 30 min on a Welch C4 column). The ligation product was obtained in 80% (9.0 mg) yield. Figure 16C An analytical HPLC chromatogram (λ = 214 nm) of the peak transition during the NCL reaction is provided. Column: Welch C4. Gradient: 5%-95% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) for 30 min.

[0149] Figure 17A It is D-9°NC-1 and D-9 oPeptide sequences of NC-2 (SEQ ID NO: 21, 22, 23); Figure 17B A synthetic route for preparing D-9°NC-7@72-mer was provided. D-9°NC-1@33-mer (5.4 mg) was dissolved in 0.27 mL of acidified ligation buffer (aqueous solution of 6 M Gn·HCl and 0.1 M NaH2PO4, pH 3.0). The mixture was cooled in an ice-salt bath (-15 °C), and 27 μl of 0.5 M NaNO2 (in acidified ligation buffer) was added. The reaction was maintained in an ice-salt bath with stirring for 20 min, after which 0.14 mL of 0.4 M MPAA (in 8 M Gn·HCl and 0.1 M Na2HPO4, pH 5.7) was added. After the addition of D-9°NC-2@39-mer (5.8 mg), the pH of the reaction mixture was adjusted to 6.6–6.8 at room temperature using NaOH solution. After 16 h, the mixture was purified by HPLC (purification conditions: on a Polaris C18 column, for 30 min, in a gradient of 20%–70% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA)). The ligation product was obtained. Figure 17C This is an analytical HPLC chromatogram of the peak transition during the NCL reaction (λ = 214 nm). Column: Welch C4. Gradient: 20%-70% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) for 30 min. Figure 17D It is an ESI-MS with MPAA attachment, specifically D-9°NC-7@72-mer. Figure 17E The connection products of MPAA-attachment are shown (observation quality: 9042; computation quality: 9042).

[0150] Figure 18A These are the peptide sequences of D-9°NN-1@(His)6+39-mer and D-9°NN-2@62-mer. Figure 18B The synthetic route for preparing D-9°NN-10@(His)6+101-mer is shown. Figure 18C The analytical HPLC chromatogram (λ = 214 nm) shows the peak transition during the NCL reaction. Column: Welch C4. Gradient: 5%-95% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) for 30 min [√ = confirm peak]. Figure 18DThe image shows the deconvolution MS (ESI-MS) spectrum of the purified D-9°NN-10@(His)6+101-mer [calculated value: 12875.8, observed value: 12876.8].

[0151] Figure 19A These are the peptide sequences of D-9°NN-7@52-mer and D-9°NN-8@56-mer. Figure 19B The synthetic route for preparing D-9°NN-13@108-mer is shown. Figure 19C An analytical HPLC chromatogram (λ = 214 nm) of the peak transition during the NCL reaction is provided. Column: Welch C4. Gradient: 5%-95% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) for 30 min [√ = peak confirmation]. Figure 19D It is purified D-9 o Deconvolutional MS (ESI-MS) spectrum of NN-13@108-mer [calculated value: 12467.52, observed value: 12467.39].

[0152] Figure 20 This provides a chemical linkage pathway for the synthesis of terminal deoxynucleotidyl transferases.

[0153] Figure 21A It is the peptide sequence of D-TdT-WT-1@(His)6+46-mer. Figure 21B An analytical HPLC chromatogram (λ=214 nm) of crude D-TdT-WT-1@(His)6+46-mer is provided. Figure 21C The image shows the deconvolution MS (ESI-MS) spectrum of purified D-TdT-WT-1@(His)6+46-mer [calculated value: 5922.97, observed value: 5922.97].

[0154] Figure 22A These are the peptide sequences of D-TdT-WT-1@(His)6+46-mer and D-TdT-WT-2@61-mer. Figure 22B The synthetic route for preparing D-TdT-WT-8@(His)6+107-mer is shown. Figure 22C Analytical HPLC chromatograms of crude D-TdT-WT-1@(His)6+46-mer, D-TdT-WT-2@61-mer, and D-TdT-WT-8@(His)6+107-mer are provided. Figure 22DThe image shows the deconvolution MS (ESI-MS) spectrum of purified D-TdT-WT-1@(His)6+107-mer [calculated value: 12663.21, observed value: 12663.23].

[0155] Figure 23 The synthetic pathway of the (D)-type mutant terminal deoxynucleotidyl transferase provided in Example 18 is shown, including a converging fragment assembly strategy.

[0156] Figure 24A Results of an analytical HPLC chromatogram (λ=214 nm) from crude D-TdT-WT-1@66-mer are provided. Column: Welch C4. Gradients: 1–30 min in ACN (0.1% TFA) 80%–30% water (0.1% TFA), 30–35 min in ACN (0.1% TFA) 30%–0% water (0.1% TFA), and 35–47 min in 0%–80% water (0.1% TFA). Figure 24B The deconvolution MS (ESI-MS) spectrum of the purified D-TdT-WT-1@66-mer is shown. Purification conditions: 1-5 min in 100%-80% water (0.1% TFA) in ACN (0.1% TFA), 5-55 min in 80%-30% water (0.1% TFA) in ACN (0.1% TFA), and 55-60 min in 30%-0% water (0.1% TFA), using a C18 semi-preparative column.

[0157] 5. Detailed Explanation

[0158] 5.1. Definition

[0159] "L-nucleic acid" or "L-nucleotide" should refer to any nucleic acid or nucleotide molecule that has L-chirality, including but not limited to L-DNA, L-RNA, and their hybrids. The nucleic acid bases forming the nucleic acid molecule can be the bases A, C, G, T, and U, and their derivatives. Derivatives of these bases are well known in the art and are illustrated in PCR systems, reagents and consumables (Perkin Elmer Catalogue 1996-1997, Roche Molecular Systems, Inc., Branchburg, New Jersey, USA).

[0160] As used herein, the term "D-polymerase" refers to a protein that is a mirror image of a primitive polymerase having L-chirality. A primitive polymerase can be a polymerase obtained from nature or produced through modification or synthesis. A primitive polymerase can be a DNA or RNA polymerase or a transferase, such as a terminal deoxynucleotide transferase.

[0161] Although bases are commonly referred to as purines or pyrimidines, those skilled in the art will understand that derivatives and analogs can be obtained without altering the ability of a nucleotide or nucleoside to undergo Watson-Crick base pairing. As used herein, the term "base" includes compounds or molecules whose core structure is the same as or very similar to that of a native base but which have chemical or physical modifications, such as allowing the derivative nucleotide or nucleoside to attach to a different or additional side group of another molecule. For example, a base can be a denitropurine.

[0162] "Hybridization" should be understood as the annealing of one single-stranded nucleic acid with another based on sequence complementarity. The tendency for nucleic acids to hybridize depends on the temperature and ionic strength of their environment, as well as the length and degree of complementarity of the nucleic acids. The influence of these parameters on hybridization is well known in the art (see Sambrook J, Fritsch EF, Maniatis T. 1989. Molecular cloning: a laboratory manual. Cold Spring Harbor Laboratory Press, New York.).

[0163] Dehybridization is understood by those skilled in the art to mean separating the hybridizing primer (or its extension) from the target nucleic acid without destroying the target nucleic acid and thus allowing a second primer to further hybridize with the target nucleic acid. In one embodiment, hybridization as used herein means strict hybridization, such as that described in Sambrook, J., Russell, DW, (2000) Molecular Cloning: A Laboratory Manual: Third Edition.

[0164] As used herein, the term "polynucleotide" refers to a linear polymer containing more than one nucleotide monomer. In some embodiments, the polynucleotide contains 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, or more nucleotide monomers. It can be DNA, RNA, or a modification thereof.

[0165] As used in this article, hybridization of primer sequences should mean full annealing so that the primers are extendable by the formation of phosphodiester bonds.

[0166] When a range of values ​​is provided, it should be understood that, unless the context explicitly specifies otherwise, each intermediate value between the upper and lower limits of the range, up to one-tenth of the unit of the lower limit (if appropriate), and any other value or intermediate value within the range, are encompassed within this invention. The upper and lower limits of these smaller ranges may be independently included within these smaller ranges and are also encompassed within this invention, subject to any specifically excluded limit values ​​within the range. Where the range includes one or two limit values, the range excluding one or both of those included limit values ​​is also included in this invention.

[0167] 5.2. Mirror nucleotides used in sequencing or synthesis of L-polynucleotides

[0168] 5.2.1. L-nucleotides and L-nucleosides

[0169] In one aspect, this disclosure provides mirror-image nucleosides or nucleotides. Mirror-image nucleosides or nucleotides can be used for the synthesis or sequencing of L-polynucleotides. L-polynucleotides may include pentose sugars, nitrogenous bases, and phosphate groups.

[0170] Pentoses can be in linear or cyclic form, and it should be understood that the L-nucleosides and L-nucleotides of this disclosure include pentose units, which can be referred to via their linear or cyclic forms. In some embodiments, the pentose is L-ribose or L-deoxyribose. In some embodiments, the pentose is (3R,4S)-3,4,5-trihydroxypentanal and (4R)-4,5-dihydroxypentanal. A nitrogenous base may be attached to the C1 position of the L-ribose or L-deoxyribose, and a phosphate group may be attached to the C5 position.

[0171] In some embodiments, the mirror-image nucleoside or nucleotide comprises at least one of (i) a cleavable protecting group (if present) at the 3' O of the pentose ring and (ii) a cleavable label. In some embodiments, the mirror-image nucleoside or nucleotide comprises neither (i) a cleavable protecting group (if present) at the 3' O of the pentose ring nor (ii) a cleavable label. It should be understood that, when referring to the pentose ring, the 3' position or 5' position of the ring may alternatively be referred to as the 3 position or 5 position.

[0172] Mirror-image nucleosides or nucleotides can be used for sequencing reactions, polynucleotide synthesis, nucleic acid amplification, nucleic acid hybridization assays, single nucleotide polymorphism studies, techniques using enzymes (such as polymerases, reverse transcriptases, terminal transferases), techniques using labeled dNTPs (e.g., nick translation, random primer labeling, terminal labeling (e.g., with terminal deoxynucleotide transferases)), reverse transcription, or nucleic acid amplification.

[0173] In some embodiments of the invention, a chemically modified mirror nucleoside or nucleotide is provided, comprising at least one of (i) a cleavable protecting group (if present) on the 3' O of the pentose sugar and (ii) a cleavable label. In embodiments in which the mirror nucleotide comprises a cleavable label, the label is not particularly limited, provided that the label provides a method for detection and R' comprises H.

[0174] 5.2.1.1 Bases

[0175] In some embodiments, the nitrogenous base is a purine or a pyrimidine. In some embodiments, the nitrogenous base is a denitropurine. In some embodiments, the nitrogenous base is selected from thymine (5-methyl-2,4-dioxapyrimidine), cytosine (2-oxo-4-aminopyrimidine), 5-methyl-cytosine (2-oxo-5-methyl-4-aminopyrimidine), uracil (2,4-dioxapyrimidine), adenine (6-aminopurine), 7-deadenine (7H-pyrrolo[2,3-d]pyrimidin-4-amine; 6-amino-7-deadenine), guanine (2-amino-6-oxypurine), 7-deadenine (2-amino-4-hydroxy-pyrrolo-[2,3-d]pyrimidine; 2-amino-7-deadenine-6-oxypurine), hypoxanthine (1,9-dihydro-6H-purine-6-one), xanthine (3,7-dihydro-1H-purine-2,6-dione), and 5-nitroindole. In some embodiments, the nitrogenous base is thymine, uracil, cytosine, adenine, guanine, 7-deadenine, or 7-deadenine.

[0176] 5.2.1.2 R' - Cleavageable Protecting Group

[0177] In some embodiments, R' is selected from H and a cleavable protecting group. The cleavable protecting group of the present invention is not particularly limited, as long as the resulting nucleotide is a highly efficient substrate for mirror DNA polymerase.

[0178] Technicians will understand how to attach a suitable protecting group to the pentose ring to block the interaction with the 3'-OH group. The protecting group can be attached directly to the 3' position or to the 2' position (the size or charge of the protecting group is sufficient to block the interaction at the 3' position). Alternatively, the protecting group can be attached to both the 3' and 2' positions and can be cleaved to expose the 3'-OH group.

[0179] A suitable protecting group will be obvious to a person skilled in the art and can be formed from any suitable protecting group disclosed in Greene & Wuts, Protective Groups in Organic Synthesis, and John Wiley & Sons. The protecting group should be removable (or modifiable) to produce a 3' OH group. The method for obtaining the 3' OH group can be any suitable chemical or enzymatic reaction.

[0180] In some embodiments, the cleavable protecting group comprises an allyl, nitrobenzyl, 1-methyl-2-alkyl disulfide, or methyl azide moiety.

[0181] In a particular embodiment, the cleavable protecting group is selected from:

[0182]

[0183] The wavy line indicates the attachment point to 3'O.

[0184] 5.2.1.3 Decomposable marker construct (R2-marker)

[0185] 5.2.1.3.1 Marking

[0186] The present invention can use conventional detectable markers, which can be modified to covalently attach to nucleotides via cleavable linkers.

[0187] Detection can be performed by any suitable method, including fluorescence spectroscopy or other optical methods. A particularly contemplated marker is a fluorophore that emits radiation at a defined wavelength after absorbing energy. Many suitable fluorescent markers are known. For example, Welch et al. (Chem. Eur. J. 5(3):951–960, 1999) disclosed dansyl-functionalized fluorescent moieties that can be used in this invention. Zhu et al. (Cytometry 28:206–211, 1997) described the use of fluorescent markers Cy3 and Cy5 that can also be used in this invention. Suitable markers are also disclosed in Prober et al. (Science 238:336–341, 1987); Connell et al. (BioTechniques 5(4):342–384, 1987); Ansorge et al. (Nucl. Acids Res. 15(11):4593–4602, 1987); and Smith et al. (Nature 321:674, 1986). Other commercially available markings include, but are not limited to: ATTO dyes (e.g., Atto 655 and Atto 647N), Quasar dyes, CF dyes, fluorescein, rhodamine (including TMR, Texas Red, and Rox), Alexa Fluor® 647, 488, 532, 594, 633; ​​Dyomics dyes, bodipyrrole, acridine, R6G, Cy3, Cy3.5, Cy5, Cy5.5, coumarin, pyrene, benzene, and anthocyanins.

[0188] Although fluorescent labeling is specifically conceived, other forms of detectable labeling will be readily available to the average person skilled in the art. For example, microparticles, including quantum dots (Empodocles et al., Nature 399:126–130, 1999), gold nanoparticles (Reichert et al., Anal. Chem. 72:6025–6029, 2000), microbeads (Lacoste et al., Proc. Natl. Acad. Sci USA 97(17):9461–9466, 2000), and tags detectable by changes in pH and voltage, can be used with spectroscopic methods such as mass spectrometry, Raman spectroscopy, and surface plasmon resonance (SPR) sensing.

[0189] i. Fluorescein

[0190] In some embodiments of the L-nucleotides disclosed herein, the label is selected from the fluorophores Rox, fluoroboron dipyrrole, fluoroboron dipyrrole-FL-510, R6G, and Cy5 and their functional derivatives.

[0191] In a particular implementation, the fluorophore is selected from:

[0192]

[0193] 5.2.1.3.2 Disintegrable linker (R2)

[0194] Some embodiments of the L-nucleotides disclosed herein include a cleavable tag attached to, for example, a nitrogenous base, a 3'O, or a 5' phosphate group via a cleavable linker. In some embodiments, the linker is attached to a nitrogenous base. In some embodiments, the linker is attached to the C8 of a purine base, the C7 of a 7-denitropurine base, or the C5 of a pyrimidine base.

[0195] In some embodiments, the cleavable linker is photocleavable, cleavable by contact with water-soluble phosphine, or cleavable by a water-soluble catalyst containing a transition metal.

[0196] Suitable linkers known to those skilled in the art include, but are not limited to, disulfide linkers, acid-unstable linkers (including dialkoxybenzyl linkers, Sieber linkers, indole linkers, tert-butyl Sieber linkers, electrophilic cleavable linkers, nucleophilic cleavable linkers, and photocleavable linkers), cleavage under reducing or oxidizing conditions, cleavage via the use of safety-catch linkers, and cleavage via elimination mechanisms.

[0197] i. Electrophilic cleavage linker

[0198] Electrophilic cleavage linkers are typically proton-cleaved and include acid-sensitive cleavage. Suitable linkers include modified benzyl systems, such as triphenylmethyl, p-alkoxybenzyl esters, and p-alkoxybenzyl amides. Other suitable linkers include tert-butoxycarbonyl (Boc) groups and acetal systems.

[0199] Alternatively, thiophilic metals, such as nickel, silver, or mercury, can be used to prepare suitable linker molecules during the cleavage of thioacetals or other sulfur-containing protecting groups.

[0200] ii. Nucleophilic cleavage linker

[0201] Nucleophilic cleavage is also a recognized method for preparing linker molecules. Groups such as those of esters that are unstable in water (i.e., can be easily cleaved at alkaline pH) and groups unstable to non-aqueous nucleophiles can be used. Fluoride ions can be used to cleave the silicon-oxygen bonds in groups such as triisopropylsilane (TIPS) or tert-butyldimethylsilane (TBDMS).

[0202] iii. Photolytically schistolytic linker

[0203] Photolytic linkers have been widely used in carbohydrate chemistry. Preferably, the light required to activate cleavage does not affect other components of the modified nucleotide. For example, if a fluorophore is used as a label, it is preferred if the wavelength of light it absorbs differs from the wavelength required to cleave the linker molecule. Suitable linkers include those based on O-nitrobenzyl and nitroveratrol compounds. Linkers based on benzoin chemistry can also be used (Lee et al., J.Org. Chem. 64:3454–3460, 1999).

[0204] iv. Pyrolysis under reducing conditions

[0205] Many linker groups are known to be readily reducible and cleavable. Catalytic hydrogenation using palladium-based catalysts has been used to cleave benzyl and benzyloxycarbonyl groups. Disulfide bond reduction is also known in this art.

[0206] v. Pyrolysis under oxidizing conditions

[0207] Oxidation-based methods are well known in the art. These include the oxidation of alkoxybenzyl groups and the oxidation of sulfur and selenium linkages. The use of aqueous iodine to cleave disulfides and other sulfur- or selenium-based linkages is also within the scope of this invention.

[0208] vi. Safety-catch type connection base

[0209] Safety handle-type linkers are those that cleave in two steps. In preferred systems, the first step is the generation of a reactive nucleophilic center, followed by a second step involving intramolecular cyclization that leads to cleavage. For example, the levulinate bond can be treated with hydrazine or photochemically to release the active amine, which is then cyclized to cleave the ester elsewhere in the molecule (Burgess et al., J. Org. Chem. 62:5165–5168, 1997).

[0210] vii. Fracturing through elimination mechanisms

[0211] Elimination reactions can also be used. For example, base-catalyzed elimination with groups such as Fmoc and cyanoethyl, and palladium-catalyzed reductive elimination with allyl groups can be used.

[0212] 5.2.1.3.3 Spacer

[0213] In addition to the cleavage site, the linker may also contain one or more spacer portions. The spacers (linkers and bridges) can be any commonly used in the synthesis of, for example, biological conjugates. The spacers maintain distance between the nucleotide bases and the cleavage site or label. The length of the linker is not important, provided that the label is sufficiently far from the nucleotide to avoid interfering with any interaction between the nucleotide and the enzyme.

[0214] In certain embodiments, in addition to additional spacer groups, the cleavable linker group also comprises an allyl, disulfide, or azide group. In some embodiments, the cleavable linker group comprises:

[0215] ,or

[0216] .

[0217] 5.2.1.3.4 Decomposable marker constructs

[0218] In some embodiments of the mirror nucleosides or nucleotides provided herein, the cleavable label comprises the label as described in section 5.2.1.3.1 and the cleavable linker as described in more detail in section 5.2.1.3.2.

[0219] In a particular implementation, the cleavable marker construct is selected from:

[0220] ;

[0221] The wavy lines indicate attachment points to mirrored nucleotides, including attachments to linker / spacer groups, which are attached to nucleotides for the purpose of enabling attachment, such as amine moieties.

[0222] 5.2.2. Implementation plan of this disclosure

[0223] In some embodiments, an L-nucleoside or L-nucleotide is provided, comprising:

[0224] Pentose sugars selected from (3R,4S)-3,4,5-trihydroxypentanal and (4R)-4,5-dihydroxypentanal;

[0225] In this sugar, C-5 is replaced by a hydroxyl group, monophosphate, diphosphate, or triphosphate; and

[0226] The C-3 of the sugar is replaced by H or O-R', where R' is H or a cleavable protecting group;

[0227] Nitrogenous bases; and

[0228] Optional detachable marker;

[0229] The L-nucleoside or L-nucleotide contains at least one selected from (i) a cleavable protecting group and (ii) a cleavable label.

[0230] In some embodiments, this disclosure provides nucleotides comprising:

[0231] Pentose sugars in cyclic form containing (3R,4S)-3,4,5-trihydroxypentanal or (4R)-4,5-dihydroxypentanal;

[0232] In this pentose, the H of the 5-hydroxy group is replaced by one or more phosphate groups; and

[0233] In (3R,4S)-3,4,5-trihydroxypentanal, the H of the 3-hydroxy group is optionally replaced by a cleavable protecting group.

[0234] The nitrogenous base linked to the 2-position of the pentose sugar, and

[0235] Optional sharding markers including shardable linker bases and markers;

[0236] It contains at least one of a cleavable protecting group and a cleavable label.

[0237] The embodiments provided herein will be further described with reference to nucleotides. However, unless otherwise stated, references to nucleotides are also intended to apply to nucleosides.

[0238] In some embodiments, an L-nucleotide having the structure according to formula A is provided:

[0239] Formula A

[0240] Wherein R' is H or a cleavable protecting group; X is H or a hydroxyl group; the base is a nitrogenous base; and wherein the nucleotide optionally contains a cleavable tag, and wherein at least one of a cleavable protecting group or a cleavable tag is present.

[0241] In some embodiments, the nitrogen-containing base is as described in section 5.2.1.1. In some embodiments, the cleavable protecting group is as described in section 5.2.1.2. In some embodiments, the cleavable label is as described in section 5.2.1.3.

[0242] In any of the described embodiments, the location of the cleavable tag is not particularly limited and can be attached to the nucleotide at any chemically feasible and / or known in the art. In some embodiments, the cleavable tag is attached to a nitrogenous base via a terminal phosphate or at 3'O, in which case the cleavable protecting group is the cleavable tag.

[0243] 5.2.2.1 (L)-nucleotide reversible terminator (L-NRT)

[0244] 5.2.2.1.1 [(L)-3'-O-R'-dNTPs] - Equation I

[0245] In one aspect of this disclosure, an L-nucleotide having a structure according to Formula I is provided:

[0246] Formula I

[0247] The bases are nitrogen-containing bases, and R' is a cleavable protecting group.

[0248] In some embodiments, the nitrogen-containing base is as described in section 5.2.1.1 of this document. In some embodiments, the cleavable protecting group is as described in section 5.2.1.2 of this document.

[0249] In some embodiments, an L-nucleotide having the following structure is provided:

[0250] and .

[0251] In some embodiments, R' comprises a cleavable protecting group as described in section 5.2.1.2 of this document. In other embodiments, the cleavable protecting group R' is selected from:

[0252] ;

[0253] The wavy line indicates the attachment point to 3'O.

[0254] 5.2.2.1.2 [(L)-3'-O-R'-dNTP-R2-marking] -Formula II

[0255] In one aspect of this disclosure, an L-nucleotide having a structure according to Formula II is provided:

[0256] Formula II

[0257] The bases are nitrogenous bases; R' is a cleavable protecting group or H; and the label -R2 together contains a cleavable label construct.

[0258] In some embodiments, the nitrogenous base is as described in section 5.2.1.1. In some embodiments, the cleavable protecting group is as described in section 5.2.1.2. In some embodiments, the cleavable labeled construct is as described in section 5.2.1.3.

[0259] In some embodiments, an L-nucleotide having a structure selected from the following is provided:

[0260]

[0261] and

[0262] .

[0263] In some embodiments, the cleavable protecting group R' is as described in section 5.2.1.2 of this document. In some embodiments, the cleavable linker R2 is as described in section 5.2.1.3.2 of this document.

[0264] In a particular implementation, R' is selected from:

[0265] H, ;

[0266] The wavy line indicates the attachment point to 3'O.

[0267] In a particular implementation, R2 includes:

[0268] ,or .

[0269] In a particular embodiment, an L-nucleotide having a structure selected from the following is provided:

[0270]

[0271] and

[0272]

[0273] Where R' is H or a cleavable protecting group, as described in section 5.2.1.2 of this document.

[0274] In some implementations, R' is selected from:

[0275] H, .

[0276] The wavy line indicates the attachment point to 3'O.

[0277] 5.2.2.2 Termination of the Mark

[0278] 5.2.2.2.1 [(L)-ddNTP-R2-label] Formula III

[0279] In one aspect of this disclosure, an L-nucleotide having a structure according to Formula III is provided:

[0280] Formula III

[0281] The bases are nitrogenous bases; R2 is a cleavable linker; and the marker-R2 together contain a cleavable marker construct.

[0282] In some embodiments, the nitrogenous base is as described in section 5.2.1.1 of this document. In some embodiments, the cleavable marker construct marker -R2 is as described in section 5.2.1.3 of this document.

[0283] In some embodiments, an L-nucleotide having a structure selected from the following is provided:

[0284] and

[0285] .

[0286] In some embodiments, the cleavable linker R2 is as described in section 5.2.1.3.2 of this document. In a particular embodiment, R2 comprises:

[0287] ,or .

[0288] In a particular embodiment, an L-nucleotide having a structure selected from the following is provided:

[0289]

[0290]

[0291] and

[0292] .

[0293] 5.3. Mirror polymerase

[0294] On the other hand, this disclosure provides a mirror-image polymerase. Specifically, the mirror-image polymerase comprises D-amino acids. In some embodiments, the mirror-image polymerase consists of D-amino acids. In some embodiments, the mirror-image polymerase comprises both D- and L-amino acids. In some embodiments, the mirror-image polymerase does not contain L-amino acids.

[0295] In some embodiments, the mirror-image nucleic acid is a mirror image of a DNA polymerase or an RNA polymerase. In some embodiments, the mirror-image nucleic acid is a mirror image of a 9°N DNA polymerase or a modification thereof.

[0296] In some embodiments, the mirror polymerase comprises a sequence having at least 90% sequence identity with SEQ ID NO: 1. In some embodiments, the mirror polymerase comprises a sequence having at least 96% sequence identity with SEQ ID NO: 1. In some embodiments, the mirror polymerase comprises a sequence having at least 96% sequence identity with SEQ ID NO: 1. In some embodiments, the mirror polymerase comprises a sequence having at least 97% sequence identity with SEQ ID NO: 1. In some embodiments, the mirror polymerase comprises a sequence having at least 98% sequence identity with SEQ ID NO: 1. In some embodiments, the mirror polymerase comprises a sequence having at least 99% sequence identity with SEQ ID NO: 1. In some embodiments, the mirror polymerase comprises the sequence of SEQ ID NO: 1.

[0297] In some embodiments, the mirror polymerase has a sequence having at least 90% sequence identity with SEQ ID NO: 1. In some embodiments, the mirror polymerase has a sequence having at least 96% sequence identity with SEQ ID NO: 1. In some embodiments, the mirror polymerase has a sequence having at least 96% sequence identity with SEQ ID NO: 1. In some embodiments, the mirror polymerase has a sequence having at least 97% sequence identity with SEQ ID NO: 1. In some embodiments, the mirror polymerase has a sequence having at least 98% sequence identity with SEQ ID NO: 1. In some embodiments, the mirror polymerase has a sequence having at least 99% sequence identity with SEQ ID NO: 1. In some embodiments, the mirror polymerase has the sequence of SEQ ID NO: 1.

[0298] In some embodiments, the mirror-image polymerase, compared to SEQ ID NO: 1, comprises one or more modifications at one or more amino acid positions disclosed in Table 1. In some embodiments, the mirror-image polymerase, compared to SEQ ID NO: 1, comprises one or more amino acid substitutions disclosed in Table 1. In some embodiments, the mirror-image polymerase comprises all amino acid substitutions disclosed in Table 1.

[0299]

[0300] In some embodiments, the mirror polymerase, compared to SEQ ID NO: 1, contains one or more modifications for natural chemical ligation (NCL). In some embodiments, compared to SEQ ID NO: 1, the mirror polymerase contains one or more modifications at one or more amino acid sites selected from E276, K317, N424, and S651. In some embodiments, compared to SEQ ID NO: 1, the mirror polymerase contains one or more substitutions selected from E276A, K317G, N424A, and S651A. In some embodiments, compared to SEQ ID NO: 1, the mirror polymerase contains substitutions of E276A, K317G, N424A, and S651A.

[0301] In some embodiments, the mirror polymerase, compared to SEQ ID NO: 1, comprises one or more substitutions of isoleucine. In some embodiments, the mirror polymerase, compared to SEQ ID NO: 1, comprises one or more modifications at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744. In some embodiments, compared to SEQ ID NO: 1, the mirror polymerase comprises one or more substitutions from Ile to Ala, Val, Leu, or Tyr at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733, and I744. In some embodiments, compared to SEQ ID NO: 1, the mirror polymerase comprises one or more substitutions selected from I80V, I127V, I171A, I176V, I191V, I228V, I256V, I264A, I268L, I400V, I597V, I610V, I618A, I630L, I642V, I715Y, I733V, and I744V.

[0302] In some embodiments, compared to SEQ ID NO: 1, the mirror polymerase contains one or more modifications to improve its interaction with the L-nucleotide or its modifications disclosed herein.

[0303] In some embodiments, compared to SEQ ID NO: 1, the mirror polymerase includes one or more modifications at one or more amino acid sites selected from M129, I130, G131, D141, E143, L408, Y409, P410, A485, T514, and I521. In some embodiments, compared to SEQ ID NO: 1, the mirror polymerase includes one or more modifications at one or more amino acid sites selected from D141, E143, Y409, and A485. In some embodiments, compared to SEQ ID NO: 1, the mirror polymerase includes one or more substitutions selected from D141A, E143A, Y409V, and A485L. In some embodiments, compared to SEQ ID NO: 1, the mirror polymerase includes substitutions of D141A, E143A, Y409V, and A485L.

[0304] In some embodiments, compared to SEQ ID NO: 1, the mirror polymerase includes one or more modifications at one or more amino acid sites selected from D141, E143, L408, Y409, P410, A485, T514, and I521. In some embodiments, compared to SEQ ID NO: 1, the mirror polymerase includes one or more substitutions selected from D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L. In some embodiments, compared to SEQ ID NO: 1, the mirror polymerase includes substitutions of D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L.

[0305] In some embodiments, compared to SEQ ID NO: 1, the mirror polymerase comprises one or more substitutions selected from M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L, or a modification of adding D between I130 and G131. In some embodiments, compared to SEQ ID NO: 1, the mirror polymerase comprises substitutions of M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, and I521L, and the addition of D between I130 and G131.

[0306] In some embodiments, the mirror polymerase comprises a sequence selected from SEQ ID Nos 1-7. In some embodiments, the polymerase has a sequence selected from SEQ ID Nos: 1-7.

[0307] Mirror-image polymerases can be obtained through chemical synthesis. In some embodiments, polypeptides or small peptides are synthesized via solid-phase peptide synthesis and then linked by chemical ligation to obtain a mirror-image polymerase. In some embodiments, the mirror-image polymerase has the same enzymatic activity as the original polymerase but acts on D-nucleotides.

[0308] 5.4. Mirror transferase

[0309] In one aspect, this disclosure provides a mirror-image transferase. In some embodiments, the mirror-image transferase is a (D)-terminal deoxynucleotidyl transferase. The (D)-terminal deoxynucleotidyl transferase can be a template-independent polymerase that catalyzes the addition of L-deoxynucleotides to the 3' hydroxyl end of a D-DNA molecule.

[0310] Specifically, the mirror-image transferase contains D-amino acids. In some embodiments, the mirror-image transferase consists entirely of D-amino acids. In some embodiments, the mirror-image transferase contains both D- and L-amino acids. In some embodiments, the mirror-image transferase does not contain L-amino acids.

[0311] In some implementations, the mirrored nucleic acid has the following SEQ ID NO: Mirror image of the terminal deoxynucleotidyl transferase of sequence 36: (NSSPSPVPGSQNVPAPAVKKISQYACQRRTTLNNYNQLFTDALDILAENDELRENEGSCLAFMRASSVLKSLPFPITSMKDTEGIPCLGDKVKSIIEGIIEDGESSEAKAVLNDERYKSFKLFTSVFGVGLKTAEKWFRMGFRTLSKIQSDKSLRFTQMQKAGFLYYEDLVSCVNRPEAEAVSMLVKEAVVTFLPDALVTMTGGFRRGKMTGHDVDFLITSPEATEDEEQQLLHKVTDFWKQQGLLLYCDILESTFEKFKQPSRKVDALDHFQKCFLILKLDHGRVHSEKSGQQEGKGWKAIRVDLVMCPYDRRAFALLGWTGSRQFERDLRRYATHERKMMLDNHALYDRTKRVFLEAESEEEIFAHLGLDYIEPWERNA)

[0312] In some embodiments, the mirror-linked transferase comprises a sequence having at least 90% sequence identity with SEQ ID NO: 36. In some embodiments, the mirror-linked transferase comprises a sequence having at least 96% sequence identity with SEQ ID NO: 36. In some embodiments, the mirror-linked transferase comprises a sequence having at least 96% sequence identity with SEQ ID NO: 36. In some embodiments, the mirror-linked transferase comprises a sequence having at least 97% sequence identity with SEQ ID NO: 36. In some embodiments, the mirror-linked transferase comprises a sequence having at least 98% sequence identity with SEQ ID NO: 36. In some embodiments, the mirror-linked transferase comprises a sequence having at least 99% sequence identity with SEQ ID NO: 36. In some embodiments, the mirror-linked transferase comprises the sequence of SEQ ID NO: 36.

[0313] In some embodiments, the mirror-linked transferase has a sequence having at least 90% sequence identity with SEQ ID NO: 36. In some embodiments, the mirror-linked transferase has a sequence having at least 96% sequence identity with SEQ ID NO: 36. In some embodiments, the mirror-linked transferase has a sequence having at least 96% sequence identity with SEQ ID NO: 36. In some embodiments, the mirror-linked transferase has a sequence having at least 97% sequence identity with SEQ ID NO: 36. In some embodiments, the mirror-linked transferase has a sequence having at least 98% sequence identity with SEQ ID NO: 36. In some embodiments, the mirror-linked transferase has a sequence having at least 99% sequence identity with SEQ ID NO: 36. In some embodiments, the mirror-linked transferase has the sequence of SEQ ID NO: 36.

[0314] In some embodiments, the mirror polymerase contains one or more modifications for the NCL (natural chemical linker) site. In some embodiments, the mirror polymerase contains one or more isoleucine (I) to valine (V) substitutions. In some embodiments, the mirror polymerase contains one or more modifications for interacting with the modified nucleotide.

[0315] Mirror-image transferases can be obtained through chemical synthesis. In some embodiments, polypeptides or small peptides are synthesized via solid-phase peptide synthesis and then linked by chemical ligation to obtain the mirror-image transferase. In some embodiments, the mirror-image transferase has the same enzymatic activity as the original transferase but acts on D-nucleotides.

[0316] In some implementations, the mirror transferase is obtained by linking more than one synthetic peptide.

[0317] 5.5. Usage Instructions

[0318] In one aspect, this disclosure provides methods for using mirror-image nucleotides and mirror-image polymerases disclosed herein. Mirror-image compositions can be used to replicate or sequence L-polynucleotides. In some embodiments, the compositions are used in sequencing-by-synthesis methods, Sanger methods, or combinations thereof.

[0319] 5.5.1. Method for copying L-shaped templates

[0320] This disclosure provides a method for replicating L-polynucleotides. The method may include the steps of incubating a mixture comprising (i) an L-polynucleotide, (ii) an L-primer, (iii) an L-dNTP, (iv) a mirror-type polymerase, and (v) a buffer, thereby inducing the replication of the L-polynucleotide. In some embodiments, the L-polynucleotide is L-type DNA or RNA. In some embodiments, the L-polynucleotide comprises at least 10, 20, 30, 50, 100, 200, 500, or more nucleotides.

[0321] In some embodiments, the mixture comprises two or more L-dNTPs. In some embodiments, the mixture comprises L-dATP, L-dGTP, L-dCTP, L-dTTP, or modifications thereof. In some embodiments, the mixture comprises L-dATP, L-dGTP, L-dCTP, and L-dTTP.

[0322] In some embodiments, the mixture contains a buffer developed for use with 9°N DNA polymerase. In some embodiments, the buffer contains Tris-HCl at concentrations of 1 mM to 200 mM, 5 mM to 150 mM, 10 mM to 100 mM, 20 mM to 100 mM, or 30 mM to 80 mM. In some embodiments, the buffer contains Tris-HCl at concentrations of about 10 mM, 20 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, 80 mM, 90 mM, or 100 mM. In some embodiments, the buffer contains MgCl2 at concentrations of 1 mM to 100 mM, 5 mM to 100 mM, 10 mM to 100 mM, 15 mM to 50 mM, or 15 mM to 30 mM. In some embodiments, the buffer contains MgCl2 at concentrations of about 10 mM, 20 mM, 30 mM, 40 mM, or 50 mM. In some embodiments, the buffer contains MnCl2 at concentrations of 1 mM to 100 mM, 5 mM to 100 mM, 10 mM to 100 mM, 15 mM to 50 mM, or 15 mM to 30 mM. In some embodiments, the buffer contains MnCl2 at concentrations of about 10 mM, 20 mM, 30 mM, 40 mM, or 50 mM. In some embodiments, the buffer contains DTT at concentrations of 0.1 mM to 10 mM, 0.5 mM to 5 mM, 0.5 mM to 3 mM, or 0.5 mM to 2 mM. In some embodiments, the buffer contains DTT at concentrations of about 0.5 mM, 1 mM, 1.5 mM, or 2 mM. In some embodiments, the buffer contains KCl at concentrations of 10 mM to 100 mM, 20 mM to 80 mM, 25 mM to 75 mM, or 30 mM to 70 mM. In some embodiments, the buffer contains KCl at concentrations of about 25 mM, 30 mM, 40 mM, 50 mM, 60 mM, 70 mM, or 100 mM. In some embodiments, the buffer has a pH of 7 to 8. In some embodiments, the buffer has a pH of about 7, 7.5, or 8.

[0323] In some embodiments, the buffer solution comprises Tris-HCl, MgCl2, DTT, and KCl. In some embodiments, the buffer solution comprises Tris-HCl, MnCl2, DTT, and KCl. In some embodiments, the buffer solution comprises Tris-HCl, MnCl2, MgCl2, DTT, and KCl. In some embodiments, the buffer solution comprises 50 mM Tris-HCl, 20 mM MgCl2, 1 mM DTT, and 50 mM KCl at pH 7.5.

[0324] 5.5.2. Sequencing Methods

[0325] This disclosure provides a method for sequencing L-polynucleotides. L-polynucleotides comprise L-type nucleotides. L-polynucleotides can be DNA or RNA.

[0326] In some embodiments, the sequencing method includes the following cycle: (a) incubating a mixture containing (i) an L-polynucleotide, (ii) an L-primer, (iii) an L-3'-O-R'-dNTP-R2-label, (iv) a mirror-type polymerase, and (v) a buffer to obtain a replication product; (b) detecting a signal from the L-3'-O-R'-dNTP-R2-label incorporated into the replication product; and (iii) inducing cleavage of the R' and R2 groups of the L-3'-O-R'-dNTP-R2-label incorporated into the replication product. The method includes (i) incorporating the L-3'-O-R'-dNTP-R2-label, (ii) identifying the incorporated nucleotide by a signal from the incorporated L-3'-O-R'-dNTP-R2-label, and (iii) cleaving the label while restarting the polymerase reaction for sequential sequencing. The L-3'-O-R'-dNTP-R2- label comprises a chemically capped 3'-OH moiety (R') and a label tethered to a base via a chemically cleavable linker (R2). Cleavage of the 3'-capped moiety (R') and the label on the reaction product allows the polymerase reaction to restart.

[0327] In some implementations, the method includes multiple loops. In some implementations, the loops are repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times. In some implementations, the loops are repeated less than 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, 1000, 2000, 3000, or 5000 times.

[0328] In some embodiments, the L-polynucleotide contains more than 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 nucleotides. In some embodiments, the L-polynucleotide contains fewer than 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, 1000, 2000, 3000, or 5000 nucleotides.

[0329] In some embodiments, the L-3'-O-R'-dNTP-R2- label includes the L-3'-O-R'-dATP-R2- label, the L-3'-O-R'-dTTP-R2- label, the L-3'-O-R'-dGTP-R2- label, or the L-3'-O-R'-dCTP-R2- label. In some embodiments, the L-3'-O-R'-dNTP-R2- label includes two or more of the L-3'-O-R'-dATP-R2- label, the L-3'-O-R'-dTTP-R2- label, the L-3'-O-R'-dGTP-R2- label, and the L-3'-O-R'-dCTP-R2- label. In some implementations, the L-3'-O-R'-dNTP-R2- tag includes the L-3'-O-R'-dATP-R2- tag, the L-3'-O-R'-dTTP-R2- tag, the L-3'-O-R'-dGTP-R2- tag, and the L-3'-O-R'-dCTP-R2- tag. The tag attached to each dNTP can be unique and identifiable.

[0330] In some embodiments, the L-3'-O-R'-dNTP-R2- tag has the structure according to Formula I as described in 5.2.2.1.2. In some embodiments, the L-3'-O-R'-dNTP-R2- tag has the structure according to Formula II as described in 5.2.2.1.2.

[0331] In some embodiments, the L-3'-O-R'-dNTP-R2-label includes a fluorescent label. In this case, the signal is a fluorescent signal. In some embodiments, each type of L-3'-O-R'-dNTP-R2-label (e.g., dATP, dGTP, dTTP, dCTP) is labeled as a unique label. In some embodiments, each type of L-3'-O-R'-dNTP-R2-label (e.g., dATP, dGTP, dTTP, dCTP) provides a unique fluorescent signal. The L-3'-O-R'-dNTP-R2-label can include any label disclosed in 5.2.1.3.1. In some embodiments, the L-3'-O-R'-dNTP-R2-label includes a non-fluorescent label.

[0332] In some embodiments, the sequencing method includes the following steps: (a) incubating a mixture containing (i) L-polynucleotide, (ii) L-primer, (iii) L-dNTP, (iv) L-ddNTP-R2-label, (v) the mirror polymerase disclosed herein, and (vi) buffer to obtain a replication product; (b) isolating the replication product; and (c) detecting the signal from the L-ddNTP-R2-label incorporated into the replication product. The incubation step may include a PCR reaction.

[0333] In a preferred embodiment of the method, a low ratio of L-ddNTP-R2-labels is added to the mixture compared to L-dNTPs. During incubation (e.g., PCR reaction), the L-ddNTP-R2-labels lacking the 3'-OH group can be randomly incorporated into the replication product by mirror polymerase. The incorporation of L-ddNTP-R2-labels terminates the replication process. The reaction can induce the production of oligonucleotide copies of the replication product, which are terminated by L-ddNTP-R2-labels of random length.

[0334] In this method, the replication products can be separated by size. In some embodiments, the replication products are separated by size via gel electrophoresis. By detecting the signal from the L-ddNTP-R2-label incorporated into the replication product, the identity of the terminal L-ddNTP-R2-label (e.g., ddATP, ddGTP, ddTTP, or ddCTP) of the replication product can be determined. The data can show the nucleotide type along the length of the L-polynucleotide. This process can allow for the determination of the L-polynucleotide sequence.

[0335] In some embodiments, L-dNTPs comprise L-dATP, L-dTTP, L-dGTP, or L-dCTP. In some embodiments, L-dNTPs comprise one or more of L-dATP, L-dTTP, L-dGTP, and L-dCTP. In some embodiments, L-dNTPs comprise L-dATP, L-dTTP, L-dGTP, and L-dCTP.

[0336] In some embodiments, the L-ddNTP-R2-label includes an L-ddATP-R2-label, an L-ddTTP-R2-label, an L-ddGTP-R2-label, or an L-ddCTP-R2-label. In some embodiments, the L-ddNTP-R2-label includes one or more of the L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label, and L-ddCTP-R2-label. In some embodiments, the L-ddNTP-R2-label includes an L-ddATP-R2-label, an L-ddTTP-R2-label, an L-ddGTP-R2-label, and an L-ddCTP-R2-label. The label attached to each ddNTP can be unique and identifiable.

[0337] In some embodiments, the L-ddNTP-R2-marker has the structure disclosed in 5.2.2.2.1. In some embodiments, the L-ddNTP-R2-marker has Formula III as defined in 5.2.2.2.1.

[0338] In some embodiments, the L-ddNTP-R2-label includes a fluorescent label. In this case, the signal is a fluorescent signal. In some embodiments, each type of L-ddNTP-R2-label (e.g., ddATP, ddGTP, ddTTP, ddCTP) is labeled as a unique label. In some embodiments, each type of L-ddNTP-R2-label (e.g., ddATP, ddGTP, ddTTP, ddCTP) provides a unique fluorescent signal. The L-ddNTP-R2-label can include any label disclosed in 5.2.1.3.1. In some embodiments, the L-ddNTP-R2-label includes a non-fluorescent label.

[0339] In some embodiments, the sequencing method includes the following cycle: (a) incubating a mixture containing (i) an L-polynucleotide, (ii) an L-primer, (iii) an L-3'-O-R'-dNTP, (iv) an L-ddNTPs-R2-label, (v) the mirror polymerase provided herein, and (vi) a buffer to obtain a replication product; (b) detecting a signal from the L-ddNTP-R2-label incorporated into the replication product; and (c) inducing cleavage of the R' group of the L-3'-O-R'-dNTP incorporated into the replication product and the R2 group of the L-ddNTPs-R2-label. In this method, the polymerase reaction is performed using a combination of a 3′-capped nucleotide reversible terminator (L-3'-O-R'-dNTP) and a cleavable fluorescent dideoxynucleotide (L-ddNTPs-R2-label). In this method, the sequence is determined by the signal of each label on the reaction product terminated by the ddNTP (L-ddNTPs-R2-label). After the 3′-OH capping group (R' group) is removed from the incorporated nucleotide reversible terminator (L-3'-O-R'-dNTP) and the label is removed from the DNA product terminated by ddNTPs (L-ddNTPs-R2-labeled), the polymerase reaction restarts to continue sequence determination.

[0340] In some implementations, the method includes multiple loops. In some implementations, the loops are repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times. In some implementations, the loops are repeated less than 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, 1000, 2000, 3000, or 5000 times.

[0341] In some embodiments, the L-polynucleotide contains more than 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 nucleotides. In some embodiments, the L-polynucleotide contains fewer than 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, 1000, 2000, 3000, or 5000 nucleotides.

[0342] In some embodiments, the L-3'-O-R'-dNTP comprises L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, or L-3'-O-R'-dCTP. In some embodiments, the L-3'-O-R'-dNTP comprises one or more of L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, and L-3'-O-R'-dCTP. In some embodiments, the L-3'-O-R'-dNTP comprises L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, and L-3'-O-R'-dCTP.

[0343] In some embodiments, L-3'-O-R'-dNTP has any of the structures provided in 5.2.2.1.1. In some embodiments, L-3'-O-R'-dNTP has Formula I as defined in 5.2.2.1.1.

[0344] In some embodiments, the L-ddNTP-R2-label includes an L-ddATP-R2-label, an L-ddTTP-R2-label, an L-ddGTP-R2-label, or an L-ddCTP-R2-label. In some embodiments, the L-ddNTP-R2-label includes one or more of the L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label, and L-ddCTP-R2-label. In some embodiments, the L-ddNTP-R2-label includes an L-ddATP-R2-label, an L-ddTTP-R2-label, an L-ddGTP-R2-label, and an L-ddCTP-R2-label. The label attached to each dNTP can be unique and identifiable.

[0345] In some embodiments, the L-ddNTP-R2-label has any of the structures provided in 5.2.2.2.1. In some embodiments, the L-ddNTP-R2-label has Formula III as defined in 5.2.2.2.1. In some embodiments, each type of L-3'-O-R'-dNTP-R2-label (e.g., dATP, dGTP, dTTP, dCTP) provides a unique fluorescent signal. The L-ddNTP-R2-label can include any label disclosed in 5.2.1.3.1. In some embodiments, the L-ddNTP-R2-label includes a non-fluorescent label.

[0346] In some embodiments, the sequencing method includes the following cycles: (a) incubating a mixture containing (i) L-polynucleotides, (ii) L-primers, (iii) L-dNTPs, (iv) a mirror-type polymerase, and (v) a buffer to obtain a reaction product; and (b) detecting the release of PPi molecules from the reaction product. In some embodiments, the sequencing method includes multiple cycles, wherein in each cycle, the mixture contains a single type of L-dNTP selected from L-dATP, L-dTTP, L-dGTP, and L-dCTP. In some embodiments, the sequencing method includes multiple cycles, wherein each cycle is performed by progressively changing the type of L-dNTP to one of L-dATP, L-dTTP, L-dGTP, and L-dCTP.

[0347] In some embodiments, the step of detecting the release of PPi molecules includes the following steps: (i) treating the reaction product to convert PPi molecules into ATP (if present) and generating a detectable signal from ATP (if present); and (ii) detecting the presence or absence of the detectable signal. In some embodiments, ATP is treated with luciferase to generate a detectable signal. In some embodiments, the detectable signal is light. In some embodiments, PPi molecules are converted into ATP by ATP-sulfatase.

[0348] 5.5.3. Methods for synthesizing L-polynucleotides

[0349] In one aspect, this disclosure provides a method for synthesizing L-polynucleotides using a mirror-linked transferase (e.g., a terminal deoxynucleotide transferase). In some embodiments, the mirror-linked transferase is used to catalyze the repeated addition of deoxyribonucleotides to oligodeoxyribonucleotides and the 3'-OH of single-stranded or double-stranded DNA. In some embodiments, the mirror-linked transferase is used for the production of synthetic homopolymers and heteropolymers, homopolymer tailing of linear double-stranded DNA with any type of 3'-OH terminus, labeling of oligonucleotides, DNA, or RNA, 5'-RACE (rapid amplification of cDNA ends), or in situ localization for apoptosis. Mirror-linked transferases can be used for any other known applications of transferases.

[0350] In some embodiments, a method for synthesizing L-polynucleotides includes the steps of incubating a mixture comprising (i) an L-primer, (ii) an L-dNTP or L-ddNTP, (iii) a mirror-image transferase disclosed herein, and (iv) a buffer, thereby inducing the synthesis of L-polynucleotides. In some embodiments, the L-polynucleotide is DNA or RNA. The L-dNTP or L-ddNTP can be any mirror-image nucleotide or combination thereof disclosed herein.

[0351] In some embodiments, the mixture comprises L-dNTPs. In some embodiments, the mixture comprises L-ddNTPs. In some embodiments, the mixture comprises L-dATP, L-dGTP, L-dCTP, or L-dTTP. In some embodiments, the L-dNTPs comprise (i) L-dATP or L-dTTP and (ii) L-dGTP or L-dCTP. In some embodiments, the L-dNTPs comprise L-dATP, L-dGTP, L-dCTP, and L-dTTP.

[0352] In some embodiments, the mixture comprises L-3'-OR-dNTP. In some embodiments, the mixture comprises L-3'-OR-dATP, L-3'-OR-dGTP, L-3'-OR-dCTP, or L-3'-OR-dTTP. In some embodiments, the mixture comprises L-3'-OR-dATP, L-3'-OR-dGTP, L-3'-OR-dCTP, and L-3'-OR-dTTP.

[0353] In some embodiments, the mixture contains radiolabeled L-ddNTPs. In some embodiments, the L-ddNTPs are radiolabeled L-ddATP, L-ddGTP, L-ddCTP, or L-ddTTP. In some embodiments, the L-ddNTPs contain L-ddNTP-R2-labeled or L-3'-O-R'-dNTP-R2-labeled as disclosed herein.

[0354] In some embodiments, the method further includes a step of removing the 3'-OR group by a deprotection reaction. In some embodiments, the deprotection reaction includes incubating the mixture with TCEP buffer. In some embodiments, the deprotection reaction includes incubating with 50 mM TCEP buffer at 37°C-55°C for 5-15 minutes. In some embodiments, R is an azide methyl group.

[0355] In some embodiments, the method further includes a step of stopping the reaction by heating or by adding a chelating agent. In some embodiments, the chelating agent is EDTA.

[0356] In some embodiments, incubation is carried out at 20°C to 40°C. In some embodiments, incubation is carried out at 37°C to 55°C. In some embodiments, incubation is carried out at 22°C to 37°C. In some embodiments, incubation is carried out at 37°C. In some embodiments, incubation is carried out at 30°C to 37°C.

[0357] In some embodiments, heating is performed at a temperature above 50°C. In some embodiments, heating is performed at a temperature above 55°C. In some embodiments, heating is performed at a temperature above 60°C. In some embodiments, heating is performed at a temperature above 60°C. In some embodiments, heating is performed at 70°C.

[0358] In some embodiments, the method includes repeating the steps provided above. In some embodiments, the method includes repeating the step of incubating the mixture for the next nucleotide addition. In some embodiments, the method includes repeating the steps of incubating the mixture and the deprotection reaction.

[0359] In some implementations, the method also includes a step of sequencing the synthesized L-polynucleotide.

[0360] 5.6. Reagent Kit

[0361] This disclosure provides kits for replicating, sequencing, or synthesizing L-polymerases. The kits contain the mirror polymerase or mirror transferase disclosed herein and optional buffers.

[0362] In some embodiments, the kit further comprises L-dNTPs. In some embodiments, the L-NTPs comprise L-dATP, L-dGTP, L-dCTP, L-dTTP, or L-UTP. In some embodiments, the L-NTPs comprise one or more of L-dATP, L-dGTP, L-dCTP, L-dTTP, and L-UTP. In some embodiments, the L-NTPs comprise L-dATP, L-dGTP, L-dCTP, and L-dTTP. In some embodiments, the L-NTPs comprise L-dATP, L-dGTP, L-dCTP, and L-UTP. In some embodiments, the L-NTPs comprise L-dATP, L-dGTP, L-dCTP, L-dTTP, and L-UTP.

[0363] In some embodiments, the kit contains an L-3'-O-R'-dNTP-R2- label. In some embodiments, the L-3'-O-R'-dNTP-R2- label comprises an L-3'-O-R'-dATP-R2- label, an L-3'-O-R'-dTTP-R2- label, an L-3'-O-R'-dGTP-R2- label, an L-3'-O-R'-dCTP-R2- label, or an L-3'-O-R'-dUTP-R2- label. In some embodiments, the L-3'-O-R'-dNTP-R2- label comprises one or more of the following: L-3'-O-R'-dATP-R2- label, L-3'-O-R'-dTTP-R2- label, L-3'-O-R'-dGTP-R2- label, L-3'-O-R'-dCTP-R2- label, and L-3'-O-R'-dUTP-R2- label. In some embodiments, the L-3'-O-R'-dNTP-R2- label comprises one or more of the following: L-3'-O-R'-dATP-R2- label, L-3'-O-R'-dTTP-R2- label, L-3'-O-R'-dGTP-R2- label, and L-3'-O-R'-dCTP-R2- label. In some embodiments, the L-3'-O-R'-dNTP-R2-label comprises one or more of the following: L-3'-O-R'-dATP-R2-label, L-3'-O-R'-dGTP-R2-label, L-3'-O-R'-dCTP-R2-label, and L-3'-O-R'-dUTP-R2-label. In some embodiments, each type of L-3'-O-R'-dNTP-R2-label (e.g., dATP, dGTP, dTTP, dCTP) provides a unique fluorescent signal.

[0364] In some embodiments, the L-3'-O-R'-dNTP-R2- tag has Formula II as defined in 5.2.2.1.2. In some embodiments, the L-3'-O-R'-dNTP-R2- tag has any of the structures disclosed in 5.2.2.1.2.

[0365] In some embodiments, the kit contains an L-ddNTP-R2-label. In some embodiments, the L-ddNTP-R2-label comprises an L-ddATP-R2-label, an L-ddTTP-R2-label, an L-ddGTP-R2-label, an L-ddCTP-R2-label, or an L-ddUTP-R2-label. In some embodiments, the L-ddNTP-R2-label comprises one or more of the following: L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label, L-ddCTP-R2-label, and L-ddUTP-R2-label. In some embodiments, the L-ddNTP-R2-label comprises an L-ddATP-R2-label, an L-ddTTP-R2-label, an L-ddGTP-R2-label, and an L-ddCTP-R2-label. In some implementations, the L-ddNTP-R2-label includes the L-ddATP-R2-label, L-ddGTP-R2-label, L-ddCTP-R2-label, and L-ddUTP-R2-label.

[0366] In some embodiments, the L-ddNTP-R2-marker has Formula III as defined in 5.2.2.2.1. In some embodiments, the L-ddNTP-R2-marker has any of the structures disclosed in 5.2.2.2.1.

[0367] In some embodiments, the kit further comprises L-3'-O-R'-dNTPs. L-3'-O-R'-dNTPs may comprise L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, or L-3'-O-R'-dCTP. In some embodiments, L-3'-O-R'-dNTPs comprise one or more of L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, and L-3'-O-R'-dCTP. In some embodiments, L-3'-O-R'-dNTPs comprise L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, and L-3'-O-R'-dCTP.

[0368] In some embodiments, L-3'-O-R'-dNTP has Formula I as defined in 5.2.2.1.1. In some embodiments, L-3'-O-R'-dNTP has any of the structures disclosed in 5.2.2.1.1.

[0369] In some embodiments, each L-nucleotide-label in the kit has a unique label. In some embodiments, some of the L-nucleotide-labels in the kit have the same label. In some embodiments, the label is a fluorescent label. In some embodiments, each L-nucleotide-label in the kit provides a unique fluorescent signal.

[0370] 6. Example

[0371] 6.1. Example 1. Synthesis of (L)-3'-O-N3-dNTP

[0372] Materials and methods

[0373] Starting materials β-L-deoxyadenosine, β-L-deoxyguanosine, β-L-deoxycytidine, and β-L-deoxythymidine are available from, for example, Chemgenes. Unless otherwise stated, all solvents and reagents are reagent-grade, commercially available, and used without further purification.

[0374] 6.1.1. Synthesis of (L) 3'-O-N3-dATP

[0375] 6.1.1.1 Scheme 1. Synthesis of (L) 3'-O-N3-dATP

[0376]

[0377] A stirred solution of the starting material (β-L-deoxyadenosine) (1.0 equivalent, 2.00 g, 7.48 mmol) was co-evaporated with anhydrous pyridine, dissolved in 15 mL of anhydrous pyridine, and sealed with a diaphragm. After stirring under argon for 5 min, trimethylsilyl chloride (TMS-Cl, 5.0 equivalent, 4.06 g, 37.4 mmol) was added via syringe. Benzoyl chloride (1.2 equivalent, 1.26 g, 8.98 mmol) was added dropwise via syringe over 20 min intervals and after 30 min, and stirring continued for 2.5 h, while a clear yellow solution formed. Then, 4 mL of H₂O was added in one addition, and after 5 min, 8 mL of ammonia solution (28%–30%) was added in one addition, followed by stirring for another 15 min. The mixture was evaporated to dryness, and the oily residue was co-evaporated twice with toluene to give a yellow solid (compound dA-1). tert-butyldimethylsilyl chloride (TBDMSCl) (765 mg; 4.92 mmol) was added to a stirred mixture of compound dA-1 (1.50 g; 3.96 mmol) and imidazole (693 mg; 9.51 mmol) in anhydrous DMF (21.0 mL). The reaction mixture was stirred at room temperature for 20 h. After evaporation, the residue was purified by rapid column chromatography using CH3OH-CH2Cl2 (1:20) as eluent to give compound dA-2 as a white solid. Acetic acid (5.5 mL) and acetic anhydride (17.6 mL) were added to a stirred solution of compound dA-2 (3.0 g; 6.38 mmol) in DMSO (12 mL). The reaction mixture was stirred at room temperature for 48 h. A saturated NaHCO3 solution (100 mL) was added and the aqueous layer was extracted with CH2Cl2 (3 × 100 mL). The combined organic extracts were washed with saturated NaHCO3 solution (100 ml) and dried over Na2SO4. After concentration, the residue was purified by rapid column chromatography (hexane / ethyl acetate, 1:1 to 1:4) to give compound dA-3 as a white powder. Cyclohexene (400 ml) and SO2Cl2 (155 ml; 1.91 mmol, redistilled) were added to a stirred solution of compound dA-3 (400 mg; 0.76 mmol) in dry CH2Cl2 (7 ml) under nitrogen. The reaction mixture was stirred at 0 °C for 2 h. The solvent was removed first under reduced pressure and then under high vacuum for 10 min. The residue was dissolved in dry DMF (5 ml) and reacted with NaN3 (400 mg; 6.6 mmol) at room temperature for 3 h. The reaction mixture was dispersed in distilled water (50 ml) and extracted with CH2Cl2 (3 × 50 ml). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure.The residue was dissolved in MeOH (5 ml) and stirred with NH4F (300 mg; 8.1 mmol) at room temperature for 24 h. The solvent was removed under reduced pressure. The reaction mixture was concentrated under reduced pressure and partitioned between H2O and CH2Cl2. The organic layers were separated and dried over Na2SO4. After concentration, the crude product was purified by rapid column chromatography (ethyl acetate / methanol, 100:0 to 98:2) to give compound dA-4 as a white powder. Compound dA-4 (123 mg; 0.3 mmol) and proton sponge (75.8 mg; 0.35 mmol) were dried overnight under vacuum in P2O5 and then dissolved in trimethyl phosphate (600 ml). Freshly distilled POCl3 (40 ml; 0.35 mmol) was then added dropwise at 0 °C, and the mixture was stirred at 0 °C for 2 h. Subsequently, at room temperature, a mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) was added in a single vortex and stirred for 30 min. Then, triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) was added, and the mixture was stirred at room temperature for 1 h. Then, concentrated NH4OH (15 ml) was added, and the mixture was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum, and the residue was diluted with 5 ml of water. The crude mixture was then purified by anion exchange chromatography on a DEAE-Sephadex A-25 at 4 °C using a gradient of TEAB (pH 8.0; 0.1–1.0 M). The crude product was further purified by reversed-phase HPLC to give (L)3'-O-N3-dATP (compound dA-5).

[0378] 6.1.2. Synthesis of (L) 3'-O-N3-dGTP

[0379] 6.1.2.1 Scheme 2. Synthesis of (L) 3'-O-N3-dGTP

[0380]

[0381]

[0382] A stirred solution of the starting material (β-L-deoxyguanosine) (1.0 equivalent, 2.00 g, 7.13 mmol) was co-evaporated with anhydrous pyridine (3 × 4 mL) and then dissolved in anhydrous pyridine (2 mL). The resulting solution was protected from moisture (drying tube), purged with argon, and placed on ice. TMS-Cl (4.51 mL, 58.4 mmol, 8.2 equivalent) was added dropwise to the ice-cold solution via a syringe. The ice bath was then removed, and the mixture was stirred for 2 hours. The solution was cooled on ice, and isobutyric anhydride (0.29 mL, 15.69 mmol, 2.2 equivalent) was added dropwise via a syringe, and the ice bath was removed. After stirring for another 2 hours at room temperature, the reaction was placed on ice again, and ice-cold water (20 mL) was slowly added, followed by concentrated ammonia solution (1.5 mL) after 15 minutes to give a final ammonia concentration of 2.5 M. The mixture was kept on ice for 30 minutes and then evaporated to dryness. The residue was co-evaporated with toluene (3 × 5 mL) to remove trace amounts of water, resuspended in MeOH, and filtered to remove the precipitate. The filtrate was then concentrated, dissolved in a small amount of MeOH, adsorbed onto silica gel, and purified by column chromatography (DCM / MeOH 95:5 to 91:9 (v / v)) to give compound dG-1 as a yellow solid. Compound dG-1 (495 mg, 1.07 mmol) was co-evaporated three times with dry pyridine, dried under high vacuum, and dissolved in an ice bath at 2 cm⁻¹. 3In dry N,N-dimethylformamide, bis(trifluoromethanesulfonic acid) di-tert-butylsilyl ester (590 mg, 1.34 mmol) was added dropwise over a 15-min time interval, and the reaction mixture was stirred at 0 °C for 30 min. Imidazole (419 mg, 6.15 mmol) was added, and the mixture was stirred at 0 °C for 15 min and then at room temperature for 15 min. Then, 241 mg of tert-butyldimethylsilyl chloride (1.59 mmol) was added, and the solution was stirred at 60 °C for another 2 h. The mixture was diluted with dichloromethane, washed with brine, dried over sodium sulfate, and evaporated. The crude product was purified by column chromatography on silica gel (methanol:dichloromethane 0:100–2:98) to a white foam (compound dG-2). Acetic acid (5.5 ml) and acetic anhydride (17.6 ml) were added to a stirred solution of compound dG-2 (3.0 g; 6.08 mmol) in DMSO (12 ml). The reaction mixture was stirred at room temperature for 48 h. A saturated NaHCO3 solution (100 ml) was added and the aqueous layer was extracted with CH2Cl2 (3 × 100 ml). The combined organic extracts were washed with a saturated NaHCO3 solution (100 ml) and dried over Na2SO4. After concentration, the residue was purified by rapid column chromatography (hexane / ethyl acetate, 1:1 to 1:4) to give compound dG-3 as a white powder. Diphenylcarbamoyl chloride (677 mg; 2.92 mmol) and DIEA (N,N-diisopropylethylamine) (1.02 ml; 5.9 mmol) were added to a stirred solution of compound dG-3 (1.0 g; 2.0 mmol) in dry pyridine (22 ml). The reaction mixture was stirred at room temperature under a nitrogen atmosphere for 3 h. The solvent was removed under high vacuum. The crude product was purified by rapid column chromatography (ethyl acetate / hexane, 1:1 to 7:3) to give compound dG-4 as a pale yellow powder. Cyclohexene (400 ml) and SO2Cl2 (155 ml; 1.91 mmol, redistilled) were added to a stirred solution of compound dG-4 (400 mg; 0.71 mmol) in dry CH2Cl2 (7 ml) under nitrogen. The reaction mixture was stirred at 0 °C for 2 h. The solvent was removed first under reduced pressure and then under high vacuum for 10 min. The residue was dissolved in dry DMF (5 ml) and reacted with NaN3 (400 mg; 6.6 mmol) at room temperature for 3 h. The reaction mixture was dispersed in distilled water (50 ml) and extracted with CH2Cl2 (3 × 50 ml). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure.The residue was dissolved in MeOH (5 ml) and stirred with NH4F (300 mg; 8.1 mmol) at room temperature for 24 h. The solvent was removed under reduced pressure. The reaction mixture was concentrated under reduced pressure and partitioned between H2O and CH2Cl2. The organic layers were separated and dried over Na2SO4. After concentration, the crude product was purified by rapid column chromatography (ethyl acetate / methanol, 100:0 to 98:2) to give compound dG-5 as a white powder. Compound dG-5 (123 mg; 0.28 mmol) and proton sponge (75.8 mg; 0.35 mmol) were dried overnight under vacuum in P2O5 and then dissolved in trimethyl phosphate (600 ml). Freshly distilled POCl3 (40 ml; 0.35 mmol) was then added dropwise at 0 °C, and the mixture was stirred at 0 °C for 2 h. Subsequently, at room temperature, a mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) was added in a single vortex and stirred for 30 min. Then, triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) was added, and the mixture was stirred at room temperature for 1 h. Then, concentrated NH4OH (15 ml) was added, and the mixture was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum, and the residue was diluted with 5 ml of water. The crude mixture was then purified by anion exchange chromatography on a DEAE-Sephadex A-25 at 4 °C using a gradient of TEAB (pH 8.0; 0.1–1.0 M). The crude product was further purified by reversed-phase HPLC to give (L)3'-O-N3-dGTP (compound dG-6).

[0383] 6.1.3. Synthesis of (L) 3'-O-N3-dCTP

[0384] 6.1.3.1 Scheme 3. Synthesis of (L) 3'-O-N3-dCTP

[0385]

[0386] The preparation procedure for (L)3'-O-N3-dCTP (compound dC-5) is similar to the synthetic scheme (5 steps) outlined above for (L)3'-O-N3-dATP. The starting material used for the synthesis of (L)3'-O-N3-dCTP is β-L-deoxycytidine (Chemgenes).

[0387] 6.1.4. Synthesis of (L) 3'-O-N3-dTTP

[0388] 6.1.4.1 Scheme 4. Synthesis of (L) 3'-O-N3-dTTP

[0389]

[0390] Tert-butyldimethylsilyl chloride (TBDMSCl) (765 mg; 4.92 mmol) was added to a stirred mixture of the starting material (β-L-deoxythymidine) (1.3 g; 3.87 mmol) and imidazole (693 mg; 9.51 mmol) in anhydrous DMF (21.0 mL). The reaction mixture was stirred at room temperature for 20 h. After evaporation, the residue was purified by rapid column chromatography using CH3OH-CH2Cl2 (1:20) as the eluent to give compound dT-1 as a white solid. Acetic acid (5.5 mL) and acetic anhydride (17.6 mL) were added to a stirred solution of compound dT-1 (3.0 g; 6.38 mmol) in DMSO (12 mL). The reaction mixture was stirred at room temperature for 48 h. A saturated NaHCO3 solution (100 mL) was added and the aqueous layer was extracted with CH2Cl2 (3 × 100 mL). The combined organic extracts were washed with saturated NaHCO3 solution (100 ml) and dried over Na2SO4. After concentration, the residue was purified by rapid column chromatography (hexane / ethyl acetate, 1:1 to 1:4) to give compound dT-2 as a white powder. Cyclohexene (400 ml) and SO2Cl2 (155 ml; 1.91 mmol, redistilled) were added to a stirred solution of compound dT-2 (400 mg; 0.76 mmol) in dry CH2Cl2 (7 ml) under nitrogen. The reaction mixture was stirred at 0 °C for 2 h. The solvent was removed first under reduced pressure and then under high vacuum for 10 min. The residue was dissolved in dry DMF (5 ml) and reacted with NaN3 (400 mg; 6.6 mmol) at room temperature for 3 h. The reaction mixture was dispersed in distilled water (50 ml) and extracted with CH2Cl2 (3 × 50 ml). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in MeOH (5 ml) and stirred with NH4F (300 mg; 8.1 mmol) at room temperature for 24 h. The solvent was removed under reduced pressure. The reaction mixture was concentrated under reduced pressure and partitioned between H2O and CH2Cl2. The organic layers were separated and dried over Na2SO4. After concentration, the crude product was purified by rapid column chromatography (ethyl acetate / methanol, 100:0 to 98:2) to give compound dT-3 as a white powder. Compound dT-3 (123 mg; 0.3 mmol) and proton sponge (75.8 mg; 0.35 mmol) were dried overnight under vacuum in P2O5 and then dissolved in trimethyl phosphate (600 ml).Freshly distilled POCl3 (40 ml; 0.35 mmol) was then added dropwise at 0 °C, and the mixture was stirred at 0 °C for 2 h. Subsequently, a mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (0.55 ml; 2.31 mmol) in anhydrous DMF (2.33 ml) was added in a single vortex at room temperature and stirred for 30 min. Triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0; 15 ml) was then added, and the mixture was stirred at room temperature for 1 h. Concentrated NH4OH (15 ml) was then added, and the mixture was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum, and the residue was diluted with 5 ml of water. The crude mixture was then purified by anion exchange chromatography on a DEAE-Sephadex A-25 at 4 °C using a gradient of TEAB (pH 8.0; 0.1–1.0 M). The crude product was further purified by reversed-phase HPLC to obtain (L) 3'-O-N3-dTTP (compound dT-4).

[0391] 6.2. Example 2. Synthesis of (L) 3'-O-R'-dNTP

[0392] Having reviewed the exemplary methods provided herein, those skilled in the art will be able to readily adapt these methods to produce compounds having various R' groups protecting 3'O. Specifically, the allyl and nitrobenzyl analogues of the (L)-3'-O-N3-dNTP described are contemplated using allyl bromide and 2-nitrobenzyl bromide, respectively, instead of the acetic acid / acetic anhydride steps in reaction schemes 1-4 above. Specific references can be found in Ju J. et al. 2006, respectively. PNAS Volume 103, Issue 52 and Wu J. et al. 2007, PNAS The methods provided in Volume 104, Issue 42, are incorporated herein by reference in their entirety (including supporting information).

[0393] 6.3. Example 3. Synthesis of (L) 3'-O-N3-dNTP-label

[0394] 6.3.1. Synthesis of (L) 3'-O-N3-dATP-ROX

[0395] 6.3.1.1 Scheme 5. Synthesis of NH2-(L)3'-O-N3-dATP

[0396]

[0397]

[0398]

[0399] 6.3.1.2 Scheme 6. Synthesis of (L) 3'-O-N3-dATP-ROX

[0400]

[0401] Azide-ROX compound (ROX-N3-linker). 2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-acetic acid linker-6 (7.0 mg, 0.019 mmol) prepared according to the literature (Milton J, Ruediger S, Liu X (2006), US Patent Application US20060160081A1) was dissolved in DMF (300 µl) and 1 M NaHCO3 aqueous solution (100 µl). A solution of ROX NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF (400 µl) was slowly added to the above reaction mixture, and then stirred at room temperature in the dark for 5 h. The crude product was purified on preparative silica gel TLC plates (CHCl3 / CH3OH, 1:4).

[0402] The L-3'-O-N3-dATP-ROX compound (L-3'-O'-N3-dATP-ROX) was added to a stirred solution of the ROX-N3-linker in dry DMF (2 ml) at room temperature. DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 µmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 µmol) were added. The reaction mixture was stirred at room temperature for 2 h. TLC showed complete conversion of the ROX-N3-linker to the compound ROX-N3-linker NHS ester, which was then used directly for coupling with L-amino-dATP (13 µmol) in NaHCO3 / Na2CO3 buffer (pH 8.7, 0.1 M) (300 µl). The reaction mixture was stirred at room temperature in the dark for 3 h. The reaction mixture was purified by preparative silica gel TLC (CH3OH / CH2Cl2, 1:1). The crude product was further purified by reversed-phase HPLC to obtain L-3'-O-N3-dATP-ROX (L-3'-O-N3-dATP-ROX).

[0403] 6.3.2. Synthesis of (L) 3'-O-N3-dGTP-Cy5

[0404] 6.3.2.1 Scheme 7. Synthesis of NH2-(L)3'-O-N3-dGTP

[0405]

[0406]

[0407] 6.3.2.2 Scheme 8. (L) 3'-O-N3-dGTP-Cy5

[0408]

[0409] Azide-Cy5 compound (Cy5-N3-linker). 2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-acetic acid linker-6 (7.0 mg, 0.019 mmol) prepared according to the literature (Milton J, Ruediger S, Liu X (2006), US Patent Application US20060160081A1) was dissolved in DMF (300 µl) and 1 M NaHCO3 aqueous solution (100 µl). A solution of Cy5 NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF (400 µl) was slowly added to the above reaction mixture, and then stirred at room temperature in the dark for 5 h. The crude product was purified on preparative silica gel TLC plates (CHCl3 / CH3OH, 1:4).

[0410] The L-3'-O-N3-dGTP-Cy5 compound (L-3'-O-N3-dGTP-Cy5) was added to a stirred solution of the Cy5-N3-linker in dry DMF (2 ml) at room temperature. DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 µmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 µmol) were added. The reaction mixture was stirred at room temperature for 2 h. TLC showed complete conversion of the Cy5-N3-linker to the compound Cy5-N3-linker NHS ester, which was then directly used for coupling with L-amino-dGTP (13 µmol) in NaHCO3 / Na2CO3 buffer (pH 8.7, 0.1 M) (300 µl). The reaction mixture was stirred at room temperature in the dark for 3 h. The reaction mixture was purified by preparative silica gel TLC (CH3OH / CH2Cl2, 1:1). The crude product was further purified by reversed-phase HPLC to obtain L-3'-O-N3-dGTP-Cy5 (L-3'-O-N3-dGTP-Cy5).

[0411] 6.3.3. Synthesis of (L) 3'-O-N3-dCTP-Bodipy-FL-510

[0412] 6.3.3.1 Scheme 9. Synthesis of NH2-(L)3'-O-N3-dCTP

[0413]

[0414]

[0415] 6.3.4. Scheme 10. Synthesis of (L) 3'-O-N3-dCTP-Bodipy-FL-510

[0416]

[0417] Azide-Bodipy-FL-510 (compound BODIPY-FL-510-N3-linker). 2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-acetic acid linker-6 (7.0 mg, 0.019 mmol) prepared according to the literature (Milton J, Ruediger S, Liu X (2006), US Patent Application US20060160081A1) was dissolved in DMF (300 µl) and 1 M NaHCO3 aqueous solution (100 µl). A solution of Bodipy-FL-510 NHS (N-hydroxysuccinimide) ester (Invitrogen) (5.0 mg, 0.013 mmol) in DMF (400 µl) was slowly added to the above reaction mixture, and then stirred at room temperature in the dark for 5 h. The crude product was purified on a preparative silica gel TLC plate (CHCl3 / CH3OH, 1:4) to obtain the BODIPY-FL-510-N3- linker.

[0418] L-3'-O-N3-dCTP-Bodipy-FL-510 (compound L-3'-O-N3-dCTP-Bodipy-FL-510). DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 µmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 µmol) were added to a stirred solution of the BODIPY-FL-510-N3-linker in dry DMF (2 ml). The reaction mixture was stirred at room temperature for 2 h. TLC showed complete conversion of the BODIPY-FL-510-N3-linker to the compound BODIPY-FL-510-N3-linker NHS ester, which was directly used for coupling with L-amino-dCTP (13 µmol) in NaHCO3 / Na2CO3 buffer (pH 8.7, 0.1 M) (300 µl). The reaction mixture was stirred at room temperature in the dark for 3 h. The reaction mixture was purified by preparative silica gel TLC (CH3OH / CH2Cl2, 1:1). The crude product was further purified by reversed-phase HPLC to give L-3'-O-N3-dCTP-Bodipy-FL-510.

[0419] 6.3.5. Synthesis of (L) 3'-O-N3-dUTP-R6G

[0420] 6.3.5.1 Scheme 11. Synthesis of NH2-(L)3'-O-N3-dUTP

[0421]

[0422]

[0423] 6.3.5.2 Scheme 12. Synthesis of (L) 3'-O-N3-dUTP-R6G

[0424]

[0425] Azide-R6G (compound R6G-N3-linker). 2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-acetic acid (7.0 mg, 0.019 mmol) prepared according to the literature (Milton J, Ruediger S, Liu X (2006), US Patent Application US20060160081A1) was dissolved in DMF (300 µl) and 1M NaHCO3 aqueous solution (100 µl). A solution of R6G NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF (400 µl) was slowly added to the above reaction mixture, and then stirred at room temperature in the dark for 5 h. The crude product was purified on preparative silica gel TLC plates (CHCl3 / CH3OH, 1:4).

[0426] L-3'-O-N3-dUTP-R6G. DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 µmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 µmol) were added to a stirred solution of the R6G-N3-linker in dry DMF (2 ml). The reaction mixture was stirred at room temperature for 2 h. TLC showed complete conversion of the R6G-N3-linker to the compound R6G-N3-linker NHS ester, which was directly used for coupling with L-amino-dUTP (13 µmol) in NaHCO3 / Na2CO3 buffer (pH 8.7, 0.1 M) (300 µl). The reaction mixture was stirred at room temperature in the dark for 3 h. The reaction mixture was purified by preparative silica gel TLC (CH3OH / CH2Cl2, 1:1). The crude product was further purified by reversed-phase HPLC to obtain L-3'-O-N3-dUTP-R6G (L-3'-O-N3-dUTP-R6G).

[0427] 6.4. Example 4. Synthesis of (L) 3'-O-allyl-dNTP-allyl-labeled

[0428] Having reviewed the exemplary methods provided herein, those skilled in the art will be able to readily adapt the provided methods to generate various R' groups with protected 3'O and alternative cleavable linkers according to the following scheme (e.g., incorporated by Ju J. et al. 2006, PNASThe compounds reported in Volume 103, Issue 52 (allyl-fluorescent linker). Specifically, the allyl analogues of the described (L)-3'-O-N3-dNTP-NH2 compounds are conceived using allyl bromide instead of the acetic acid / acetic anhydride steps in reaction schemes 5, 7, 9, and 11 above. See Ju J. et al. 2006 for details. PNAS The methods provided in Volume 103, Issue 52, the entire contents of which (including supporting information) are incorporated herein by reference.

[0429]

[0430]

[0431]

[0432] 6.5. Example 5. Synthesis of mirror polymerase

[0433] The amino acid sequence of the 9°N polymerase is divided into multiple fragments. Each peptide fragment is synthesized using a solid-phase peptide synthesis method (Fmoc-SPPS) based on a strategy using a 9-fluorenylmethoxycarbonyl (Fmoc) group as a protecting group. Once synthesized, the peptide fragments are separated and purified using semi-preparative grade reversed-phase high-performance liquid chromatography (RP-HPLC). The separated peptide fragments are ligated using a natural chemical ligation method from the C-terminus to the N-terminus. After the chemical ligation reaction is complete, the target product is separated using semi-preparative grade RP-HPLC.

[0434] The synthesized mirror-image polymerase was folded and refolded. Circular dichroism and mass spectrometry were used to confirm that the mirror-image polymerase folded correctly.

[0435] 6.6. Example 6. Activity of mirror polymerase

[0436] The mirror polymerase was mixed with (i) polymerase reaction buffer; (ii) L-primer; (iii) L-polynucleotide template; and (iv) four 0.4 mM (L) dNTPs. The reaction mixture was incubated at 37 °C for 4 hours, and the reaction was terminated by adding 1 μl of 0.5 M EDTA. The activity of the mirror polymerase was measured based on the production of a polynucleotide fragment complementary to the template or by using a double-stranded DNA intercalation dye (e.g., EvaGreen dye) and measuring an increase in fluorescence.

[0437] 6.7. Example 7. Mirror Polymerase Chain Reaction

[0438] The mirror polymerase was mixed with (i) polymerase reaction buffer; (ii) 2.5 μM L-primers; (iii) 2.5 μM L-polynucleotide template; and (iv) four 0.4 mM (L) dNTPs. The initial cycle consisted of: 5 min at 95 °C, 5 min at 50 °C (during which polymerase and BSA were added), and 5 min at 70 °C. Subsequent PCR cycles consisted of: 1 min at 93 °C, 1 min at 50 °C, and 5 min at 70 °C. After 0, 13, 23, and 40 cycles, 20 μL of a 100 μL volume was collected and subjected to agarose gel electrophoresis with ethidium bromide present to quantify the template sequence amplification. The reaction produced a polynucleotide fragment complementary to the template.

[0439] 6.8. Example 8. A general procedure for specific single-base extension, deprotection, and restart of extension of L-primers using (L)-3'-OR-dNTPs, (L)-3'-OR-dNTPs-R-labeled, (L)-ddNTPs-R2-labeled, and -D-polymerase to illustrate sequencing-by-synthesis (SBS).

[0440] To obtain de novo DNA sequencing data on L-primers / L-DNA templates immobilized on solid surfaces, precise and specific single-base extensions were first validated in each polymerase extension reaction using a combination of solution A (3'-O-N3-dCTP (3 mM), 3'-O-N3-dTTP (3 mM), 3'-O-N3-dATP (3 mM), and 3'-O-N3-dGTP (0.5 mM)) and solution B (ddCTP-N3-Bodipy-FL-510 (50 nM), ddUTP-N3-R6G (100 nM), ddATP-N3-ROX (200 nM), and ddGTP-N3-Cy5 (100 nM). For example, along with a modified L-nucleotide reversible terminator, 60 pmol of self-initiated DNA template, 1X Thermopol II reaction buffer, 40 nmol of MnCl2, and 1 unit of D-polymerase were added to a total reaction volume of 20 ml. The reaction consisted of incubation at 94 °C for 5 min, at 4 °C for 5 min, and at 65 °C for 20 min. Subsequently, the extension products were analyzed by fluorescence-based gel electrophoresis (and alternatively, MALDI-TOF MS could be used as an alternative) to confirm the specific incorporation of the correct nucleotides. For the cleavage of DNA extension products carrying 3'-O-N3-dNTP and ddNTP-N3-fluorophores, the DNA product was resuspended in 50 ml of 100 mM TCEP solution (pH 9.0) at 65 °C for 15 min, and then analyzed by gel electrophoresis or MALDI-TOF MS to confirm the cleavage of the R-labeled / protecting group, thus allowing the restart of the next base extension. The above describes a complete single cycle of SBS (extension, label detection, and cleavage).

[0441] Typically, separate solutions are used in the polymerase extension reaction: "Solution A" consists of four L-3'-O-R'-dNTPs (each with dATP, dTTP, dGTP, or dCTP) and "Solution B" consists of four L-ddNTP-R2-labeled molecules (each with ddATP, ddTTP, ddGTP, or ddCTP). Solutions A and B are mixed with mirror polymerase, L-primers, buffer, and L-polynucleotide template at specific ratios (i.e., 7:3 v / v, 9:1 v / v), and the mixture is incubated for multiple cycles of sequencing-by-synthesis (SBS). During the SBS cycles, the mirror polymerase synthesizes the complementary sequence to the L-polynucleotide using a combination of a 3'-capped nucleotide reversible terminator (L-3'-O-R'-dNTP) and a cleavable fluorescent dideoxynucleotide (L-ddNTPs-R2-labeled). The replication product terminated by ddNTPs (L-ddNTPs-R2-labeled) is detected using a signal from a label specific to ddATP, ddTTP, ddGTP, or ddCTP. Following signal detection, the 3'-OH capping group (R' group) from the incorporated nucleotide reversible terminator (L-3'-O-R'-dNTP) and the label from the DNA product terminated by ddNTPs (L-ddNTPs-R2-labeled) are cleaved, and the polymerase reaction is restarted. Since each label conjugated to dATP, dTTP, dGTP, or dCTP is unique, the fluorescent signal from the label indicates the nucleotide corresponding to the termination site. Based on the signal, the sequence of the L-polynucleotide template is determined.

[0442] L-polynucleotide templates are also sequenced using four L-3'-O-R'-dNTP-R2-labels (each with dATP, dTTP, dGTP, or dCTP). The L-polynucleotide template is mixed with the four L-3'-O-R'-dNTP-R2-labels (each with dATP, dTTP, dGTP, or dCTP), L-primers, D-polymerase, and buffer under similar reaction conditions described above. The mixture is incubated for multiple cycles of sequencing-by-synthesis (SBS). During the SBS step, the L-3'-O-R'-dNTP-R2-labels are incorporated into the synthetic product. The L-3'-O-R'-dNTP-R2-label comprises a 3'-OH capping chemical moiety (R') and a label tethered to a base via a chemically cleavable linker (R2). After incorporation, a fluorescent signal from the label is detected, and then the 3'-capped portion (R') on the reaction product and the label are cleaved to restart the polymerase reaction. Since each label conjugated to dATP, dTTP, dGTP, or dCTP is unique, the fluorescent signal from the label indicates the nucleotide corresponding to the termination site. Based on the signal, the sequence of the L-polynucleotide template is determined.

[0443] 6.9. Example 9. Sequencing of L-polynucleotides using a method similar to Sanger sequencing

[0444] L-polynucleotide templates are sequenced using four L-ddNTP-R2-labels (each with ddATP, ddTTP, ddGTP, or ddCTP) (similar to Sanger sequencing). The four L-ddNTP-R2-labels are mixed with the L-polynucleotide template, L-primers, the four L-dNTPs (L-dATP, L-dGTP, L-dCTP, and L-dTTP), D-polymerase, and buffer under similar reaction conditions described above. In the mixture, the L-ddNTP-R2-labels are added in a much smaller amount compared to the L-dNTPs. PCR (cycle sequencing – generation of DNA ladder bands) is performed using the mixture. Replication products are separated by size using gel electrophoresis, and signals from the L-ddNTPs-R2-labels incorporated into the replication products are detected. Since each label conjugated to dATP, dTTP, dGTP, or dCTP is unique, the fluorescent signal from the label indicates the nucleotide corresponding to the termination site. The sequence of the L-polynucleotide template was determined based on fluorescence signal and fragment mobility (based on size).

[0445] 6.10. Example 10. Complete synthesis of (L)-3'-O-azidomethyl-dNTP

[0446] As shown in schemes 13–16, four target molecules and their synthetic routes were designed. L-3'-O-N3-dTTP (also known as L-3'-O-azidomethyl-dTTP) (dT-4) and L-3'-O-N3-dCTP (dC-5) were prepared and synthesized via... 1 H, 31 Characterized by P NMR and HRMS. For the synthesis of L-3'-O-N3-dATP (dA-5) and L-3'-O-N3-dGTP (dG-6), we have synthesized intermediates dA-3 and dG-5, respectively.

[0447] Materials and methods

[0448] Unless otherwise stated, all solvents and reagents are reagent-grade, commercially available, and used without further purification. All chemicals were purchased from Sigma-Aldrich, Fisher Scientific, TCI, etc. 1 H NMR from Bruker Ascend, Chapman University TMRecorded on a (400 MHz) spectrometer and reported in parts per million (ppm) of CDCl3 (7.26 ppm) or D2O. Data are reported as follows: (s = singlet, d = doublet, t = triplet, td = triplet-doublet, q = quartet, m = multiplet, dt = doublet-triplet, dd = doublet-doublet, J = coupling constant, in Hz, integral). Proton decoupling... 31 P NMR spectra from Bruker Ascend, Chapman University TM Recorded on a 121.4 MHz spectrometer. High-resolution mass spectrometry (HRMS) was obtained from the Analytical Chemistry Instrumentation Facility of Chapman University School of Pharmacy and University of California, Riverside. Starting materials β-L-deoxythymidine, β-L-deoxycytidine, β-L-deoxyadenosine, and β-L-deoxyguanosine were purchased from Chemgenes. Analytical (Polaris 180A C18-A, 4.6 x 250 mm, 5 μm) and semi-preparative (Polaris 180A C18-A, 4.6 x 250 mm, 5 μm) HPLC columns were purchased from Agilent. 3'-O-modified nucleotides were purified by reversed-phase HPLC on a 4.6 x 250 mm C18 column (Polaris) using the following mobile phases: A, 25 mM TEAB buffer in water; B, 25 mM TEAB buffer in acetonitrile. Elution was performed under isocratic conditions as described in each procedure.

[0449] 6.10.1. Synthesis of (L) 3'-O-azidomethyl-dTTP

[0450] 6.10.1.1 Scheme 13. Synthesis of (L) 3'-O-N3-dTTP (dT-4)

[0451]

[0452] 6.10.1.1.1 Experimental Procedure:

[0453] dT-1 synthesis: At 0°C, under nitrogen atmosphere, to L-Deoxythymidine (1.5 g, 6.18 mmol) was added to a solution of anhydrous N,N-dimethylformamide (DMF) (37.5 mL) with imidazole (633 mg, 9.30 mmol) and tert-butyldimethylsilyl chloride (1.02 g, 6.81 mmol). After stirring at room temperature for 3 h, ice-cold water was added to the solution and extraction was performed with EtOAc (2 × 50 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was dissolved in CH3OH and silica gel was added. The solution was dried under reduced pressure until dry. The resulting residue was purified by column chromatography (2:98 to 5:95, CH3OH–CH2Cl2) to give dT-1 (1.8 g, 82%) as a white solid. R f 0.35 (1:19 CH3OH–CH2Cl2). The product was confirmed by TLC.

[0454] Synthesis of dT-2: Acetic acid (9 ml) and acetic anhydride (27 ml) were added to a stirred solution of dT-1 (1.9 g; 5.33 mmol) in DMSO (18 ml) at room temperature. The reaction mixture was stirred at room temperature for 48 h. At 0 °C, a saturated NaHCO3 solution was added and stirred for 30 min, and the aqueous layer was extracted with CH2Cl2 (2 × 100 ml). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by rapid column chromatography (1:1, hexane–EtOAc) to give dT-2 (2.0 g, 90%) as a pale yellow syrup. R f 0.55 (1:1 hexane–EtOAc); 1 H NMR (400 MHz, CDCl3): δ 9.36 (s, 1H, N H ), 7.48 (d, 1H, 4 J = 1.1 Hz, H-6), 6.31 (dd, 1H, J 1',2'a = 5.6 Hz, J 1',2'b = 8.6 Hz, H-1'), 4.68 (d, 1H, J gem = 11.8 Hz, C H 2 S), 4.61 (d, 1H, J gem = 11.8 Hz, C H2 S), 4.47 (app dt, 1H, J 3',2'b = 5.9 Hz, J 3',2'a =1.9 Hz, H-3'), 4.12–4.08 (m, 1H, H-4'), 3.89 (dd, 1H, J 5'a,4' = 2.6 Hz, J gem =11.3 Hz, H-5'a), 3.80 (dd, 1H, J 5'a,4' = 2.9 Hz, J gem =11.3 Hz, H-5'b), 2.41(ddd, 1H, J 2'a,3' = 1.9 Hz, J 2'a,1' = 5.6 Hz, J gem = 13.6 Hz, H-2'a), 2.16 (s, 3H, SC H 3 ), 1.99 (ddd, 1H, J 2'b,3' = 5.9 Hz, J 2'b,1' = 8.6 Hz, J gem = 13.6 Hz, H-2'b),1.92 (d, 3H, 4 J = 1.1 Hz, C H 3 ), 0.93 (s, 9H, (C H 3 )3CSi), 0.12 (s, 6H, (C H 3 )2Si));HRMS (ESI) m / z for C 18 H 33 Calculated value of N₂O₅SSi [M + H] + 417.1879; measured value 417.1881.

[0455]

[0456] Synthesis of dT-3: Cyclohexene (2.1 mL) and SO2Cl2 (1.0 M DCM solution) (6.48 mL, 6.48 mmol) were added to a stirred solution of dT-2 (2.0 g, 4.80 mmol) in dry CH2Cl2 (45 mL). The reaction mixture was stirred at 0 °C for 3 h. Volatile substances were removed under reduced pressure. The residue was dissolved in dry DMF (30 mL) and reacted with NaN3 (1.88 g, 28.8 mmol) at room temperature for 2 h. The reaction mixture was dispersed in cold distilled water (100 mL) and extracted with EtOAc (2 × 200 mL). The combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure. The resulting residue was dissolved in CH3CN (10 mL) and reacted with 2 M HCl (2-3 mL) at 0 °C for 5 h. A saturated Na₂CO₃ solution was added and the mixture was extracted with CH₂Cl₂ (2 × 50 mL), dried over Na₂SO₄, and concentrated. The organic layer was washed with water, dried over Na₂SO₄, and concentrated. The resulting residue was purified by rapid column chromatography (hexane / ethyl acetate, 3:7 to 1:4) to give dT-3 as a white powder. R f 0.20 (1:4 hexane–EtOAc); 1 H NMR (400 MHz, CDCl3): δ 8.63 (s, 1H, N H ), 7.38 (d, 1H, 4 J = 1.1 Hz, H-6), 6.13 (app t, 1H, J 1',2'a = J 1',2'b = 7.0 Hz, H-1'), 4.77 (d, 1H, J gem = 9.0 Hz, C H 2 N3), 4.70 (d, 1H, J gem = 9.0 Hz, C H 2 N3), 4.50 (app dt, 1H, J 3',2'b = 6.0 Hz, J 3',2'a = 3.5 Hz, H-3'),4.16–4.12 (m, 1H, H-4'), 3.98 (dd, 1H, J5'a,4' = 2.7 Hz, J gem =12.0 Hz, H-5'a),3.84 (dd, 1H, J 5'a,4' = 2.8 Hz, J gem = 12.0 Hz, H-5'b), 2.51–2.38 (m, 3H, H-2'a,H-2'b, 5'-OH), 1.94 (d, 3H, 4 J = 1.1 Hz, C H 3 ); HRMS (ESI) m / z for C 11 H 16 Calculated value of N5O5S [M + H] + 298.1151; Measured value 298.1144

[0457]

[0458] Synthesis of dT-4: dT-3 (120 mg, 0.403 mmol) was dried overnight in a vacuum desiccator using P2O5. POCl3 (94.3 uL, 1.00 mmol) was added dropwise to a solution of dT-3 in trimethyl phosphate (5 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h, and then a thoroughly vortexed mixture of tributylammonium pyrophosphate (552 mg) and tributylamine (738 uL, 3.10 mmol) in anhydrous DMF (2 mL) was added. The mixture was stirred at room temperature for 1 h, and then 0.1 M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.5, 20 mL) was added, and the mixture was stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure, and the residue was diluted with 10 mL of water. The crude mixture was extracted with CH2Cl2 (2 × 10 mL), and the aqueous layer was concentrated under reduced pressure. The residue was then purified by anion exchange chromatography on a DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.5; 0.1–0.8 M). Fractions containing the product (0.3 M–0.4 M) were collected and concentrated under reduced pressure. The residue was diluted with ddH2O and subjected to C18 HPLC (4% isocratic, 25 mM TEAB in acetonitrile: 25 mM TEAB in water). The product (retention time: 19 min) was collected and concentrated under reduced pressure to give dT-4 (14 mg, 6.5%) as a syrup. R f0.28 (1:2 TEAB–ACN); 1 H NMR (400MHz, D2O): δ 7.61 (d, 1H, 4 J = 1.0 Hz, H-6), 6.19 (dd, 1H, J 1',2'a = 5.8 Hz, J 1',2'b = 8.6 Hz, H-1'), 4.74 (d, 1H, J gem = 8.8 Hz, C H 2 N3), 4.69 (d, 1H, J gem =8.8 Hz, C H 2 N3), 4.53–4.48 (m, 1H, H-3'), 4.25–4.20 (m, 1H, H-4'), 4.12–4.00 (m, 2H, H-5'a, H-5'b), 2.37 (ddd, 1H, J 2'a,1' = 5.8 Hz, J 2'a,3' = 2.0 Hz, J gem =14.4 Hz, H-2'a), 2.25 (ddd, 1H, J 2'a,1' = 8.8 Hz, J 2'a,3' = 5.9 Hz, J gem = 14.4Hz, H-2'b), 1.78 (d, 3H, 4 J = 1.0 Hz, C H 3 ); 31 P NMR (121.4 MHz, D2O): δ –10.95(bs, 1P), –11.75 (d, J = 20.0 Hz, 1P), –23.36 (bs, 1P); HRMS (ESI) m / z for C 11 H 17 N5O 14 P3 – The calculated value [M – H]– 535.9990; measured value 535.9993.

[0459] dT-4:

[0460] 6.10.2. Synthesis of L-3'-O-azidomethyl-dCTP:

[0461] 6.10.2.1 Scheme 14. Synthesis of L-3'-O-N3-dCTP(dC-5)

[0462]

[0463]

[0464] dC-1 synthesis: Under nitrogen atmosphere, -L-deoxycytidine (1 g, 4.40 mmol) was co-evaporated with anhydrous pyridine (2 × 10 mL) and then dissolved in anhydrous pyridine (15 mL). Trimethylsilyl chloride (TMSCl, 2.79 mL, 22.0 mmol) was slowly added and the mixture was stirred at room temperature for 1 h, followed by the addition of benzoyl chloride (2.56 mL, 22.0 mmol) and stirring at room temperature for another 24 h. After cooling to 0 °C, water (10 mL) was added and the mixture was stirred at 0 °C for 20 min. Then, concentrated ammonia solution (15 mL) was added and the solution was stirred for another 1 h while warming to room temperature. The solvent was evaporated under reduced pressure, and the resulting crude product was purified by column chromatography (19:1, CH2Cl2–CH3OH) to give dC-1 (520 mg, 36%) as a white solid. R f 0.35 (1:19 CH3OH–CH2Cl2).

[0465] Synthesis of dC-2: Imidazole (172 mg, 2.53 mmol) and tert-butyldimethylsilyl chloride (204 mg, 1.35 mmol) were added to a solution of dC-1 (280 mg, 0.84 mmol) in anhydrous DMF (5.0 mL) at 0 °C. The solution was stirred at 0 °C under nitrogen for 3 hours. After the reaction was complete (monitored by TLC), cold water (10 mL) was added and the mixture was extracted with EtOAc (3 × 20 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (19:1 to 9:1, CH2Cl2–CH3OH) to give dC-2 (280 mg, 74%) as a colorless oil. R f0.32 (1:19 CH3OH–CH2Cl2).

[0466] Synthesis of dC-3. Acetic acid (3 ml) and acetic anhydride (9 ml) were added to a stirred solution of dC-2 (240 mg, 0.538 mmol) in DMSO (6 ml) at room temperature. The reaction mixture was stirred at room temperature for 72 h. At 0 °C, a saturated NaHCO3 solution was added and stirred for 30 min, and the aqueous layer was extracted with CH2Cl2 (2 × 30 ml). The combined organic extracts were dried over Na2SO4, filtered, and concentrated. The crude product was purified by rapid column chromatography (1:1, hexane–EtOAc) to give dC-3 (163 mg, 60%) as a white powder. R f 0.22 (1:1 hexane–EtOAc); 1 H NMR (400 MHz, CDCl3): δ8.42 (d, 1H, J = 7.5 Hz), 7.92 (d, 2H, J = 7.5 Hz, ArH), 7.67–7.58 (m, 1H,ArH), 7.56–7.38 (m, 3H, ArH), 6.29 (app t, 1H, J 1',2'a = J 1',2'b = 6.0 Hz, H-1'), 4.69 (d, 1H, J gem = 11.7 Hz, C H 2 S ), 4.61 (d, 1H, J gem = 11.7 Hz, C H 2 S ), 4.50(app dt, 1H, J 3',2'a = J 3',4' = 4.0 Hz, J 3',2'b = 6.0 Hz, H-3'), 4.21–4.16 (m, 1H,H-4'), 4.00 (dd, 1H, J 5'a,4' = 3.2 Hz, J gem=11.8 Hz, H-5'a), 3.84 (dd, 1H, J 5'a,4' = 2.6 Hz, J gem = 11.8 Hz, H-5'b), 2.79 (ddd, 1H, J 2'a,1' = 6.0 Hz, J 2'a,3' =4.0 Hz, J gem = 13.7 Hz, H-2'a), 2.21–2.13 (m, 4H, H-2'b, SC H 3 ), 0.95 (s, 9H, (C H 3 )3CSi); 0.15 (s, 3H, C H 3 Si), 0.14 (s, 3H, C H 3 Si); HRMS (ESI) m / z for C 24 H 36 Calculated value of N3O5SSi [M + H] + 506.2145; measured value 506.2150.

[0467] Synthesis of dC-4. Cyclohexene (200 μL) was added to a stirred solution of dC-3 (220 mg, 0.67 mmol) in dry CH2Cl2 (6 mL), and a CH2Cl2 solution of SO2Cl2 (1.0 M, 0.6 mL) was added. After stirring at 0 °C for 1.5 h, the volatiles were removed under reduced pressure. At room temperature, NaN3 (169 mg, 2.61 mmol) was added to a solution of the residue in dry DMF (3 mL) for 3 h. The reaction mixture was dispersed in cold distilled water (30 mL) and extracted with EtOAc (2 × 30 mL). The combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure. The resulting residue was subjected to TBDMS removal by 2 M HCl for 1–2 h at room temperature. The mixture was neutralized with saturated NaHCO3 solution and diluted with EtOAc. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by rapid column chromatography (1:2, hexane–EtOAc to 100% EtOAc) to give dC-4 (73 mg, 56%) as a colorless oil. R f0.3 (1:4 hexane–EtOAc); 1 H NMR (400 MHz, CDCl3): δ 8.99 (bs, 1H, N H ), 8.32 (d, 1H, J = 7.5 Hz), 7.87 (d, 2H, J = 7.5 Hz, ArH), 7.65–7.53 (m, 2H), 7.52–7.45 (m, 2H, ArH), 6.18 (app t, 1H, J 1',2'a = J 1',2'b = 6.3 Hz, H-1'), 4.79 (d, 1H, J gem = 9.2 Hz, C H 2 N3), 4.68 (d, 1H, J gem = 9.2 Hz, C H 2 N3), 4.52 (app dt, 1H, J 3',2'a = J 3',4' = 3.9 Hz, J 3',2'b = 6.3 Hz, H-3'), 4.26–4.22 (m, 1H, H-4'), 4.04 (dd, 1H, J 5'a,4' = 2.8 Hz, J gem = 12.0 Hz, H-5'a), 3.89 (dd, 1H, J 5'a,4' = 2.8 Hz, J gem = 12.0 Hz, H-5'b), 3.45 (bs, 1H, 5'-OH), 2.68 (ddd, 1H, J 2'a,1' = 6.3 Hz, J 2'a,3' = 3.9 Hz, J gem = 13.5 Hz, H-2'a), 2.44 (app dt, 1H, J2'b,1' = J 2'b,3' = 6.3 Hz, J gem = 13.5Hz, H-2'b); HRMS (ESI) m / z for C 17 H 19 Calculated value of N6O5 [M + H] + 387.1417; measured value 387.1426.

[0468] Synthesis of dC-5: dC-4 (100 mg, 0.259 mmol) was dried overnight in a vacuum desiccator using P2O5. POCl3 (60.5 μL, 0.647 mmol) was added dropwise to a solution of dC-4 in trimethyl phosphate (5 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h, and then a thoroughly vortexed mixture of tributylammonium pyrophosphate (355 mg, 0.647 mmol) and tributylamine (473.8 μL, 1.99 mmol) in anhydrous DMF (2 mL) was added. The mixture was stirred at room temperature for 1.5 h, and then 0.1 M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.5, 0.1 M, 15 mL) was added, and the mixture was stirred at room temperature for 1 h. Then, concentrated ammonium hydroxide (10 mL) was added to the mixture, and the mixture was stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure, and the residue was diluted with 30 mL of water. The crude mixture was extracted with CH2Cl2 (2 × 20 mL) and the aqueous layer was concentrated under reduced pressure. The residue was then purified by anion exchange chromatography on a DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.5; 0.2–0.8 M). Fractions containing the product (0.3 M – 0.4 M) were collected and concentrated under reduced pressure. The residue was diluted with ddH2O and subjected to C18 HPLC (2% isocratic, 25 mM TEAB in ACN : 25 mM TEAB in water). The product (retention time: 13.2 min) was collected and concentrated under reduced pressure to give dC-5 (18 mg, 13.4%) as a syrup. R f 0.43 (1:2 TEAB–ACN); 1 H NMR (400MHz, D2O): δ 8.00–7.88 (m, 1H), 6.23 (dd, 1H, J 1',2'a = 5.6 Hz, J 1',2'b= 7.9 Hz,H-1'), 6.18–6.06 (m, 1H) 4.85–4.74 (m, 2H, C H 2 N3), 4.56–4.51 (m, 1H, H-3'), 4.35–4.29 (m, 1H, H-4'), 4.18–4.09 (m, 2H, H-5'a, H-5'b), 2.55–2.46 (m, 1H,H-2'a), 2.31–2.19 (m, 1H, H-2'b); HRMS (ESI) m / z for C 10 H 16 N5O 13 P3 – The calculated value [M –H] – 520.9994; Measured value 520.9990.

[0469] 6.10.3. Synthesis of L-3'-O-azidomethyl-dATP:

[0470] 6.10.3.1 Scheme 15. Synthesis of L-3'-O-N3-dATP (dA-5)

[0471]

[0472] Experimental procedure:

[0473] dA-1 synthesis: -L-deoxyadenosine (2.0 g, 7.96 mmol) was co-evaporated with anhydrous pyridine (twice: 10 mL + 10 mL) and dissolved in anhydrous pyridine (20 mL). The resulting solution was cooled to 0 °C and trimethylchlorosilane (TMSCl, 5.06 mL, 39.8 mmol) was added dropwise via syringe. The mixture was stirred at 0 °C for 1 hour. Benzoyl chloride (4.62 mL, 39.8 mmol) was added dropwise via syringe and the ice bath was removed. After stirring at room temperature for 3 hours, the flask was placed on an ice bath and concentrated ammonia solution (15 mL) was added. The solution was stirred at 0 °C for 30 min and then evaporated to dryness. The residue was dissolved in CH3OH and silica gel was added. The mixture was dried under reduced pressure. The residue was purified by column chromatography (49:1 to 19:1, CH2Cl2–CH3OH) to give dA-1 (2.12 g, 75%) as a white solid.

[0474] Synthesis of dA-2: Imidazole (608 mg, 8.95 mmol) and tert-butyldimethylsilyl chloride (989 mg, 6.56 mmol) were added to a solution of dA-1 (2.12 g, 5.98 mmol) in anhydrous DMF (30 mL) at 0 °C under nitrogen atmosphere. The reaction mixture was stirred at 0 °C for 3 hours. After the reaction was complete, cold water (50 mL) was added and the mixture was extracted with EtOAc (3 × 50 mL). The combined organic layers were dried over anhydrous Na₂SO₄, filtered, and concentrated. The resulting residue was purified by column chromatography (1:2, hexane–EtOAc to 100% EtOAc) to give compound dA-2 (1.92 g, 69%) as a colorless oil.

[0475] Synthesis of dA-3: Acetic acid (12 ml) and acetic anhydride (36 ml) were added to a stirred solution of dA-2 (1.8 g; 3.83 mmol) in DMSO (24 ml) at room temperature. After stirring at room temperature for 72 hours, a saturated NaHCO3 solution was added to the solution at 0 °C and stirred for 30 min. The mixture was extracted with CH2Cl2 (2 × 100 ml). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The residue was purified by rapid column chromatography (1:1, hexane–EtOAc) to give dA-3 (1.27 g, 63%) as a white foam. R f 0.28 (1:7 hexane–EtOAc); 1 H NMR (400 MHz, CDCL3): δ9.28 (bs, 1H, N H ), 8.77 (s, 1H), 8.33 (s, 1H), 8.02 (d, 2H, J = 7.5 Hz, ArH),7.62–7.55 (m, 1H, ArH), 7.53–7.47 (m, 2H, ArH), 6.51 (dd, 1H, J 1',2'a = 7.2 Hz, J 1',2'b = 6.0 Hz, H-1'), 4.74–4.64 (m, 3H, C H 2 S, H-3'), 4.25–4.18 (m, 1H, H-4'), 3.89 (dd, 1H, J 5'a,4' = 4.4 Hz, Jgem =11.0 Hz, H-5'a), 3.82 (dd, 1H, J 5'a,4' = 3.3 Hz, J gem = 11.0 Hz, H-5'b), 2.79 (ddd, 1H, J 2'a,1' = 7.2 Hz, J 2'a,3' = 6.0Hz, J gem = 13.7 Hz, H-2'a), 2.63 (ddd, 1H, J 2'b,1' = 6.0 Hz, J 2'b,3' = 3.0 Hz, J gem = 13.7 Hz, H-2'b), 2.18 (s, 3H, SC H 3 ), 0.91 (s, 9H, (C H 3 )3CSi); 0.10 (s, 6H,(C H 3 ) 2 Si); HRMS (ESI) m / z for C 25 H 36 Calculated value of N5O4SSi [M + H] + 530.2157; measured value 530.2132.

[0476] Synthesis of dA-4: SO2Cl2 (1.0 M DCM solution) (0.84 mL, 0.849 mmol, 1.5 equivalent) was added to a stirred solution of dA-3 (300 mg; 1.0 equivalent, 0.566 mmol) in dry CH2Cl2 (10 mL) at –30 °C. The reaction mixture was stirred at –30 °C for 20–30 min. After starting material depletion (TLC), cyclohexene (400 μL) was added, and the mixture was allowed to reach room temperature. Volatiles were removed under reduced pressure (water bath temperature <10 °C) and dried under high pressure for 10 min. The residue was dissolved in dry DMF (8 mL) and reacted with NaN3 (320 mg, 4.924 mmol, 8.7 equivalent) at room temperature for 3 h. The reaction mixture was dispersed in cold distilled water (10 mL) and extracted with EtOAc (2 × 10 mL). The combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure. The resulting residue was used in the next reaction without further purification. The crude product (300 mg) was dissolved in dry THF (6 mL) and reacted with 1 M TBAF (0.84 mL, 0.849 mmol, 1.5 equivalence) at 0 °C for 1 h. The reaction was quenched by adding MeOH (5 mL) and the solvent was removed under reduced pressure. The residue was suspended in water (5 mL) and extracted with CH2Cl2 (2 × 5 mL). The combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure. The crude product was purified by rapid column chromatography (hexane / ethyl acetate, 1:4 to 100% ethyl acetate) to give dA-4 (203 mg, 87.5%) as a white foam. R f 0.40 (2:98 MeOH–EtOAc); 1 H NMR (500 MHz, CDCl3): δ 9.09 (s, 1H), 8.79 (s, 1H), 8.08 (s, 1H), 8.03 (d, 2H, J = 7.5 Hz),7.65 – 7.59 (m, 1H), 7.54 (t, 1H, J = 7.7 Hz), 6.34 (dd, 1H, J = 9.5, 5.5Hz), 5.83 (d, 1H, J = 10.2 Hz), 4.78 (s, 1H), 4.68 (d, 1H, J = 5.4 Hz), 4.37(s, 1H), 4.02 (d, 1H, J= 12.9 Hz), 3.86 – 3.78 (m, 1H), 3.16 – 3.06 (m, 1H), 2.52 (dd,1H, J = 13.7, 5.5 Hz).

[0477] Synthesis of dA-5: dA-4 (60 mg, 0.146 mmol) and proton sponge (37.59 mg, 0.175 mmol) were dried overnight in a vacuum desiccator using P2O5. POCl3 (21 μL, 0.219 mmol) was added dropwise to a solution of dA-4 in trimethyl phosphate (0.49 mL) at 0 °C. The mixture was stirred at 0 °C for 1 h, and then a thoroughly vortexed mixture of tributylammonium pyrophosphate (304 mg) and tributylamine (0.27 mL, 2.31 mmol) in anhydrous DMF (1.2 mL) was added. The mixture was stirred at room temperature for 20 min, and then 0.1 M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.0, 0.1 M, 4 mL) was added, and the mixture was stirred at room temperature for 3 h. Then, concentrated ammonium hydroxide (15 mL) was added to the mixture, and the mixture was stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure, and the residue was diluted with 30 mL of water. The crude mixture was extracted with CH2Cl2 (2 × 20 mL), and the aqueous layer was concentrated under reduced pressure. The residue was then purified by preparative HPLC (C18 column) followed by anion exchange chromatography on a DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.0; 0.05–1.0 M). Fractions containing the product (0.3 M – 0.4 M) were collected, concentrated under reduced pressure, and lyophilized with ddH2O to give dA-5 (50 mg, 62%) as a foamy solid. 1 H NMR (500 MHz, D2O): δ8.36 (s, 1H), 8.08 (s, 1H), 6.34 (m, 1H), 4.82-4.73 (m, 1H), 4.64-4.61 (m,1H), 4.35–4.30 (m, 1H), 4.12-3.96 (m, 1H), 2.77-2.67 (m, 1H), 2.65-2.55 (m, 1H).; 31 P NMR (202 MHz, D2O): δ -9.76 (brs, 1P), -10.82 (d, 1P, J = 18.4 Hz), -22.56 (brs, 1P). HRMS (ESI) m / z for C 11 H16 N8O 12 P3 – The calculated value [M – H] – 545.0106; measured value 545.0105.

[0478] 6.10.4. Synthesis of L-3'-O-azidomethyl-dGTP:

[0479] 6.10.4.1 Scheme 16. Synthesis of L-3'-O-N3-dGTP (dG-6)

[0480]

[0481]

[0482] Experimental procedure:

[0483] dG-1 Synthesis: β-L-deoxyguanosine (2 g, 7.48 mmol) was co-evaporated (2 × 12 mL), and then pyridine (30 mL) was added, and the suspension was cooled to 0 °C. Trimethylsilyl chloride (4.8 mL, 37.4 mmol) was added dropwise via syringe. The ice bath was then removed, and the mixture was stirred for 1 hour. The solution was cooled to 0 °C, and isobutyric anhydride (6.2 mL, 37.4 mmol) was added dropwise via syringe. The ice bath was removed, and the resulting solution was stirred at room temperature for 3 hours. After stirring for 3 hours, the mixture was cooled to 0 °C, and ice-cold water (5 mL) was slowly added and stirred for 15 min, followed by the addition of concentrated ammonia solution (10 mL) to a final ammonia concentration of 2.5 M. The mixture was stirred on an ice bath for 1 hour, and then evaporated to dryness. The residue was redissolved in CH3OH, and silica gel was added and concentrated to dryness. The mixture was concentrated to dryness. The resulting residue was purified by column chromatography (19:1 to 9:1, CH2Cl2–CH3OH) to give dG-1 (1.85 g, 73%) as a white solid.

[0484] Synthesis of dG-2: Imidazole (544 mg, 8.0 mmol) and tert-butyldimethylsilyl chloride (885 mg, 5.87 mmol) were added to a solution of dG-1 (1.8 g, 5.33 mmol) in anhydrous DMF (30 mL) at 0 °C under nitrogen atmosphere, and the mixture was heated to room temperature. After stirring at room temperature for 8 h, ice-cold water was added to the solution and the mixture was extracted with EtOAc (3 × 100 mL). The combined organic layers were dried over anhydrous Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (19:1 to 9:1, CH2Cl2–CH3OH) to give dG-2 (1.9 g, 79%) as a white foam.

[0485] Synthesis of dG-3: Acetic acid (6 ml) and acetic anhydride (18 ml) were added to a stirred solution of dG-2 (1.7 g; 3.76 mmol) in DMSO (12 ml). After stirring at room temperature for 48 h, a saturated NaHCO3 solution was added to the solution at 0 °C and stirred for 30 min. The aqueous layer was extracted with EtOAc (2 × 100 ml). The combined organic layers were dried over Na2SO4, filtered, and concentrated. The crude product was purified by rapid column chromatography (1:1 to 1:3, hexane–EtOAc) to give dG-3 (1.2 g; 65%) as a pale yellow foam. R f 0.26 (1:4 hexane–EtOAc); 1 H NMR (400 MHz, CDCL3): δ 12.1 (s,1H, N H ), 9.40 (s, 1H, N H ), 7.98 (s, 1H), 6.18 (app t, 1H, J 1',2'a = J 1',2'b = 6.9Hz, H-1'), 4.71–4.61 (m, 3H, C H 2 S, H-3'), 4.15–4.09 (m, 1H, H-4'), 3.78 (d,2H, J = 3.9 Hz, H-5'a, H-5'b), 2.74 (sep, 1H, J = 7.0 Hz, COC H (CH3)2), 2.57–2.46 (m, 2H, H-2'a, H-2'b), 2.15 (s, 3H, SC H3 ), 1.25 (app t, 6H, J = 7.0 Hz, COCH(C H 3 )2), 0.89 (s, 9H, (C H 3 )3CSi); 0.08 (s, 3H, (C H 3 ) 2 Si), 0.07 (s, 3H, (C) H 3 ) 2 Si); HRMS (ESI) m / z for C 22 H 38 Calculated value of N5O5SSi [M + H] + 512.2363; measured value 512.2360.

[0486] Synthesis of dG-4. At room temperature, diphenylcarbamoyl chloride (814 mg; 3.52 mmol) and DIPEA (N,N-diisopropylethylamine) (1.23 ml; 7.03 mmol) were added to a stirred solution of dG-3 (1.20 g; 2.35 mmol) in dry pyridine (25 ml). After stirring at room temperature for 3 hours, the solvent was removed under high vacuum. The residue was diluted with EtOAc and washed with 2M HCl and saturated NaHCO3. The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by rapid column chromatography (1:1 to 1:3, hexane–EtOAc) to give dG-4 (1.5 g, 90%) as a foamy, pale yellow powder. R f 0.5 (1:1 hexane–EtOAc); 1 H NMR (400 MHz, CDCL3): δ 8.25 (s, 1H, N H ), 8.05 (s, 1H), 7.50–7.33 (m, 8H, ArH), 7.29–7.21 (m, 2H, ArH), 6.41 (app t, 1H, J 1',2'a = J 1',2'b = 6.7 Hz, H-1'), 4.76–4.66 (m, 3H, C H 2 S, H-3'), 4.19–4.13 (m, 1H, H-4'), 3.88 (dd, 1H,J 5'a,4' = 4.5 Hz, J gem = 11.1 Hz, H-5'a), 3.81 (dd, 1H, J 5'b,4' = 3.6 Hz, J gem = 11.1 Hz, H-5'b), 2.96 (bs, 1H, COC H (CH3)2), 2.75 (ddd, 1H, J 2'b,1 = 6.7 Hz, J 2'a,3' = 7.2 Hz, J gem = 13.6 Hz, H-2'a), 2.57 (ddd, 1H, J 2'a,1 =6.7 Hz, J 2'a,3' = 3.2 Hz, J gem = 13.6 Hz, H-2'b), 2.18 (s, 3H, SC H 3 ), 1.28 (d,6H, J = 6.8 Hz, COCH(C H 3 )2), 0.92 (s, 9H, (C H 3 )3CSi); 0.11 (s, 3H, (C H 3 ) 2 Si), 0.10 (s, 3H, (C) H 3 ) 2 Si); HRMS (ESI) m / z for C 35 H 47 Calculated value of N6O6SSi [M + H] + 707.3047; Measured value 707.3048.

[0487] Synthesis of dG-5: At 0 °C, cyclohexene (2.1 mL) and a CH2Cl2 solution of 1.0 M SO2Cl2 (1.39 mL, 1.39 mmol) in a stirred solution of dG-4 (490 mg, 0.694 mmol) in dry CH2Cl2 (14 mL) were added. After stirring at 0 °C for 1 h, the volatiles were removed under reduced pressure. At room temperature, NaN3 (271 mg, 4.61 mmol) was added to a solution of the residue in dry DMF (14 mL). After stirring at room temperature for 2 h, the reaction mixture was dispersed in distilled water (200 mL) and extracted with EtOAc (2 × 200 mL). The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. At 0 °C, 2 M HCl (7 mL) was added to a solution of the residue in acetonitrile (14 mL). The reaction mixture was stirred for 30 min. The solution was diluted with EtOAc (100 mL) and washed with saturated NaHCO3. The organic layer was washed with water (2 × 50 mL), dried over Na2SO4, filtered, and concentrated under reduced pressure. The resulting residue was purified by rapid column chromatography (1:2 to 0:1, hexane–EtOAc) to give dG-4 as a colorless oil. R f 0.32 (1:4 hexane–EtOAc); 1 H NMR (400 MHz, CDCL3): δ 8.38 (s, 1H, N H ), 8.08 (s, 1H), 7.46–7.39 (m, 4H, ArH), 7.37–7.32 (m, 4H, ArH), 7.27–7.20 (m, 2H, ArH), 6.22 (app t, 1H, J 1',2'a = J 1',2'b = 6.6 Hz, H-1'), 5.04–4.98 (m, 1H, H-3'), 4.77 (s, 2H, C H 2 N3), 4.19–4.14(m, 1H, H-4'), 3.88 (dd, 1H, J 5'a,4' = 2.9 Hz, J gem = 12.6 Hz, H-5'a), 3.80 (dd,1H, J 5'b,4' = 2.9 Hz, J gem= 12.6 Hz, H-5'b), 2.99 (app dt, 1H, J 2'b,1 = J 2'a,3' =6.6 Hz, J gem = 13.7 Hz, H-2'a), 2.79–2.66 (m, 1H, COC H (CH3)2), 2.48 (ddd, 1H, J 2'a,1 = 6.6 Hz, J 2'a,3' = 3.8 Hz, J gem = 13.7 Hz, H-2'b); HRMS (ESI) m / z for C 28 H 30 Calculated value of N9O6 [M + H] + 588.2219; measured value 588.2177.

[0488] Synthesis of dG-6: dG-5 (40 mg, 0.146 mmol) and proton sponge (37.59 mg, 0.175 mmol) were dried overnight in a vacuum desiccator using P2O5. POCl3 (21 μL, 0.219 mmol) was added dropwise to a solution of dG-5 in trimethyl phosphate (0.49 mL) at 0 °C. The mixture was stirred at 0 °C for 1 h, and then a thoroughly vortexed mixture of tributylammonium pyrophosphate (304 mg) and tributylamine (0.27 mL, 2.31 mmol) in anhydrous DMF (1.2 mL) was added. The mixture was stirred at room temperature for 20 min, and then 0.1 M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.0, 0.1 M, 4 mL) was added, and the mixture was stirred at room temperature for 3 h. Then, concentrated ammonium hydroxide (15 mL) was added to the mixture, and the mixture was stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure, and the residue was diluted with 30 mL of water. The crude mixture was extracted with CH2Cl2 (2 × 20 mL), and the aqueous layer was concentrated under reduced pressure. The residue was then purified by preparative HPLC (C18 column) followed by anion exchange chromatography on a DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.0; 0.05–1.0 M). Fractions containing the product (0.3 M – 0.4 M) were collected, concentrated under reduced pressure, and lyophilized with ddH2O to give dG-6 (2.7 mg, 4.5%) as a foamy solid. 1H NMR (400 MHz, D2O): δ 8.09 (s, 1H), 6.21 (m, 1H, H-1'), 4.83 (d, 1H, J = 11.5 Hz, C H 2 N3), 4.77 (d, 1H, J = 11.5 Hz, C H 2 N3), 4.64–4.61 (m, 1H), 4.37–4.31 (m, 1H), 4.16–4.03 (m,2H, H-5'a, H-5'b), 2.81–2.72 (m, 1H, H-2'a), 2.61-2.50 (m, 1H, H-2'b); 31 P NMR (121.4 MHz, D2O): δ –10.8 (bs, 1P), -11.5 (d, 1P, J = 18.4 Hz), -23.2 (bs, 1P). HRMS (ESI) m / z for C 11 H 17 N8O 13 Calculated value of P3 [M]: 562.0128; Measured value [M – H]: – 561.0072.

[0489] 6.11. Example 11. Synthesis of (L) 3'-O-azidomethyl-dNTP-labeled intermediate

[0490] The synthesis of L-3'-O-azidomethyl-dNTP-FL was designed and divided into three two-part processes: synthesis of the linker (Scheme 17), synthesis of the NH2-L-3'-O-N3-dNTP intermediate (Schemes 18-23), and then coupling of the intermediate with the fluorophore, as described in Example 3 above.

[0491] Currently, the synthesis of the linker (Scheme 17) provides compound 6, referred to as intermediate linker-6, which is obtained through... 1Characterization by ¹H NMR and HRMS. Attaching iFluor488 to linker-6 yields linker-7 for the coupling reaction of the NH₂-L⁻³'-O⁻N₃-dCTP intermediate. The first NH₂-L⁻³'-O⁻N₃-dNTP of interest is the synthesis of NH₂-L⁻³'-O⁻N₃-dUTP (Scheme 18). Currently, the synthesis of NH₂-L⁻³'-O⁻N₃-dUTP is complete up to step 3, yielding the intermediate “dUTP-FL-3”. Synthetic schemes for other L-3'-O-azidomethyl-dNTP-FL intermediates are also attached below (Schemes 19-21).

[0492] Given the compatibility of fluorophore-attached dNTPs with 9°N DNA polymerase, we also synthesized NH2-(L)3'-O-N3-7-denitro-dGTP (Scheme 26) and NH2-(L)3'-O-N3-7-denitro-dATP (Scheme 23). Starting materials for the nucleotide bases (6-chloro-7-denitroguanine and 6-chloro-7-denitro-7-iodopurine) and 2-deoxysugars (1-chloro-3,5-di-O-p-toluyl-2-L-deoxyfuranose) were commercially available. Denitro-dATP-FL-8 and denitro-dGTP-FL-9 were synthesized and characterized by NMR and HRMS.

[0493] 6.11.1. Scheme 17. Synthesis of Linking Groups

[0494]

[0495]

[0496]

[0497]

[0498] Synthesis of linker-1

[0499]

[0500] At room temperature, potassium carbonate (5.53 g, 40 mmol), sodium iodide (1.2 g, 0.4 mmol), and 2-bromomethyl-1,3-dioxolane (8.3 mL, 80 mmol) were added to a solution of ethyl-3-hydroxybenzoate (3.32 g, 20 mmol) in anhydrous DMF (8 mL). The mixture was heated to 120 °C and stirred overnight. The mixture was then heated to room temperature and evaporated under reduced pressure. The residue was diluted with CH2Cl2 (250 mL) and washed with water. The aqueous layer was washed twice with CH2Cl2 (2 × 50 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated. The residue was purified by column chromatography (hexane–EtOAc, 19:1 to 2:1) to obtain linker-1 (4.6 g, 91%). R f 0.54 (100% CH2Cl2); 1 H NMR (400 MHz, CDCL3): δ 7.67 (dt, 1H, J = 1.1, 7.1 Hz, ArH), 7.61 (dd, 1H, J = 1.5, 2.6 Hz,ArH), 7.35 (t, 1H, J = 7.8 Hz, ArH), 7.15 (ddd, 1H, J = 1.0, 2.7, 8.6 Hz,ArH), 5.32 (t, 1H, J = 4.0 Hz, C H ), 4.38 (q, 2H, J = 7.2 Hz, OC H 2 CH3), 4.12–3.96 (m, 6H, OC H 2 C H 2 O, ArOC H 2 ), 1.40 (t, 3H, J gem = 7.2 Hz, OCH2C H 3 ).

[0501] Synthesis of Linker-2

[0502]

[0503] At room temperature, 80 μL of tin chloride (IV) was added to a solution of linker-1 (2.70 g, 10.7 mmol) in azidotrimethylsilane (1.55 mL, 11.8 mmol). The mixture was stirred at room temperature for 2 h, and then 10 mL of 2% aqueous methanol was added. The mixture was stirred at room temperature for 30 min, and then concentrated under reduced pressure. The residue was co-evaporated with EtOH (2 × 30 mL), and the resulting residue was purified by column chromatography (hexane–EtOAc, 2:1 to 1:1) to obtain linker-2 (1.42 g, 45%). R f 0.32 (2:1 hexane–EtOAc); 1 H NMR (400 MHz, CDCl3): δ 7.70 (dt,1H, J = 1.1, 7.6 Hz, ArH), 7.61 (dd, 1H, J = 1.4, 2.5 Hz, ArH), 7.38 (t, 1H, J = 7.8 Hz, ArH), 7.15 (ddd, 1H, J = 0.9, 2.5, 8.1 Hz, ArH), 4.90 (t, 1H, J =5.3 Hz, C H N3), 4.40 (q, 2H, J = 7.2 Hz, OC H 2 CH3), 4.25 (dd, 1H, J = 5.3, 10.2Hz, ArOC H 2 ), 4.17 (dd, 1H, J = 5.3, 10.2 Hz, ArOC H 2 ), 4.06–4.00 (m, 1H, OC H 2 ), 3.91–3.73 (m, 3H, OC) H 2 C H 2 OH), 1.40 (t, 3H, J gem = 7.2 Hz, OCH2C H 3 ); HRMS (ESI) m / z for C 15H 18 Calculated values ​​of N3O7 [M – H] – 352.1236; measured value 352.1165.

[0504] Synthesis of Linker-3

[0505]

[0506] At room temperature, 4N NaOH (5 mL) was added to a solution of linker-2 (1.42 g, 4.8 mmol) in EtOH (5 mL). The mixture was stirred at room temperature for 3 hours, then concentrated under reduced pressure and acidified with 2N HCl (20 mL) and extracted twice with CH2Cl2 (2 × 50 mL). The combined organic layers were dried over MgSO4, filtered, and concentrated to obtain linker-3 (1.21 g, 94%). R f 0.41 (19:1 EtOAc–CH3OH); 1 H NMR (400 MHz, CDCL3): δ 7.78 (dt, 1H, J = 1.1, 7.6 Hz, ArH), 7.67 (dd, 1H, J = 1.4, 2.5 Hz, ArH), 7.43 (t, 1H, J = 7.8 Hz, ArH), 7.21 (ddd, 1H, J = 0.9, 2.5, 8.1 Hz,ArH), 4.92 (t, 1H, J = 5.2 Hz, C H N3), 4.26 (dd, 1H, J = 5.2, 10.1 Hz, ArOC H 2 ),4.17 (dd, 1H, J = 5.2, 10.1 Hz, ArOC H 2 ), 4.08–4.01 (m, 1H, OC H 2 ), 3.89–3.77 (m, 3H, OC) H 2 C H 2 OH); HRMS (ESI) m / z for C 11 H 12Calculated values ​​of N3O5 [M – H] – 266,0867; measured value 266.0794.

[0507] Synthesis of linker-4

[0508]

[0509] At 0 °C, 60% NaH (188 mg, 4.71 mmol) was added to a solution of linker-3 (320 mg, 1.57 mmol) in THF (5 mL). The mixture was stirred at 0 °C for 10 min, and then ethyl 2-bromoacetate (382 mg, 4.71 mmol) was added to the mixture. L, 3.45 mmol). The mixture was heated to room temperature and stirred for 4 hours. After stirring for 4 hours, cold water (50 mL) was poured into the reaction mixture and the resulting mixture was extracted with CH2Cl2 (50 mL). The organic layer was discarded and the aqueous layer was acidified by adding 2N HCl (25 mL). The resulting mixture was extracted with CH2Cl2 (2 × 50 mL), and the collected organic layer was dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (98:2, CH2Cl2–CH3OH) to obtain linker-4 (120 mg, 22%) as a pale yellow oil. R f 0.67 (1:1:0.5 EtOAc–hexane–AcOH); 1 H NMR (400 MHz, CDCl3): δ 7.75 (app dt, 1H, J = 1.1, 7.5 Hz, ArH), 7.64 (dd, 1H, J = 1.4, 2.5 Hz, ArH), 7.39 (t, 1H, J = 7.8 Hz, ArH), 7.19 (ddd, 1H, J = 0.8,2.6, 8.2 Hz, ArH), 4.96 (app t, 1H, J = 5.0 Hz, C H N3), 4.28–4.12 (m, 6H, ArOC) H 2 OC H 2 CH3, OC H 2 C=O), 4.05 (app dt, 1H, J= 4.1, 11.4 Hz, OC H 2 ), 3.97–3.87 (m, 1H, OC) H 2 ), 3.82 (appt, 2H, J = 4.8 Hz, OC H 2 ), 1.29 (t, 3H, J = 7.2 Hz, OCH2C H 3 HRMS (ESI) m / z for C 15 H 18 Calculated values ​​of N3O7 [M – H] – 352.1236; measured value 352.1165.

[0510] Synthesis of Linker-5

[0511]

[0512]

[0513] At room temperature, DSC (104 mg, 0.407 mmol) and DMAP (49.8 mg, 0.407 mmol) were added to a solution of linker-4 (120 mg, 0.339 mmol) in DMF (1 mL). The mixture was stirred at room temperature for 10 min, and then N-(2-aminoethyl)-2,2,2-trifluoroacetamide (78.5 mg, 0.407 mmol) and DIPEA (142 mg, 0.339 mmol) were added to the mixture at room temperature. L, 0.815 mmol). The mixture was stirred overnight at room temperature and quenched with 1N Na2HPO4 solution (25 mL). The mixture was extracted with CH2Cl2 until no product was observed in the aqueous layer. The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (1:1, hexane–EtOAc) to obtain linker-5 (42 mg, 25%) as a colorless oil. R f 0.62 (1:1:0.5 EtOAc–hexane–AcOH); 1 H NMR (400 MHz, CDCl3): δ8.25 (bs, 1H, N H ), 7.42–7.30 (m, 4H, Ar H , N H), 7.07 (ddd, 1H, J = 0.9, 2.5, 8.0 Hz, Ar H ), 4.90 (app t, 1H, J = 4.9 Hz, C H N3), 4.25–4.07 (m, 6H, ArOC H 2 ,OC H 2 CH3, OC H 2 C=O), 4.05–3.99 (m, 1H, OC H 2 ), 3.89–3.82 (m, 1H, OC H 2 ), 3.79(appt, 2H, J = 4.2 Hz, OC H 2 ), 3.70–3.62 (m, 2H, NC H 2 CH2N), 3.61–3.54 (m, 2H,NCH2C H 2 N), 1.28 (t, 3H, J = 7.2 Hz, OCH2C H 3 ); HRMS: [M]: C 19 H 24 F3N5O7, calculated value 491.1646; measured value [M – H] – 490.1574.

[0514] Synthesis of Linker-6

[0515]

[0516]

[0517] At room temperature, 4N NaOH (2 mL) was added to a solution of linker-5 (42 mg, 0.113 mmol) in EtOH (2 mL). The mixture was stirred at room temperature for 2 hours, then evaporated under reduced pressure and the residue was redissolved in 15 mL of water. The mixture was extracted with CH2Cl2 (2 × 15 mL). The aqueous layer was acidified to pH 2 by adding 2N HCl. The solution was then extracted with CH2Cl2 (3 × 15 mL). The aqueous layer was neutralized to pH 8 by adding 1N NaOH and then evaporated to dryness under reduced pressure. The white solid was ground with CH2Cl2 / CH3OH (1:1, 2 × 15 mL). The solid was filtered and the filtered solution was evaporated to dryness. 10% CH3OH in CH2Cl2 (15 mL) was added to the resulting gel, and the white solid was filtered off. The filtered solution was evaporated to dryness to give linker-6 (32 mg, 83%) as a colorless oil; 1 H NMR (400MHz, CDCl3): δ 8.25 (bs, 1H, N H ), 7.41–7.32 (m, 2H, Ar H ), 7.31–7.27 (m, 1H,Ar H ), 7.17–7.10 (m, 1H, Ar H ), 5.02 (app t, 1H, J = 4.4 Hz, C H N3), 4.19 (d,2H, J = 4.4 Hz, ArOC H 2 ), 4.01–3.95 (m, 1H, OC H 2 ), 3.87–3.78 (m, 3H, OC H 2 C=O,OC H 2 ), 3.67–3.63 (m, 2H, OC H 2 ), 3.60 (app t, 2H, J = 6.0 Hz, NC H 2 CH2N), 3.01(app t, 2H, J = 6.0 Hz, NCH2C H 2 N); HRMS:M:C 15 H20 N3NaO6, calculated value 389.1312; measured value [M + H] + 390.1386.

[0518] Synthesis of Linker-7

[0519]

[0520] Linker-7 (1.1 mg, 0.0030 mmol) was dissolved in DMF (50 μL) and 1 M NaHCO3 aqueous solution (15 μL). A solution of fluorophore (1.98 mg, 0.0021 mmol) in DMF (60 μL) was slowly added to the reaction mixture, and then stirred for 5 h at room temperature in the dark. The crude product was purified by preparative HPLC. (HPLC gradient: A, 100% 0.1 M TEAB; B, 100% MeCN: 0–2 min, 5% B (flow rate 2–10 ml / min); 2–19 min, 5%–45% B (flow rate 10 ml / min); 19–21 min, 45%–95% B (flow rate 10 ml / min); 21–24 min, 95% B (flow rate 10 ml / min); 24–26 min, 95%–5% B (flow rate 10 ml / min, yielding the compound with a retention time of 17.85 min); 26–30 min, 5% B (flow rate 10–2 ml / min). The title compound with a retention time of 17.85 min as water was obtained.) HRMS (ESI): m / z 993.2051 [M – Na] – .

[0521] 6.11.2. Scheme 18. Synthesis of NH2-(L)-3'-O-azidomethyl-dUTP

[0522]

[0523] Experimental procedure:

[0524] Synthesis of dUTP-FL-1: β-L-deoxyuridine (2.4 g, 10.51 mmol, 1.0 equivalent) was suspended in methanol (30 mL) and stirred for 10 min. Iodine (8.0 g, 31.55 mmol, 3 equivalent) and silver nitrate (5.3 g, 31.55 mmol, 3 equivalent) were then added, and the reaction mixture was stirred at 40 °C for 3 h. After the reaction was complete, the reaction mixture was filtered, and the filtrate was subjected to column chromatography on silica gel using MeOH / CH2Cl2 at a ratio of 1:19 to 1:9 to obtain compound dUTP-FL-1 (1.8 g, 48%) as a white solid. R f 0.65 (9:1 DCM–MeOH), 1 HNMR (500 MHz, DMSO-d6) δ11.63 (s, 1H), 8.36 (s, 1H), 6.05 (t, J = 6.5 Hz, 1H), 4.24 – 4.12 (m, 2H), 3.79 – 3.69 (m, 2H), 3.61 – 3.46 (m, 2H), 2.15 – 1.96 (m, 2H).

[0525]

[0526] Synthesis of dUTP-FL-2: Compound 1 (400 g, 1.12 mmol, 1.0 equivalent) was dissolved in anhydrous DMF (20 mL). Imidazole (114.3 mg, 1.68 mmol, 1.5 equivalent) and tert-butyldimethylsilyl chloride (185.6 mg, 1.23 mmol, 1.1 equivalent) were added to the solution under nitrogen atmosphere at 0 °C. The reaction mixture was stirred at room temperature for 12 h. Cold water was then added and the mixture was extracted with EtOAc (3 × 50 mL). The combined organic layers were dried over anhydrous Na₂SO₄, concentrated, and the resulting residue was subjected to column chromatography using 2%–5% MeOH in CH₂Cl₂ as the eluent to give compound dUTP-FL-2 (348 mg, 63%) as a foamy white solid. R f 0.45 (19:1 DCM–MeOH) 1 HNMR (500 MHz, ) δ8.38 (s, 1H), 8.10 (s, J = 4.0 Hz, 1H), 6.29 (dd, J = 8.1, 5.6 Hz, 1H), 4.47(dd, J= 5.4, 3.1 Hz, 1H), 4.08 (q, J = 2.3 Hz, 1H), 3.86 (ddd, J = 40.5,11.4, 2.5 Hz, 2H), 2.41 (ddd, J = 13.4, 5.6, 2.1 Hz, 1H), 2.09 (ddd, J =13.6, 8.1, 5.7 Hz, 2H), 1.98 (d, J = 3.5 Hz, 1H), 0.92 (s, J = 2.9 Hz, 9H),0.15(s, 3H) – 0.13(s, 3H).

[0527]

[0528] Synthesis of dUTP-FL-3: Acetic acid (4 ml) and acetic anhydride (12 ml) were added to a stirred solution of 2 (108 mg; 0.23 mmol) in DMSO (8 ml). The reaction mixture was stirred at room temperature for 48 h. At 0 °C, a saturated NaHCO3 solution (50 ml) was added and stirred for 30 min, and the aqueous layer was extracted with EtOAc (3 × 100 ml). The combined organic extracts were dried over Na2SO4 and concentrated. The crude product was purified by rapid column chromatography (ethyl acetate / hexane, 1:1 to 7:3) to give dUTP-FL-3 (106 mg, 87%) as a foamy white solid. R f 0.85 (1:1 hexane–EtOAc) 1 HNMR (500MHz, ) δ 8.86 (s), 8.09 (s), 7.27 (s), 6.23 (dd, J = 8.3, 5.5 Hz), 5.42 –5.25 (m), 4.36 (d, J = 5.9 Hz), 4.17 (d, J = 2.1 Hz), 3.87 (ddd, J = 56.2,11.5, 2.3 Hz), 2.54 (ddd, J = 13.6, 5.5, 1.7 Hz), 2.12 (s), 2.04 (ddd, J=14.0, 8.3, 5.9 Hz), 1.36 – 1.21 (m, 1H), 0.95 (s, 9H), 0.17(s, 3H), 0.16(s,3H).

[0529]

[0530] 6.11.3. Scheme 18. Synthesis of NH2-(L)-3'-O-azidomethyl-dUTP

[0531]

[0532]

[0533]

[0534] Experimental procedure:

[0535]

[0536] Synthesis of dUTP-FL-1: Sodium azide (854 mg, 13.14 mmol) and N-iodosuccinimide (1.48 g, 6.57 mmol) were added to a solution of β-L-deoxyuridine (1 g, 4.38 mmol) in H2O (10 mL), and the mixture was stirred at room temperature for 19 hours. After complete reaction, the reaction mixture was filtered off and washed with water. The remaining filtrate was extracted with DCM×2 and the combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (1:10 to 1:3, CH3OH:CH2Cl2) to give dUTP-FL-1 (1.27 g, 80%) as a white solid. 1 HNMR (400 MHz, CD3OD) δ 8.54 (s, 1H), 6.24 (app t, J = 6.5 Hz, 1H), 4.42 (app dt, J =3.4 Hz, J =6.1 Hz, 1H), 3.97–3.93 (m, 1H), 3.84 (dd, J = 12.0 Hz, J = 3.0 Hz, 1H), 3.75 (dd, J = 12 Hz, J = 3.3 Hz, 1H), 2.37–2.18 (m, 2H). HRMS (ESI): m / z for C9H 11Calculated value of IN₂O₅ [M]: 353.9717; Measured value [M – H]: – 352.9644.

[0537]

[0538] Synthesis of dUTP-FL-2: CuI (6.5 mg, 0.34 mmol) and triethylamine (50 μL) were added to a solution of dUTP-FL-1 (60 mg, 0.17 mmol) in N,N-dimethylformamide (3 mL), and the mixture was stirred at room temperature for several minutes to activate the CuI. Then, 2,2,2-trifluoro-N-(prop-2-ynyl)acetamide (76.8 mg, 0.51 mmol) and tetrakis(triphenylphosphine)palladium (0) (19.6 mg, 0.0017 mmol) were added. The reaction was allowed to run at room temperature for 24 h and concentrated. The resulting residue was purified by column chromatography (1:10:10 to 1:4:4, CH3OH: CH2Cl2: EtOAc) to give dUTP-FL-2 (32 mg, 50%) as a yellow oil. 1 HNMR (400 MHz, CD3OD ) δ 8.34 (s, 1H), 6.25 (app t, J =6.5 Hz, 1H), 4.6 (s, 1H), 4.43-4.39 (m, 1H), 4.29 (s, 2H), 4.42 (app dt, J =7.0 Hz, J = 7.1 Hz, 1 H), 3.97–3.94 (m, 1H), 3.82 (dd, J = 12.0 Hz, J = 3.0Hz, 1H), 3.75 (dd, J = 12 Hz, J = 3.5 Hz, 1H); HRMS (ESI): m / z for C 14 H 14 Calculated value of F3N3O6 [M]: 377.0825; Measured value [M + H]: + 378.0893.

[0539]

[0540] Synthesis of dUTP-FL-3: Imidazole (160.32 mg, 2.35 mmol) and tert-butyldimethyl chloride (260.29 mg, 1.73 mmol) were added to a solution of dUTP-FL-2 (590 mg, 1.57 mmol) in N,N-dimethylformamide (10 mL) at 0 °C, and then the temperature was raised to room temperature. After stirring at room temperature for 10 h, ice water was added to the solution and the mixture was extracted with ethyl acetate × 2. The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (1:3 to 1:1, hexane:EtOAc) to give dUTP-FL-3 (210 mg, 43%) as a yellow oil. 1 HNMR (400MHz, CDCl3) δ 8.69 (s,1H), 8.11 (s, 1H), 6.19–6.24 (m, 1H), 4.64 (app dt, J =11.9 Hz, J =14.6 Hz, 1H), 4.47 (d, 1H), 4.16–4.05 (m, 1H), 3.92 (dd, J = 2.5Hz, J = 11.4 Hz, 1H), 3.80 (dd, J = 2.4 Hz, J = 11.5 Hz, 1H), 2.15 (s, 3H), 0.95 (s, 9H), 0.17 (d, 6H); HRMS (ESI): m / z for C 20 H 28 The calculated value of F3N3O6Si [M] is 491.1699; the measured value is [M + H]. + 492.1778.

[0541]

[0542] Synthesis of dUTP-FL-4: At room temperature, AcOH (200 uL) and acetic anhydride (200 uL) were added to a solution of dUTP-FL-3 (164 mg, 0.46 mmol) in DMSO (1 mL). After stirring at room temperature for 24 hours, ice water and solid sodium bicarbonate were added to the solution. The aqueous layer was extracted twice with ethyl acetate. The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (3:1 to 1:1, hexane:EtOAc) to give dUTP-FL-4 (70 mg, 27%) as a colorless oil. 1HNMR (400 MHz, CDCl3) δ 9.35(s,1H), 7.96 (s, 1H), 7.49 (s,1H), 6.10 (dd, J = 5.7 Hz, 1H), 4.33–3.32 (m, 1H), 4.24–4.13 (m, 2H), 4.02(d, 1H), 3.83 (dd, J = 2.4 Hz, J = 11.5 Hz, 1H), 3.75 (dd, J = 2.0 Hz, J =11.4 Hz, 1H), 2.38–2.33 (m, 1H), 2.01 (s, 3H), 0.78 (s, 9H), 0.01 (d, 6H); HRMS (ESI): m / z for C 22 H 32 Calculated value of F3N3O6SSi [M]: 551.1733; Measured value [M + H]: + 552.1825.

[0543]

[0544] Synthesis of dUTP-FL-5: Cyclohexene (30 μL, 0.3 mmol) was added to a solution of dUTP-FL-4 (50 mg, 0.63 mmol) in dry CH2Cl2 at 4 °C, followed by dropwise addition of sulfonyl chloride (1 M CH2Cl2 solution, 0.11 mL, 0.11 mmol) under N2. After 40 min, TLC indicated complete consumption of dUTP-FL-4, the solvent was evaporated, and the residue was subjected to high vacuum for 20 min. It was then redissolved in dry DMF (1 mL) and treated with NaN3 (30 mg, 0.45 mmol). The resulting suspension was stirred at room temperature for 2 h. The reaction was quenched with CH2Cl2, and the organic layer was washed with a saturated aqueous NaCl solution. After solvent removal, the resulting yellow gel was redissolved in 2 M HCl:acetonitrile (2:1) and stirred at room temperature for 30 min. The solvent was removed, and the reaction was post-treated with CH2Cl2 and a saturated aqueous NaHCO3 solution. The aqueous layer was extracted three times with CH2Cl2. The solution was purified by chromatography on silica (EtOAc:heptane 1:1 to 100% EtOAc) to obtain dUTP-FL-5, which was a pale yellow foam. 1 HNMR (400 MHz, CDCl3) δ 8.32 (s, 1H), 6.22 (dd, J= 6.2,7.4 Hz, 1H, H-1'), 4.83–4.76 (m, 2H, C H 2 N3), 4.62 (bs, 1H), 4.47–4.42 (m, 1H), 4.29 (s, 2H, C H 2 N), 4.14–4.09 (m, 1H), 3.84 (dd, J = 3.3 Hz, J = 12.0 Hz, 1H,H-5'a), 3.77 (dd, J = 3.3 Hz, J = 12.0 Hz, 1H, H-5'b), 2.50 (ddd, 1H, J =3.0, 6.1, 13.8 Hz, H-2'a), 2.35–2.25 (m, 1H, H-2'b); HRMS (ESI): m / z for C 15 H 15 Calculated value of F3N6O6 [M]: 432.1005; Measured value [M + Na]: + 455.0887.

[0545] Synthesis of dUTP-FL-6: dUTP-FL-6 (174 mg, 0.402 mmol) and proton sponge (103 mg, 0.483 mmol) were dried over the weekend. A solution of dUTP-FL-6 (174 mg, 0.402 mmol) and proton sponge (103 mg, 0.483 mmol) in trimethyl phosphate (1 mL) was stirred for 30 min. Phosphorus oxychloride (56.4 μL, 0.604 mmol) was added directly to the solution of the mixture at 0 °C. The mixture was stirred at 0 °C for two hours. Then, at room temperature, a well-vortexed mixture of tributylammonium pyrophosphate (834 mg) and tributylamine (834 μL, 3.10 mmol) in anhydrous DMF (2 mL) was added to the mixture. The mixture was stirred at room temperature for 1 hour. Triethylammonium bicarbonate buffer (TEAB buffer, 20 mL, 0.1 M, pH 8.5) was added to the mixture and stirred at room temperature for 1 h. Ammonium solution was added and stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure and the residue was diluted with 30 mL of water. The crude mixture was extracted with CH2Cl2 (2 × 20 mL) and the aqueous layer was concentrated under reduced pressure. The residue was then purified by preparative HPLC (C18 column) followed by anion exchange chromatography on a DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.0; 0.05–1.0 M). Fractions containing the product (0.3 M – 0.4 M) were collected and concentrated under reduced pressure and lyophilized using ddH2O to give dUTP-FL-6 (27 mg, 21%) as a foamy solid. 1 HNMR (400 MHz, D2O) δ 8.32 (s, 1H), 6.20 (t, J = 6.6 Hz, 1H, H-1'), 4.81–4.73 (m, 2H, C H 2 N3), 4.57–4.52 (m, 1H), 4.34–4.29(m, 1H), 4.24–4.08 (m, 2H, H-5'a, H-5'b), 3.91(s, 2H, C H 2 N), 2.52 (ddd, 1H, J = 3.0, 6.1, 13.8 Hz, H-2'a), 2.38–2.25 (m, 1H, H-2'b); 31 P NMR (121.4 MHz, D2O)δ –10.8 (bs, 1P), –11.7 (d, 1P,J = 19.2 Hz), –23.1 (bs, 1P); HRMS (ESI): m / z for C 13 H 19 N6O 14 Calculated value of P3 [M] 576.0172; Measured value [M – Na] – 575.0108.

[0546] 6.11.4. Scheme 19. Synthesis of NH2-L-3'-O-azidomethyl-dCTP

[0547]

[0548]

[0549] 6.11.5. Scheme 19b. Synthesis of NH2-L-3'-O-azidomethyl-dCTP

[0550]

[0551]

[0552] Experimental procedure:

[0553] Synthesis of dCTP-FL-1: Add the following to a dry round-bottom flask: -L-deoxycytidine (1 g, 4.40 mmol), iodine (1.67 g, 60 mmol), and mCPBA (0.75 g, 4.40 mmol) were dissolved in 15 mL of DMF. The reaction was stirred at room temperature for 2 h, and then evaporated to dryness under reduced pressure. The resulting crude product was purified by column chromatography (1:9, DCM-MeOH) to give dCTP-FL-1 (0.83 g, 53% yield) as an orange solid. 1 H NMR (500 MHz, CD3OD) δ 8.49 (s,1H), 6.17 (t, J = 6.3 Hz, 1H), 4.36 (dt, J = 6.3, 4.1 Hz, 1H), 3.92 (dd, J =6.9, 3.4 Hz, 1H), 3.80 (dt, J = 15.6, 4.9 Hz, 1H), 3.71 (dd, J = 12.0, 3.4Hz, 1H), 2.34 (ddd, J= 13.6, 6.2, 4.2 Hz, 1H), 2.20–2.07 (m, 1H). HRMS (ESI): m / z for C9H 12 Calculated value of IN3O4 [M]: 352.9900; Measured value [M + Na]: + 375.9765.

[0554] Synthesis of dCTP-FL-2: dCTP-FL-1 (650 mg, 1.84 mmol) was added to a light-protected round-bottom flask and dissolved in DMF (10 ml). Subsequently, CuI (70 mg, 0.36 mmol), triethylamine (0.5 ml), 2,2,2-trifluoro-N-prop-2-ynylacetamide (833.7 mg, 5.52 mmol), and finally Pd(PPh3)4 (212.5 mg, 0.184 mmol) were added to the mixture, and the mixture was stirred at room temperature under an argon atmosphere for 18 h. Bicarbonate resin (100 mg) was added, and the mixture was stirred for another 1 h. The reaction mixture was filtered through diatomaceous earth, and the filtrate was evaporated under reduced pressure to obtain the residue. The residue was purified by silica gel chromatography (CH2Cl2: EtOAc: MeOH 4.5:4.5:1) to give the desired product (490 mg, 71% yield) as a beige solid. 1 H NMR (500 MHz, D2O) δ 8.04 (s, 1H), 6.08 (t, J = 6.5Hz, 1H), 4.37 – 4.23 (m, 1H), 4.23 (s, 2H), 3.94 (d, J = 4.3 Hz, 1H), 3.73(dd, J = 12.6, 3.0 Hz, 1H), 3.64 (dd, J = 12.3, 5.0 Hz, 1H), 2.39–2.28 (m, 1H), 2.19–2.13 (m, 1H). HRMS (ESI): m / z for C 14 H 15 Calculated value of F3N4O5 [M]: 376.1000; Measured value [M + H]: + 377.1066.

[0555] Synthesis of dCTP-FL-3: Imidazole (82.12 mg, 1.195 mmol) and tert-butyldimethylsilyl chloride (144.14 mg, 0.956 mmol) were added to a solution of dCTP-FL-2 (300 mg, 0.797 mmol) in anhydrous DMF (5.0 mL) at 0 °C. The solution was stirred at 0 °C under nitrogen for 2 h. After the reaction was complete (monitored by TLC), cold water (10 mL) was added and the mixture was extracted with EtOAc (3 × 30 mL). The combined organic layers were dried over Na2SO4, filtered, and evaporated under reduced pressure. The resulting residue was purified by column chromatography (100% EtOAc to EtOAc:MeOH 9.5:0.5) to give dCTP-FL-3 (210 mg, 54%) as a beige solid. 1 H NMR (500 MHz, DMSO-d6) δ 9.92 (t, J = 5.3 Hz,1H), 7.97 (s, 1H), 7.83 (s, 1H), 6.87 (d, J = 22.9 Hz, 1H), 6.11 (dd, J =7.2, 6.1 Hz, 1H), 5.27 (d, J = 4.1 Hz, 1H), 4.24 (d, J = 5.3 Hz, 2H), 4.17(td, J = 6.0, 3.1 Hz, 1H), 3.88 (q, J = 2.8 Hz, 1H), 3.81 (dd, J = 11.5, 2.6Hz, 1H), 3.72 (dd, J = 11.6, 3.1 Hz, 1H), 2.19–2.17 (m, 1H), 1.96–1.88 (m, 1H), 0.86 (s, 9H), 0.07 (s, 6H). HRMS (ESI): m / z for C 20 H 29 The calculated value of F3N4O5Si [M] is 490.1900; the measured value is [M + Na]. + 513.1751.

[0556] Synthesis of dCTP-FL-4: Acetic acid (2 ml) and acetic anhydride (2 ml) were added to a stirred solution of dCTP-FL-3 (210 mg, 0.428 mmol) in DMSO (6 ml) and the mixture was stirred at room temperature for 48 h. After the reaction was complete, a saturated NaHCO3 solution was added at 0 °C and the mixture was stirred for 30 min. The aqueous layer was extracted with EtOAc (3 × 30 ml). The combined organic extracts were dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude product was purified by column chromatography (1:1, hexane–EtOAc) to give dCTP-FL-4 (130 mg, 52%) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ 8.32(s, 1H), 8.14 (s, 1H), 6.15 (t, J = 6.4 Hz, 1H), 4.62 (dd, J = 32.9, 11.7 Hz,2H), 4.42 (dt, J = 5.7, 2.8 Hz, 1H), 4.37 (t, J = 4.2 Hz, 2H), 4.18 (dd, J =4.8, 2.3 Hz, 1H), 3.94 (dd, J = 11.5, 2.6 Hz, 1H), 3.79 (dd, J = 11.5, 2.2Hz, 1H), 2.66 (ddd, J = 13.7, 5.9, 2.9 Hz, 1H), 2.57 (s, 3H), 2.13 (s, 3H), 2.02 (s, 1H), 0.88 (s, 9H), 0.10 (s, 6H). HRMS (ESI): m / z for C 24 H 35 Calculated value of F3N4O6SSi [M]: 592.2000; Measured value [M + H]: + 593.2071.

[0557] Synthesis of dCTP-FL-5: Cyclohexene (1 mL) was added to a stirred solution of dCTP-FL-4 (130 mg, 0.67 mmol) in anhydrous CH2Cl2 (6 mL), and a CH2Cl2 solution of SO2Cl2 (1.0 M, 0.4 mL) was added. After stirring at 0 °C for 1 h, the volatiles were removed under reduced pressure. Dry DMF (3 mL) and NaN3 (169 mg, 2.61 mmol) were added to the residue, and the mixture was stirred at room temperature for 6 h. The reaction mixture was dispersed in cold distilled water (30 mL) and extracted with EtOAc (2 × 30 mL). The combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure. Acetonitrile (10 mL) and 2M HCl (3 drops) were added to the resulting residue, and the reaction was stirred at room temperature for 1–2 h. The mixture was neutralized with saturated NaHCO3 solution and diluted with EtOAc. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (hexane–EtOAc (1:1) to 100% EtOAc to EtOAc-MeOH (19:1)) to give dCTP-FL-5 (25 mg, 28%) in orange oil. 1 H NMR (500 MHz, DMSO-d6) δ 9.89 (t, J = 4.8 Hz, 1H), 8.06 (s,1H), 7.80 (s, 1H), 6.84 (s, 1H), 6.00 (dd, J = 7.3, 6.0 Hz, 1H), 5.11 (s,1H), 4.76 (s, 2H), 4.25 (dt, J = 6.0, 3.0 Hz, 1H), 4.20 (d, J = 5.1 Hz, 2H), 3.93–3.88 (m, 1H), 3.59–3.47 (m, 1H), 2.32–2.23 (m, 1H), 2.14–2.01 (m, 1H). HRMS (ESI): m / z for C 15 H 16 Calculated value of F3N7O5 [M]: 431.1200; Measured value [M + H]: + 432.1237.

[0558] 6.11.6. Scheme 20. Synthesis of NH2-L-3'-O-N3-dATP

[0559]

[0560] 6.11.7. Scheme 20a. Alternative synthesis of NH2-L-3'-O-N3-dATP

[0561]

[0562]

[0563] 6.11.8. Scheme 21. Synthesis of NH2-L-3'-O-N3-dGTP

[0564]

[0565]

[0566] 6.11.9. Scheme 21a. Alternative synthesis of NH2-L-3'-O-N3-dGTP

[0567]

[0568] 6.12. Example 12. Synthesis of (L) 3'-O-N3-7-denitrified dNTP-labeled

[0569] 6.12.1. Scheme 23. Synthesis of NH2-(L) 3'-O-N3-7-denitrification-dATP

[0570]

[0571]

[0572]

[0573]

[0574] 6.12.1.1 Experimental Procedure

[0575] At room temperature, powdered KOH (85%, 0.5 g, 7.57 mmol) and TDA-1 (0.075 mL, 0.24 mmol) were added to a solution of 4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (1.0 g, 3.58 mmol) in CH3CH (60 mL). After stirring for 10 min, a halogenose (1.7 g, 4.37 mmol) was introduced and stirring was continued for another 10 min. The insoluble material was filtered off and washed several times with hot acetone. The combined filtrates were evaporated to dryness. The residue was subjected to rapid chromatography (silica gel, column 5 x 15 cm, eluted with petroleum ether–EtOAc, 4:1). The combined fractions containing the product were evaporated to give 4-chloro-7-[2-deoxy-3,5-di-O-(4-methylbenzoyl- -L-erythro-furanopentosyl)-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (2.02 g, 89%). A solution of the intermediate (1.8 g, 2.85 mmol) in NH3 / CH3OH (saturated at 0 °C, 145 mL) was stirred at room temperature for 24 hours and the solvent was evaporated. Rapid chromatography (silica gel, column 4 x 16 cm, CH2Cl2 / CH3OH, 9:1) gave the compound 7-denitro-dA-2 (0.45 g, 40%) as a colorless solid. f0.45 (CH2Cl2 / CH3OH, 9:1). CuI (24 mg, 0.1260 mmol) and triethylamine (250 μL) were added to a solution of 7-denitro-dA-2 (236 mg, 0.6298 mmol) in N,N-dimethylformamide (5 mL), and the mixture was stirred at room temperature for several minutes. Then, 2,2,2-trifluoro-N-(prop-2-ynyl)acetamide (285 mg, 1.89 mmol) and tetrakis(triphenylphosphine)palladium (0) (72.78 mg, 0.06 mmol) were added. The reaction was allowed to run at room temperature for 24 h and concentrated. The resulting residue was purified by column chromatography (1:10:10 to 1:4:4, CH3OH:CH2Cl2:EtOAc) to give 7-denitro-dA-3 (164.2 mg, 65.3%) as a yellow solid. tert-butyldimethylsilyl chloride (TBDPSCl) (510 mg, 3.28 mmol) was added to a stirred mixture of 7-denitro-dA-3 (1.00 g, 2.83 mmol) and imidazole (462 mg, 6.79 mmol) in anhydrous DMF (14.0 mL). The reaction mixture was stirred at room temperature for 20 h. After evaporation, the residue was purified by rapid column chromatography (CH2Cl2–CH3OH, 20:1) to give 7-denitro-dA-4 (1.18 g, 89%) as a white solid. A solution of 7-denitro-dA-4 (128 mg, 0.2492 mmol) was dissolved in a mixture of CH3OH:N,N-dimethylacetal (10:1) and stirred at 40 °C. The reaction, monitored by TLC, was completed after 2 h. The solvent was removed under vacuum. 7-Denitro-dA-5 (99.5 mg, 70.2%) was purified by chromatography on silica (EtOAc: CH3OH = 15:1 to 10:1) to give a clear yellow oil. Acetic acid and acetic anhydride were added to a stirred solution of 7-denitro-dA-5 in DMSO. The reaction mixture was stirred at room temperature for 48 h. A saturated NaHCO3 solution was added and the aqueous layer was extracted with CH2Cl2. The combined organic extracts were washed with a saturated NaHCO3 solution and dried over Na2SO4. After concentration, the residue was purified by rapid column chromatography (hexane / ethyl acetate, 1:1 to 1:4) to give 7-denitro-dA-6. Cyclohexene and SO2Cl2 were added to a stirred solution of 7-denitro-dA-6 in dry CH2Cl2 under nitrogen. The reaction mixture was stirred at 0 °C for 2 h. The solvent was removed first under reduced pressure and then under high vacuum for 10 min. The residue was dissolved in dry DMF and reacted with NaN3 at room temperature for 3 h.The reaction mixture was dispersed in distilled water (50 ml) and extracted with CH2Cl2 (3 × 50 ml). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was dissolved in MeOH and stirred with NH4F at room temperature for 24 h. The solvent was removed under reduced pressure. The reaction mixture was concentrated under reduced pressure and partitioned between H2O and CH2Cl2. The organic layers were separated and dried over Na2SO4. After concentration, the crude product was purified by rapid column chromatography (ethyl acetate / methanol, 100:0 to 98:2) to give compound 7-denitro-dA-7. Compound 7-denitro-dA-7 and the proton sponge were dried overnight in a vacuum desiccator over P2O5 and then dissolved in trimethyl phosphate. Freshly distilled POCl3 was then added dropwise at 0 °C and the mixture was stirred at 0 °C for 2 h. Subsequently, a well-vortexed mixture of tributylammonium pyrophosphate and tributylamine in anhydrous DMF was added in one go at room temperature and stirred for 30 min. Then, triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0) was added, and the mixture was stirred at room temperature for 1 h. Concentrated NH4OH was then added, and the mixture was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum, and the residue was diluted with 5 mL of water. The crude mixture was then purified by anion exchange chromatography on a DEAE-Sephadex A-25 at 4 °C using a gradient of TEAB (pH 8.0; 0.1–1.0 M). The crude product was further purified by reversed-phase HPLC to give (L) 3'-O-N3-7-denitro-dATP (compound 7-denitro-dA-8).

[0576] 6.12.2. Scheme 23a. Alternative synthesis of NH2-(L)3'-O-azidomethyl-7-denitro-dATP

[0577]

[0578]

[0579]

[0580]

[0581]

[0582] 6.12.3. Alternative synthesis of scheme 23b. NH2-(L)3'-O-azidomethyl-7-denitro-dATP

[0583]

[0584]

[0585] Synthesis of 7dN-dATP-FL-2: At room temperature, powdered KOH (1.8 g, 7.57 mmol) and TDA-1 (0.3 ml, 0.86 mmol) were added to a solution of 4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (4.0 g, 14.4 mmol) in MeCN (200 ml). After stirring for 30 min, 1-Cl-2-deoxy sugar (8.6 g, 18.7 mmol) was introduced and stirring was continued for another 30 min. The insoluble material was filtered off and washed several times with hot acetone. The combined filtrates were evaporated to dryness. The residue was subjected to purification by chromatography on silica (EtOAc:hexane 1:1 to 100% EtOAc) to give an intermediate (4.22 g, 89%) as a yellow solid. The intermediate (1.2 g, 1.89 mmol) was suspended in a mixture of 28% NH3 aqueous solution and dioxane (1:1, 100 ml) and stirred under reflux for 17 h. The residue was applied to silica purified by chromatography (EtOAc:hexane:CH3OH 1:1:0.1) to give 7-dN-dATP-FL-2 (296 mg, 41.4%) as a yellow solid. HRMS (ESI): m / z against C 11 H 13 Calculated value of IN4O3 [M]: 376.0039; Measured value [M + H]: + 377.0113.

[0586]

[0587] Synthesis of 7dN-dATP-FL-3: CuI (24 mg, 0.1260 mmol) and triethylamine (250 μL) were added to a solution of 7-dN-dATP-FL-2 (236 mg, 0.6298 mmol) in N,N-dimethylformamide (5 mL), and the mixture was stirred at room temperature for several minutes. Then, 2,2,2-trifluoro-N-(prop-2-ynyl)acetamide (285 mg, 1.89 mmol) and tetrakis(triphenylphosphine)palladium (0) (72.78 mg, 0.06 mmol) were added. The reaction was allowed to run at room temperature for 24 h and concentrated. The resulting residue was purified by column chromatography (1:10:10 to 1:4:4, CH3OH:CH2Cl2:EtOAc) to give 7-dN-dATP-FL-3 (164.2 mg, 65.3%) as a yellow solid. 1HNMR (400 MHz, MeOH-d6) δ 10.10 (s, 1H), 8.12 (s,1H), 7.76 (s, 1H), 6.53-6.46 (m, 1H), 5.26 (d, 1H), 5.08 (t, J = 5.6 Hz, 1H),4.35 – 4.26 (m, 2H), 3.84 – 3.81(m, 1H), 2.59(dd, J = 4.8Hz, J = 0.8Hz, 1H), 2.49-2.43 (m, 1H), 2.21-2.16 (m, 1H); HRMS (ESI): m / z for C 16 H 16 Calculated value of F3N5O4 [M]: 399.1154; Measured value [M+H]: + 400.1234

[0588]

[0589] Synthesis of 7dN-dATP-FL-4: Imidazole (39.8 mg, 0.586 mmol) and tert-butyl(chloro)diphenylsilane (118.12 mg, 3.25 mmol) were added to a solution of 7dN-dATP-FL-3 (156 mg, 0.3907 mmol) in N,N-dimethylformamide (4 mL) at 0 °C, and the mixture was then heated to room temperature. After stirring at room temperature for 10 h, the solution was added to ice water and extracted with ethyl acetate × 2. The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (1:2 to 100%, hexane:EtOAc) to give 7dN-dATP-FL-4 (128 mg, 63.8%) as a yellow oil. 1 HNMR (400 MHz, MeOH-d6) δ 8.11 (s, 1H), 7.66 (d, 4H), 7.51 (s, 1H),7.42-7.33 (m, 6H), 6.56 (t, J = 6.5 Hz, 1H), 4.63-4.59 (m, 1H), 4.28 (s, 2H), 4.01-4.00 (m, 1H), 3.90 (dd, J = 11.5 Hz, J = 3.15 Hz, 1H), 3.81 (dd, J =11.4Hz, J= 3.82 Hz, 1H), 2.57-2.38 (m, 2H), 1.05 (s, 9H); HRMS (ESI): m / z for C 32 H 34 The calculated value of F3N5O4Si [M] is 637.2362; the measured value is [M + H]. + 638.2438

[0590]

[0591] Synthesis of 7dN-dATP-FL-5: A solution of 7dN-dATP-FL-4 (128 mg, 0.2492 mmol) was dissolved in a mixture of CH3OH:N,N-dimethyl acetal (10:1) and stirred at 40 °C. The reaction, monitored by TLC, was completed after 2 h. The solvent was removed under vacuum. Purification by chromatography on silica (EtOAc: CH3OH = 15:1 to 10:1) yielded 7dN-dATP-FL-4 (99.5 mg, 70.2%) as a clear yellow oil. 1 H NMR (400 MHz, CDCl3) δ8.77 (s, 1H C H =N), 8.42 (s, 1H), 7.70–7.60 (m, 4H, ArH), 7.54 (s, 1H), 7.48–7.32 (m, 6H, ArH), 6.90 (bs, 1H, CH2N H ), 6.67 (t, 1H, J = 6.5 Hz, H-1'), 4.69-4.62 (m, 1H), 4.41–4.29 (m, 2H, C H 2 NH), 4.05-3.98 (m, 1H), 3.91–3.80 (m, 2H,H-5'a, H-5'b), 3.20 (s, 3H, (C H 3 )2N), 3.18 (s, 3H, (C H 3 )2N), 2.56-2.39 (m, 2H,H-2'a, H-2'b), 1.07 (s, 9H, TBDPS) t Bu); HRMS (ESI): m / z for C 35 H 39 The calculated value of F3N6O4Si [M] is 692.2788; the measured value is [M + H].+ 693.2865.

[0592]

[0593] Synthesis of 7dN-dATP-FL-6: At room temperature, AcOH (800 μL) and acetic anhydride (800 μL) were added to a solution of 7dN-dATP-FL-5 (99.5 mg, 0.175 mmol) in DMSO (4 mL). After stirring at room temperature for 18 hours, ice-cold water and solid sodium bicarbonate were added to the solution. The aqueous layer was extracted twice with ethyl acetate. The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (hexane: EtOAc = 3:1 to 1:1) to give 7dN-dATP-FL-6 (48 mg, 40%) as a white solid. 1 H NMR (400 MHz, CDCl3) δ 9.57 (bs,1H), 8.30 (s, 1H), 7.67 (s, 1H), 7.60–7.50 (m, 4H), 7.42-7.33 (m, 6H), 6.56(t, J = 6.5 Hz, 1H), 4.65-4.62 (m, 1H), 4.61–4.54 (m, 2H, C H 2 N), 4.28–4.23 (m, 2H, C) H 2 S), 4.15-4.10 (m, 1H), 3.92–385 (m, 1H, H-5'a), 3.83–3.73 (m, 1H, H-5'b), 2.68–2.57 (m, 1H, H-2'a), 2.51-2.38 (m, 1H, H-2'b), 2.10 (s, 3H, SC H 3 ), 1.05 (s, 9H, TBDPS) t Bu); HRMS (ESI): m / z for C 35 H 39 The calculated value of F3N6O4Si [M] is 752.2821; the measured value is [M + H]. + 753.2897.

[0594]

[0595] Synthesis of 7dN-dATP-FL-7: Cyclohexene (0.03 ml, 0.16 mmol) was added to a solution of 7dN-dATP-FL-6 (20 mg, 0.032 mmol) in dry CH2Cl2 at 4 °C, followed by dropwise addition of sulfonyl chloride (1 M CH2Cl2 solution, 0.03 ml, 0.031 mmol) under N2. After 40 min, TLC indicated complete consumption of compound 6, the solvent was evaporated, and the residue was subjected to high vacuum for 20 min. It was then redissolved in dry DMF (1 ml) and treated with NaN3 (30 mg, 0.45 mmol). The resulting suspension was stirred at room temperature for 2 h. The reaction was quenched with CH2Cl2, and the organic layer was washed with a saturated aqueous NaCl solution. After solvent removal, the resulting yellow gel was redissolved in 2 M HCl:acetonitrile (2:1) and stirred at room temperature for 30 min. The solvent was removed and the reaction was post-treated with CH2Cl2 and a saturated aqueous solution of NaHCO3. The aqueous layer was extracted three times with CH2Cl2. Purification by chromatography on silica (EtOAc:heptane 1:1 to 100% EtOAc) yielded 7dN-dATP-FL-7 (15 mg, 73%) as a pale yellow foam. 1 HNMR (400 MHz, MeOH-d6 ) δ8.86 (s), 8.09 (s), 6.44 (app dt, J =2.5 Hz, J =6.0 Hz, 1H) 4.86-4.80 (m,2H), 4.57-4.54 (m, 1H),4.19-4.16 (m, 1H), 3.81 (dd, J =3.6 Hz, 12 Hz, 1H), 3.75 (dd, J = 12.1 Hz, J = 3.3 Hz, 1H), 2.73-2.66 (m, 1H), 2.53 (dd, J =2.3Hz, J = 5.7 Hz, 1H), 2.49 (dd, J =2.31 Hz, J = 5.9 Hz, 1H); HRMS (ESI): m / z for C 17 H 17 Calculated value of F3N8O4 [M]: 454.1341; Measured value [M + H]: + 455.1415.

[0596]

[0597] Synthesis of 7dN-dATP-FL-8: 7-dN-dATP-FL-7 (71 mg, 0.156 mmol) was dried overnight in a vacuum desiccator using P2O5. At 0 °C, 7dN-dATP-FL-7 was reacted with trimethyl phosphate (700 mg / L) to form 7dN-dATP-FL-8. POCl3 (22.4%) was added dropwise to the solution in l). l, 0.22 mmol). The mixture was stirred at 0 °C for 2 hours, and then tributylammonium pyrophosphate (343.1 mg) and tributylamine (286 mg) were added. l) The mixture was thoroughly vortexed in anhydrous DMF (1 mL). The mixture was stirred at room temperature for 1 hour, and then 0.1 M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.5, 20 mL) was added, and the mixture was stirred at room temperature overnight. The resulting mixture was concentrated under reduced pressure and the residue was diluted with 10 mL of water. The crude mixture was extracted with CH2Cl2 (2 × 10 mL) and the aqueous layer was concentrated under reduced pressure. The residue was then purified by anion exchange chromatography on a DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.5; 0.1–0.8 M). Fractions containing the product (0.3 M–0.4 M) were collected and concentrated under reduced pressure. The residue was resuspended in H2O (5 mL) and lyophilized to give 7dN-dATP-FL-8 (22 mg, 23.7%). 1 H NMR (400 MHz, D2O) δ 7.88 (s, 1H),7.77 (bs, 1H), 6.46 (app t, 1H, J = 7.6 Hz, H-1'), 4.88–4.77 (m, 2H, C H 2 N3),4.63–4.57 (m, 1H), 4.36–4.28 (m, 1H), 4.14–4.01 (m, 2H, H-5'a, H-5'b), 3.97(s, 2H, C H 2 NH2), 2.60–2.43 (m, 2H, H-2'a, H-2'b); 31 P NMR (121.4 MHz, D2O) δ –10.6 (d, 1P, J = 18.9 Hz), –11.3 (d, 1P, J= 18.9 Hz), –23.1 (t, 1P, J = 18.9Hz); HRMS (ESI): m / z for C 17 H 17 Calculated value of F3N8O4 [M]: 598.0492; Measured value [M – H]: – 597.0460.

[0598] 6.12.4. Scheme 25. Synthesis of (L) 3'-O-N3-7-denitrified-dATP-ROX

[0599]

[0600] Azide-ROX compound (ROX-N3-linker). 2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-acetic acid linker-6 (7.0 mg, 0.019 mmol) prepared according to the literature (Milton J, Ruediger S, Liu X (2006), US Patent Application US20060160081A1) was dissolved in DMF (300 µl) and 1 M NaHCO3 aqueous solution (100 µl). A solution of ROX NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF (400 µl) was slowly added to the above reaction mixture, and then stirred at room temperature in the dark for 5 h. The crude product was purified on preparative silica gel TLC plates (CHCl3 / CH3OH, 1:4).

[0601] The L-3'-O-N3-7-denitro-dATP-ROX compound (L-3'-O-N3-7-denitro-dATP-ROX) was added to a stirred solution of the ROX-N3-linker in dry DMF (2 ml) at room temperature. DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 µmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 µmol) were added. The reaction mixture was stirred at room temperature for 2 h. TLC showed complete conversion of the ROX-N3-linker to the compound ROX-N3-linker NHS ester, which was then directly used for coupling with L-amino-7-denitro-dATP (13 µmol) in NaHCO3 / Na2CO3 buffer (pH 8.7, 0.1 M) (300 µl). The reaction mixture was stirred at room temperature in the dark for 3 h. The reaction mixture was purified by preparative silica gel TLC (CH3OH / CH2Cl2, 1:1). The crude product was further purified by reversed-phase HPLC to obtain L-3'-O-N3-dATP-ROX (L-3'-O-N3-dATP-ROX).

[0602] 6.12.5. Scheme 26. Synthesis of NH2-(L)3'-O-azidomethyl-7-denitro-dGTP

[0603]

[0604] 6.12.5.1 Experimental Procedure

[0605] At room temperature, 2-amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (842 mg, 4.99 mmol) was added to a suspension of powdered KOH (85%, 1.15 g, 17.42 mmol) and TDA-1 (0.2 mL, 0.63 mmol) in CH3CN (60 mL). After stirring for 5 min, a halogenated sugar (2.53 g, 6.51 mmol) was introduced over 15 min, and stirring was continued for 30 min. The insoluble material was filtered off and washed several times with CH3CN. The combined filtrates were evaporated to dryness. The residue was subjected to rapid chromatography (silica gel, 6 x 12 cm column, eluted with CH2Cl2). The combined fraction containing the product was evaporated to give a colorless solid (2.21 g, 85%). A solution of the intermediate (1.04 g, 2.00 mmol) in 0.5 M NaOCH3 / CH3OH (60 mL) was stirred under reflux for 3 hours. The mixture was neutralized with AcOH and evaporated. The residue was subjected to rapid chromatography (silica gel, CH2Cl2 / CH3OH, 95:5) to give a colorless solid of compound 7-deoxy-dG-1 (400 mg, 71%). The stirred solution of 7-deoxy-dG-1 was co-evaporated with anhydrous pyridine (3 × 4 mL) and then dissolved in anhydrous pyridine (2 mL). The resulting solution was dehydrated (in a desiccator), purged with argon, and placed on ice. TMS-Cl (4.51 mL, 58.4 mmol, 8.2 equivalents) was added dropwise to the ice-cold solution via a syringe. The ice bath was then removed, and the mixture was stirred for 2 hours. The solution was cooled on ice, and isobutyryl chloride (0.29 mL, 15.69 mmol, 2.2 equivalence) was added dropwise via syringe, followed by removal of the ice bath. After stirring at room temperature for 2 hours, the reaction was placed back on ice, and ice-cold water (20 mL) was slowly added, followed by concentrated ammonia solution (1.5 mL) after 15 minutes to obtain a final ammonia concentration of 2.5 M. The mixture was kept on ice for 30 minutes and then evaporated to dryness. The residue was co-evaporated with toluene (3 × 5 mL) to remove trace amounts of water, resuspended in MeOH, and filtered to remove the precipitate. The filtrate was then concentrated, dissolved in a small amount of MeOH, adsorbed onto silica gel, and purified by column chromatography (DCM / MeOH 95:5 to 91:9 (v / v)) to give compound 7-denitro-dG-2. Compound 7-denitro-dG-2 was co-evaporated three times with dry pyridine, dried under high vacuum, and dissolved in 2 cm⁻¹ water in an ice bath. 3N,N-dimethylformamide was dried. Imidazole (419 mg, 6.15 mmol) was added, and the mixture was stirred at 0 °C for 15 min and then at room temperature for 15 min. Then, 241 mg of tert-butyldimethylsilyl chloride (1.59 mmol) was added, and the solution was stirred at 60 °C for another 2 h. The mixture was diluted with dichloromethane, washed with brine, dried over sodium sulfate, and evaporated. The crude product was purified by column chromatography on silica gel (methanol:dichloromethane 0:100–2:98) to a white foam (compound 7-denitro-dG-3). NIS was added to a vigorously stirred solution of 7-denitro-dG-3 in anhydrous DMF. The reaction mixture was stirred at room temperature for 22 h, and then most of the solvent was removed under vacuum. Diethyl ether and saturated NaHCO3 were added. The organic layer was washed with saturated NaCl and dried over Na2SO4. After evaporation, the residue was purified by rapid column chromatography to give 7-denitro-dG-4. Acetic acid (5.5 ml) and acetic anhydride (17.6 ml) were added to a stirred solution of compound 7-denitro-dG-4 in DMSO (12 ml). The reaction mixture was stirred at room temperature for 48 h. A saturated NaHCO3 solution (100 ml) was added, and the aqueous layer was extracted with CH2Cl2 (3 × 100 ml). The combined organic extracts were washed with a saturated NaHCO3 solution (100 ml) and dried over Na2SO4. After concentration, the residue was purified by rapid column chromatography to give compound 7-denitro-dG-5. Cyclohexene and SO2Cl2 were added to a stirred solution of compound 7-denitro-dG-5 in dry CH2Cl2 under nitrogen. The reaction mixture was stirred at 0 °C for 2 h. The solvent was removed first under reduced pressure and then under high vacuum for 10 min. The residue was dissolved in dry DMF and reacted with NaN3 at room temperature for 3 h. The reaction mixture was dispersed in distilled water (50 ml) and extracted with CH2Cl2 (3 × 50 ml). The combined organic layers were dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by rapid column chromatography (ethyl acetate / methanol, 100:0 to 98:2) to give compound 7-denitro-dG-6. Tetra(triphenylphosphine)palladium(O) and CuI were added to a solution of 7-denitro-dG-6 in anhydrous DMF. The reaction mixture was stirred at room temperature for 10 min. Then N-propynetrifluoroacetamide and Et3N were added to the above reaction mixture. The reaction was stirred at room temperature in the absence of air and light for 1.5 h. After evaporation, the residue was dissolved in ethyl acetate. The mixture was washed with saturated aqueous solutions of NaHCO3 and NaCl and dried over anhydrous Na2SO4. After evaporation, the residue was purified by rapid column chromatography to give 7-denitro-dG-7.NaI and trimethylchlorosilane were added to a stirred solution of 7-denitro-dG-7 in anhydrous CH3CN. The reaction was stirred at room temperature for 1 h, and then at 50 °C for 12 h. The solvent was evaporated, and the residue was dissolved in THF. A solution of tetrabutylammonium fluoride (TBAF) in THF was added, and the reaction was stirred at room temperature for 1 h. The solvent was evaporated, and the residue was dissolved in EtOAc. The solution was washed with a saturated aqueous NaCl solution and dried over anhydrous Na2SO4. After solvent evaporation, the residue was purified by rapid column chromatography (EtOAc–CH3OH) to give 7-denitro-dG-8. Compound 7-denitro-dG-8 and a proton sponge were dried overnight in a vacuum desiccator over P2O5, and then dissolved in trimethyl phosphate. Freshly distilled POCl3 was then added dropwise at 0 °C, and the mixture was stirred at 0 °C for 2 h. Subsequently, a mixture of tributylammonium pyrophosphate and tributylamine in anhydrous DMF was added in a single, fully vortexed manner at room temperature and stirred for 30 min. Then, triethylammonium bicarbonate solution (TEAB) (0.1 M; pH 8.0) was added, and the mixture was stirred at room temperature for 1 h. Concentrated NH4OH was then added, and the mixture was stirred overnight at room temperature. The resulting mixture was concentrated under vacuum, and the residue was diluted with 5 mL of water. The crude mixture was then purified by anion exchange chromatography on a DEAE-Sephadex A-25 at 4 °C using a gradient of TEAB (pH 8.0; 0.1–1.0 M). The crude product was further purified by reversed-phase HPLC to give (L) 3'-O-N3-7-denitro-dATP (compound 7-denitro-dG-9).

[0606] 6.12.6. Scheme 26a. Alternative synthesis of NH2-(L)3'-O-azidomethyl-7-denitro-dGTP

[0607]

[0608] 6.12.7. Scheme 26b. Alternative synthesis of NH2-(L)3'-O-N3-7-denitrified-dGTP

[0609]

[0610]

[0611]

[0612]

[0613] Experimental procedure:

[0614]

[0615] Synthesis of 7dN-dGTP-FL-1: At room temperature, 2-amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (421 mg, 2.50 mmol) was added to a suspension of KOH (0.68 g, 8.71 mmol) and TDA-1 (0.1 mL, 0.32 mmol) in CH3CN (30 mL). After stirring for 5 min, 1-Cl sugar (1.27 g, 3.25 mmol) was added, and stirring was continued for 30 min. The insoluble material was filtered off, the precipitate was washed with CH3CN, and the filtrate was evaporated to dryness. The residue was purified by column chromatography (hexane–EtOAc, 4:1 to 1:1) to obtain a product (1.10 g, 82%) as a white foam. At room temperature, a solution of NaOCH3 in CH3OH (0.5 M, 30 mL) was added to the starting material (1.10 g, 2.00 mmol). The solution was heated to reflux and stirred for 3 hours. The mixture was cooled to room temperature and neutralized by adding AcOH. The mixture was then evaporated to dryness. The residue was purified by column chromatography (hexane–EtOAc, 1:1 to 100% EtOAc) to obtain 7-dN-dGTP-FL-1 (420 mg, 75%) as a pale yellow foam. R f 0.21 (100% EtOAc), 1 HNMR (400 MHz, CD3OD) δ 7.00(d, J = 3.7 Hz, 1H), 6.37 (dd, J 1',2'b = 5.9 Hz, J 1',2'a = 8.6 Hz, 1H, H-1'), 6.32 (d, J = 3.7 Hz, 1H), 4.51 (app dt, J 3',2'a = 5.8 Hz, J 3',2'b = J 3',4' = 2.3Hz, 1H, H-3'), 4.05–3.97 (m, 4H, OCH3, H-4'), 3.81 (dd, J 5'a,4' = 3.4 Hz, J gem =12.1 Hz, 1H, H-5'a), 3.72 (dd, J5'b,4' = 3.5 Hz, J gem = 12.1 Hz, 1H, H-5'b),2.70 (ddd, J 2'a,3' = 5.9 Hz, J 2'a,1' = 8.6 Hz, J gem = 14.5 Hz, 1H, H-2'a), 2.24(ddd, J 2'b,3' = 2.3 Hz, J 2'b,1' = 5.9 Hz, J gem = 14.5 Hz, 1H, H-2'b).

[0616]

[0617] Synthesis of 7dN-dGTP-FL-2: At room temperature, butyric anhydride (1.22 mL, 7.50 mmol) was added to a solution of 7dN-dGTP-FL-1 (420 mg, 1.50 mmol) in pyridine (5 mL). After stirring for 24 h, the reaction mixture was quenched with CH3OH. The mixture was then evaporated and extracted with EtOAc, washed with 2N HCl and saturated NaHCO3 aqueous solution. The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (1:1, hexane–EtOAc) to obtain an intermediate (412 mg, 81%). Benzoyl chloride (705 μL, 6.07 mmol) was added to a solution of the intermediate (412 mg, 1.21 mmol) in pyridine at room temperature. After stirring for 2 h, the mixture was quenched with CH3OH and evaporated. The residue was diluted with EtOAc and washed with 2N HCl and saturated NaHCO3 aqueous solution. The organic layer was dried over Na2SO4, then filtered and concentrated. The residue was purified by column chromatography (3:1, hexane–EtOAc) to obtain 7dN-dGTP-FL-2 (425 mg, 67%) as a white foam. R f 0.53 (3:1, hexane–EtOAc), R f 0.52 (3:1 hexane–EtOAc), 1H NMR (400 MHz, CDCl3) δ7.89–7.82 (m, 4H, ArH), 7.54–7.46 (m, 2H, ArH), 7.41–7.35 (m, 4H, ArH), 7.17 (d, J = 3.7 Hz, 1H), 6.50 (d, J = 3.7 Hz, 1H), 6.39 (dd, J 1',2'b = 6.8 Hz, J 1',2'a = 8.5 Hz, 1H, H-1'), 5.25 (app dt, J 3',2'a = 6.2 Hz, J 3',2'b = J 3',4' = 2.2 Hz, 1H, H-3'), 4.35–4.29 (m, 2H, H-5'a, H-5'b), 4.24–4.19(m, 1H, H-4'), 3.79 (s, 3H, OCH3), 2.67–2.53 (m, 2H, (CH3)2C H ), 2.48–2.39 (m,1H, H-2'a), 2.33–2.24 (m, 1H, H-2'b), 1.25–1.16 (m, 12H, (C H 3 )2CH).

[0618]

[0619] Synthesis of 7dN-dGTP-FL-3: At room temperature, NIS (200 mg, 0.892 mmol) was added to a solution of 7dN-dGTP-FL-2 (425 mg, 0.81 mmol) in DMF (8.0 mL). After stirring overnight, the mixture was diluted with EtOAc and precipitated with saturated NaHCO3. 3(水溶液) Wash. Dry the organic layer with Na2SO4, filter, and concentrate. Purify the residue by column chromatography (hexane–EtOAc, 10:1 to 3:1) to obtain 7-dN-dGTP-FL-3 (330 mg, 63%) as a pale yellow foam. R f 0.42 (3:1, hexane–EtOAc), 1HNMR (400 MHz, CDCl3) δ 7.84–7.72 (m, 4H, ArH), 7.52–7.43 (m, 2H, ArH), 7.41–7.31 (m, 4H, ArH), 7.28 (s, 1H), 6.31 (dd, J =6.2 Hz, 8.0 Hz, 1H, H-1'), 5.24–5.19 (m, 1H, H-3'), 4.33–4.27 (m, 2H, H-5'a,H-5'b), 4.22–4.17 (m, 1H, H-4'), 3.77 (s, 3H, OCH3), 2.59 (septet, J = 6.9Hz, 2H, (CH3)2C H ), 2.35–2.19 (m, 2H, H-2'a, H-2'b), 1.21–1.19 (m, 6H, (C H 3 )2CH), 1.19–1.16 (m, 6H, (C H 3 )2CH); HRMS (ESI) m / z for C 34 H 35 The calculated value of IN4O8 [M] is 754.1518; the measured value is [M + H]. + 755.1591.

[0620]

[0621] Synthesis of 7dN-dGTP-FL-4: At room temperature, a 0.5 M CH3ONa solution in CH3OH (2 mL) was added to a solution of 7dN-dGTP-FL-3 (330 mg, 0.508 mmol) in CH3OH (8.0 mL). After stirring for 1.5 h, the mixture was neutralized by adding AcOH (100 uL) and then concentrated. The residue was purified by column chromatography (hexane–EtOAc, 3:1 to 100% EtOAc) to obtain 7dN-dGTP-FL-4 (240 mg, 75%) as a white solid. R f 0.23 (100% EtOAc); 1HNMR (400 MHz, CD3OD) δ 8.01–7.95 (m, 2H, ArH), 7.66–7.59 (m, 2H, ArH,H-6), 7.57–7.50 (m, 2H, ArH), 6.66 (dd, J = 6.7, 6.6 Hz, 1H, H-1'), 4.59–4.51(m, 1H, H-3'), 4.10 (s, 3H, OC H 3 ), 3.99–3.94 (m, 1H, H-4'), 3.79 (dd, J =3.8, 12.0 Hz, 1H, H-5'a), 3.73(dd, J = 4.4, 12.0 Hz, H-5'b), 2.68–2.57 (m,1H, H-2'a), 2.38(ddd, J = 3.4, 6.1, 13.4 Hz, H-2'b); HRMS (ESI) m / z for C 19 H 19 Calculated value of IN4O5 [M]: 510.0409; Measured value [M + H]: + 511.0482.

[0622]

[0623] Synthesis of 7dN-dGTP-FL-5: CuI (15 mg, 0.095 mmol) and Et3N (0.5 mL) were added to a solution of 7dN-dGTP-FL-4 (200 mg, 0.392 mmol) in DMF (5.0 mL) at room temperature. After stirring under N2 for 5 min at room temperature, alkyne (177.5 mg, 1.18 mmol) and Pd(PPh3)4 (45 mg, 0.039 mmol) were added to the mixture at room temperature. After stirring for 24 h, the mixture was concentrated and the residue was purified by column chromatography (hexane–EtOAc, 1:1 to hexane–EtOAc–CH3OH, 2:2:1) to obtain 7dN-dGTP-FL-5 (185 mg, 88%) as a pale yellow oil. R f 0.11 (2:2:1, hexane–EtOAc–CH3OH); 1HNMR (400 MHz, CD3OD) δ 8.03–7.91 (m, 2H, ArH),7.71–7.50 (m, 4H, ArH, H-6), 6.64 (dd, J = 6.7, 6.6 Hz, 1H, H-1'), 4.58–4.52(m, 1H, H-3'), 4.33 (s, 2H, C H 2 N), 4.10 (s, 3H, OC H 3 ), 3.99–3.94 (m, 1H, H-4'), 3.79 (dd, J = 3.8, 12.0 Hz, 1H, H-5'a), 3.73 (dd, J = 4.4, 12.0 Hz, H-5'b), 2.67–2.57 (m, 1H, H-2'a), 2.39 (ddd, J = 3.4, 6.1, 13.4 Hz, H-2'b); HRMS(ESI) m / z for C 24 H 22 Calculated value of F3N5O6 [M]: 533.1534; Measured value [M + Na]: + 556.1428.

[0624]

[0625] Synthesis of 7dN-dGTP-FL-6: TBDPSCl (118 uL, 0.454 mmol) was added to a solution of 7dN-GTP-FL-5 (220 mg, 0.412 mmol) in pyridine (2.0 mL) at 0 °C, and the mixture was slowly heated to room temperature. After stirring for 72 h, the mixture was quenched by adding CH3OH (5 mL) and then concentrated. The residue was diluted with EtOAc and then washed with 2N HCl and saturated NaHCO3. The organic layer was dried over Na2SO4, then filtered and concentrated. The residue was purified by column chromatography (hexane–EtOAc, 4:1 to 1:1) to obtain 7dN-dGTP-FL-6 (235 mg, 74%) as a white foam. R f 0.34 (1:1, hexane – EtOAc); 1HNMR (400 MHz, CD3OD) δ 7.98–7.90 (m,2H, ArH), 7.74–7.31 (m, 14H, ArH, H-6), 6.65 (dd, J = 6.7, 6.6 Hz, 1H, H-1'), 4.71–4.64 (m, 1H, H-3'), 4.33 (s, 2H, C H 2 N), 4.10 (s, 3H, OC H 3 ), 4.05–3.98(m, 1H, H-4'), 3.96–3.83 (m, 2H, H-5'a, H-5'b), 2.70–2.54 (m, 1H, H-2'a), 2.49–2.37 (m, 1H, H-2'b), 1.06 (s, 9H, (C H 3 )3C); HRMS (ESI) m / z for C 40 H 40 The calculated value of F3N5O6Si [M] is 771.2713; the measured value is [M + H]. + 772.2789.

[0626]

[0627] Synthesis of 7dN-dGTP-FL-7: At room temperature, AcOH (1.5 mL) and Ac2O (3 mL) were added to a solution of 7dN-dGTP-6 (180 mg, 0.233 mmol) in DMSO (6.0 mL). After stirring for 24 hours, the mixture was transferred to a solution containing saturated NaHCO3. 3(水溶液) Quenching. The mixture was stirred for 30 min and diluted with EtOAc, then washed with saturated NaHCO3. The organic layer was dried over Na2SO4, then filtered and concentrated. The residue was purified by column chromatography (hexane–EtOAc, 4:1 to 1:1) to obtain 7dN-dGTP-FL-7 (154 mg, 79%) as a white foam. R f 0.67 (1:1, hexane–EtOAc); 1 HNMR (400 MHz, CD3OD) δ 7.97–7.90 (m, 2H, ArH), 7.70–7.32 (m,14H, ArH, H-6), 6.58 (dd, J= 6.2, 7.3 Hz, 1H, H-1'), 4.83–4.78 (m, 1H, H-3'), 4.77–4.67 (m, 2H, C H 2 S), 4.30 (s, 2H, C H 2 N), 4.12–4.04 (m, 4H, H-4',OC H 3 ), 3.88 (dd, J = 4.2, 11.9 Hz, 1H, H-5'a), 3.81 (dd, J = 4.2, 11.9 Hz,1H, H-5'b), 2.69–2.58 (m, 1H, H-2'a), 2.50 (ddd, J = 3.1, 6.2, 13.7 Hz, 1H,H-2'b), 2.12 (s, 3H, SC H 3 ), 1.06 (s, 9H, (C H 3 )3C); HRMS (ESI) m / z for C 42 H 44 The calculated value of F3N5O6SSi [M] is 831.2751; the measured value is [M + H]. + 832.2826.

[0628]

[0629] Synthesis of 7dN-dGTP-FL-8: Cyclohexene (500 μL) and 1 M sulfonyl chloride (555 μL, 0.555 mmol) were added to a solution of 7dN-dGTP-FL-7 (154 mg, 0.185 mmol) in CH2Cl2 (2.0 mL) at 0 °C. After stirring for 30 min, the mixture was concentrated. The residue was then dissolved in DMF (2.0 mL), and NaN3 (120 mg, 1.85 mmol) was added to the mixture at room temperature. After stirring for 1 h, the mixture was diluted with EtOAc and washed with H2O. The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (hexane–EtOAc, 4:1 to 1:1) to obtain 7dN-dGTP-FL-8 (142 mg, 93%) as a colorless oil. R f 0.67 (1:1, hexane–EtOAc);1 HNMR (400 MHz, CD3OD) δ 7.96–7.89 (m, 2H, ArH), 7.69–7.29 (m, 14H, ArH, H-6), 6.55 (app t, J = 6.7 Hz, 1H, H-1'), 4.83 (d, J = 9.0 Hz, 1H,C H 2 N3), 4.78–4.73 (m, 2H, H-3', C H 2 N3), 4.30 (s, 2H, C H 2 N), 4.15–4.06 (m, 4H,H-4', OC H 3 ), 3.90 (dd, J = 4.2, 11.3 Hz, 1H, H-5'a), 3.84 (dd, J = 4.4, 11.3Hz, 1H, H-5'b), 2.79–2.69 (m, 1H, H-2'a), 2.55 (ddd, J = 3.7, 6.3, 13.6 Hz,1H, H-2'b), 1.06 (s, 9H, (C H 3 )3C); HRMS (ESI) m / z for C 41 H 41 The calculated value of F3N8O6Si is [M] 826.2897; the measured value is [M + H]. + 827.2973.

[0630]

[0631] Synthesis of 7dN-dGTP-FL-9: At room temperature, NH4F (13.4 mg, 0.363 mmol) was added to a solution of 7dN-GTP-8 (98 mg, 0.121 mmol) in MeOH (2.0 mL). After stirring for 24 hours, the mixture was concentrated and the residue was diluted with EtOAc and then washed with H2O. The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (hexane–EtOAc, 4:1 to 1:1) to obtain 7dN-dGTP-FL-9 (46 mg, 67%) as a colorless oil. R f0.27 (1:1, hexane – EtOAc); 1 HNMR (400 MHz, CD3OD) δ 8.00–7.94 (m, 2H,ArH), 7.67 (s, 1H, H-6), 7.65–7.60 (m, 1H, ArH), 7.57–7.51 (m, 2H, ArH), 6.58(dd, J = 6.2, 7.4 Hz, 1H, H-1'), 4.86–4.79 (m, 2H, C H 2 N3), 4.70–4.64 (m, 1H, H-3'), 4.33 (s, 2H, C H 2 N), 4.15–4.04 (m, 4H, H-4', OC H 3 ), 3.83–3.71 (m, 2H, H-5'a, H-5'b), 2.77–2.68 (m, 1H, H-2'a), 2.55 (ddd, J = 3.0, 6.3, 13.6 Hz, 1H, H-2'b); HRMS (ESI) m / z for C 25 H 23 Calculated value of F3N8O6 [M]: 588.1705; Measured value [M + H]: + 589.1776.

[0632] 6.12.8. Scheme 27. Synthesis of (L) 3'-O-azidomethyl-7-denitro-dGTP-Cy5

[0633]

[0634]

[0635] Azide-Cy5 compounds (Cy5-N3-linker). 2-{2-[3-(2-amino-ethylcarbamoyl)-phenoxy]-1-azido-ethoxy}-ethoxy)-acetic acid linker-6 (7.0 mg, 0.019 mmol) prepared according to the literature (Milton J, Ruediger S, Liu X (2006), US Patent Application US20060160081A1) was dissolved in DMF (300 µl) and 1 M NaHCO3 aqueous solution (100 µl). A solution of Cy5 NHS (N-hydroxysuccinimide) ester (Invitrogen) (0.013 mmol) in DMF (400 µl) was slowly added to the above reaction mixture, and then stirred at room temperature in the dark for 5 h. The crude product was purified on preparative silica gel TLC plates (CHCl3 / CH3OH, 1:4).

[0636] The L-3'-O-N3-7-denitrified-dGTP-Cy5 compound (L-3'-O-N3-7-denitrified-dGTP-Cy5) was added to a stirred solution of the Cy5-N3-linker in dry DMF (2 ml) at a concentration of DSC (N,N'-disuccinimidyl carbonate) (3.4 mg, 13.2 µmol) and DMAP (4-dimethylaminopyridine) (1.6 mg, 13.2 µmol). The reaction mixture was stirred at room temperature for 2 h. TLC showed that the Cy5-N3-linker was completely converted to the compound Cy5-N3-linker NHS ester, which was then used directly for coupling with L-amino-7-denitrified-dGTP (13 µmol) in NaHCO3 / Na2CO3 buffer (pH 8.7, 0.1 M) (300 µl). The reaction mixture was stirred at room temperature in the dark for 3 h. The reaction mixture was purified by preparative silica gel TLC (CH3OH / CH2Cl2, 1:1). The crude product was further purified by reversed-phase HPLC to give L-3'-O-N3-dGTP-Cy5 (L-3'-O-N3-dGTP-Cy5).

[0637] 6.13. Example 13. Chemical synthesis of (D)-type 9°N DNA polymerase via solid-phase peptide synthesis and natural chemical linking

[0638] Figure 1 The amino acid sequence of the (D)-type mutant 9°N DNA polymerase (candidate #1-1) is provided, in which all amino acids are D-type amino acids. Rhombus = mutation introduced for NCL; circle = NCL site (start of the "second" strand); square = potential substitution site for isoleucine; triangle = mutation introduced for modified nucleotide.

[0639]

[0640] The 9°N DNA polymerase was used to cleave the DNA at the cleavage site between K466 and M467 into two fragments (466-aa 9°NN fragment and 310-aa 9°NC fragment). The 466-aa 9°NN fragment was then synthesized into nine synthetic peptides (D-9°NN-1 to D-9°NN-9) via solid-phase peptide synthesis. Figure 2A ), which is linked at certain cysteine ​​residues, such as Figure 2B As shown in the diagram. Similarly, the 310-aa 9°NC fragment was split into six synthetic peptides ( Figure 3A ), which is synthesized via solid-phase peptide synthesis and then linked at certain cysteine ​​residues, such as Figure 3B As shown in the image.

[0641] Experimental methods:

[0642] Materials. 2-Chlorotriphenylmethyl chloride resin (loading = 0.98 mmol g) -1 The following were purchased from Purepep: Fmoc-D-amino acids, D-4-thiazolidinic acid, hydrazine hydrate, ethyl cyanoglyoxylate-2-oxime (Oxyma), N,N'-diisopropylcarbodiimide (DIC), trifluoroacetic acid, N,N-dimethylformamide (DMF), dichloromethane, piperidine, anisole, triisopropylsilane, 1,2-ethylenedithiol, and trifluoroacetic acid were commercially available from Chempep, Sigma-Aldrich, Alfa Aesar, TCI, etc. The reagents used in the NCL reaction, namely guanidine hydrochloride (Gn·HCl), Na2HPO4·12H2O, NaH2PO4·2H2O, sodium nitrite (NaNO2), sodium hydroxide (NaOH), sodium 2-mercaptoethanesulfonate hydrochloride, 4-mercaptophenylacetic acid (MPAA), tris(2-carboxyethyl)phosphine hydrochloride (TCEP·HCl), DL-1,4-dithiothreitol (DTT), 2,2′-azobis[2-(2-imidazolin-2-yl)propane] dihydrochloride (VA-044), glutathione (reduced form), and palladium chloride (PdCl2), were commercially available from Sigma-Aldrich, Alfaaesar, TCI, Duksan, etc.

[0643] Fmoc-based SPPS. All peptides were synthesized using Fmoc-based SPPS on a Liberty Blue automated microwave peptide synthesizer (CEM) and a PurePep® Chorus automated peptide synthesizer. All peptidylhydrazides were synthesized on hydrazine-2-chlorotriphenylmethyl chloride resin. For each peptidylhydrazide, the first residue was attached to the hydrazine-2-chlorotriphenylmethyl chloride resin via a double coupling method using 5 equivalents of amino acid, 10 equivalents of DIC, and 5 equivalents of Oxymapure. All resins were swollen in DMF for 30 min prior to coupling. The Fmoc groups of the assembled amino acids were removed by treatment with a DMF solution of 20% piperidine and 0.1 M Oxyma at 85 °C. Coupling of amino acids other than Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH was performed at 85 °C for 2 min using 5 equivalents of amino acid, 5 equivalents of Oxymapure, and 10 equivalents of DIC. The coupling reaction for Fmoc-Cys(Trt)-OH and Fmoc-His(Trt)-OH was carried out at 50 °C for 10 min to avoid side reactions at high temperatures. Trifluoroacetylthiazolidin-4-carboxylic acid-OH was coupled overnight at room temperature using 5 equivalents of Oxymapure and 10 equivalents of DIC. For peptides with more than 20 amino acids, a dual coupling strategy was used. After peptide chain assembly was completed, the peptide was cleaved from the resin using H2O / thioanisole / triisopropylsilane / 1,2-ethylenedithiol / trifluoroacetic acid (0.5 / 0.5 / 0.5 / 0.25 / 8.25) (v / v). The cleavage reaction was carried out at 27 °C with stirring for 2.5 h. Cold ether was added to precipitate the crude peptide. After centrifugation, the supernatant was discarded and the precipitate was washed twice with ether. The crude peptide was dissolved in CH3CN / H2O, analyzed by RP-HPLC, and purified by semi-preparative HPLC. The collected peptide fractions were analyzed by electrospray ionization mass spectrometry (ESI-MS).

[0644] Natural chemical bonds (NCLs).

[0645] Method A: The C-terminal peptidylhydrazine fragment was dissolved in an acidified ligation buffer (aqueous solution of 6 M Gn·HCl and 0.1 M NaH2PO4, pH 3.0). The mixture was cooled in an ice-salt bath (-15°C), and 10 equivalents of NaNO2 were added to the acidified ligation buffer (pH 3.0). The activation reaction system was maintained in an ice-salt bath with stirring for 20 min, followed by the addition of 40 equivalents of MPAA and 1 equivalent of N-terminal cysteine ​​peptide to the ligation buffer, and the pH of the solution was adjusted to 6.6–6.8 at room temperature. After overnight reaction, 150 mM TCEP was added to the ligation buffer (pH adjusted to 7.0) to dilute the system twice, and the reaction system was maintained at room temperature with stirring for 30 min. Finally, the ligation product was analyzed by HPLC and purified by semi-preparative HPLC. The purified ligation fraction was analyzed by ESI-MS.

[0646] Method B: General Method. In a 1.5 mL Eppendorf tube, dissolve peptidylhydrazine at a specific concentration (typically 1-20 mg / mL) in 6 M Gn.HCl containing 200 mM MPAA. This forms a heterogeneous suspension, and sonication / vortexing should break up any large pieces of solid MPAA. If necessary, adjust the pH to pH 3. Prepare a stock solution of acetylacetone (acac) in water (10×-20×) and add 1 to 5 equivalents of aacac to the peptide mixture. Finally, add a small stir bar to the tube and allow the mixture to stir for 2-4 hours.

[0647] After 2–4 hours, dissolve an equimolar amount of the Cys-fragment peptide (the C-terminal fragment of the ligation product) in 6 Mgn.HCl with 200 mM Na2HPO4 and 50 mM TCEP (TCEP is used to reduce disulfides between Cys fragments and is not necessary) (pH 8.5) to a volume equal to the volume in which the thioesterification reaction occurs. Combine the two solutions; the resulting pH will be approximately 5–7 and the MPAA emulsion will dissolve. The solution should be clear or slightly yellow. Then adjust the pH of the combined solution to pH 7–7.4 by adding 1 M NaOH. The ligation reaction can then be stirred for 4–18 hours.

[0648] Synthesis of Tfa-D-Thz-OH:

[0649]

[0650] Triethylamine (2.62 mL, 18.772 mmol) was added to a solution of D-4-thiazolidinic acid (1 g, 7.509 mmol) suspended in MeOH (40 mL), and the suspension was stirred for 10 min. Then, ethyl trifluoroacetate (0.98 mL, 8.2599 mmol) was added dropwise, and the mixture was stirred at room temperature for 48 h. The mixture was then concentrated and subjected to silica gel chromatography using MeOH / CH2Cl2 (1:5) to obtain Tfa-(D)-Thz-OH (i.e., (…)) as a pale yellow oil. S )-3-(2,2,2-trifluoroacetyl)thiazolidin-4-carboxylic acid)(0.9 g, 3.92 mmol, 52.3%) Figure 4A ). 1 H NMR (500 MHz, CDCl3): δ (ppm) 9.35 (s, 1H), 5.09 (dd, J = 6.9, 4.2 Hz, 1H), 4.86-4.77 (m, 1H, rotational isomer), 4.75-4.61 (m, 1H, rotational isomer), 3.52-3.28 (m, 2H, rotational isomer) Figure 4B It should be understood that Thz can also be called Cys (Thz) because it is a protected cysteine ​​residue.

[0651] SPPS based on Fmoc ( Figure 5 ):

[0652] 1) 102 mg of 2-Cl-(Trt)-Cl resin (0.98 mmol g) -1 Weigh it into the peptide synthesis container.

[0653] 2) Add 5 ml of DMF to the resin from the top of the reaction vessel, stir gently for 10 seconds, and then drain it.

[0654] 3) Repeat step 2 twice more.

[0655] 4) Add 5 ml of DCM to the resin, stir gently for 10 seconds, and then drain it.

[0656] 5) Repeat step 4 twice more.

[0657] 6) Repeat step 2 three more times.

[0658] 7) Swell the resin in 4 ml of 50% (volume / volume) DMF / DCM for 30 min, and then drain it.

[0659] 8) Add 4 ml of 5% (v / v) NH2NH2.H2O to the resin for hydrazination. Gently stir the mixture in a constant temperature shaker at 30°C for 30 min, and then filter the solution through a vacuum filter.

[0660] 9) Add 4 ml of DMF to the resin, stir gently for 10 seconds, and then drain it.

[0661] 10) Repeat step 8.

[0662] 11) Wash the resin twice by repeating steps 2–6.

[0663] 12) Add 4 ml of 5% (volume / volume) MeOH / DMF to the resin, stir gently for 10 min, and then drain it onto the unreacted sites on the resin to be encapsulated.

[0664] 13) Repeat steps 2–6 to thoroughly wash the resin.

[0665] 14) Add 4 ml of DCM to the resin, stir gently for 10 seconds, and then drain. Use the resin directly for the next coupling step.

[0666] 15) For storage, the resin was dried under high vacuum for 2 h, and the dried 2-Cl-(Trt)-NHNH2 resin was stored at -20°C under argon for up to 2 weeks.

[0667] Solid-phase peptide synthesis of D-9°NN-6@5-mer: Tfa-Thz-AVYE-NHNH2 (SEQ ID NO: 11) Figure 6A D-9°NN-6@5-mer was synthesized on a PurePep® Chorus automated peptide synthesizer following the conditions described in the experimental method. The D-9°NN-6@5-mer synthesized by this method was analyzed by HPLC chromatogram, and the results are provided. Figures 6B-6C middle.

[0668] Solid-phase peptide synthesis of D-9°NN-6@11-mer: CVFGKPKEKVY-NHNH2 (SEQ ID NO: 20): D-9°NN-6@11-mer was synthesized on a PurePep® Chorus automated peptide synthesizer following the conditions described in the experimental method. The D-9°NN-6@11-mer synthesized by this method was analyzed by HPLC chromatogram, and the results are provided. Figures 7B-7D middle.

[0669] Solid-phase peptide synthesis of D-9°NN-6@24-mer: CEEIAQAWESGEGLERVARYSMED-NHNH2 (SEQ ID NO: 13) Figure 8A D-9°NN-6@24-mer was synthesized on a PurePep® Chorus automated peptide synthesizer following the conditions described in the experimental method. The D-9°NN-6@24-mer synthesized by this method was analyzed by HPLC chromatogram, and the results are provided. Figures 8B-8C middle.

[0670] Solid-phase peptide synthesis of D-9°NC-3@9-mer: CDTDGLHAT-NHNH2 (SEQ ID NO: 14) Figure 9A D-9°NC-3@9-mer was synthesized on a Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) following the conditions described in the experimental method. The D-9°NC-3@9-mer synthesized by this method was analyzed by HPLC chromatogram, and the results are provided. Figures 9B-9C middle.

[0671] Solid-phase peptide synthesis of custom D-9°NC-1@33-mer: MKATVDPLEK KLLDYRQRLI KILANSFYGYYGY-NHNH2 (SEQ ID NO: 46). The D-9°NC-1@33-mer synthesized by this method was analyzed by HPLC chromatogram, and the results are provided. Figures 10A-10B middle.

[0672] Solid-phase peptide synthesis of custom D-9°NC-2@39-mer: CKARWY-C(Acm)-KE-C(Acm) AESVTAWGREYIEMVIRELE EKFGFKVLY-NHNH2 (SEQ ID NO: 47). The D-9°NC-2@39-mer synthesized by this method was analyzed by HPLC chromatogram, and the results are provided. Figure 11A-11B middle.

[0673] Solid-phase peptide synthesis of D-9°NC-3@21-mer: TFA-Thz-DTDGLHATIPGADAETVKKK-NHNH2 (SEQ ID NO: 15). D-9oN-C-3@21-mer was synthesized on a Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) following the conditions described in the experimental method. The product was observed to be TFA-deprotected D-9°NC-3@21-mer, as shown in the image. Figure 12B and 12C Provided in [the document / source].

[0674] Solid-phase peptide synthesis of D-9°NC-3@Cys35-mer: CKEFLKYINP KLPGLLELEY EGFYVRGFFVTKKKY-NHNH2. (SEQ ID NO: 16) D-9 was synthesized on a Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) by following the conditions described in the experimental method. o NC-3@Cys35-mer. The product was observed to be of the expected quality, such as... Figure 13B-13D Provided in [the document / source].

[0675] Solid-phase peptide synthesis of D-9°NC-3@56-mer: CDTDGLHATIPGADAETVKKKAKEFLKYINPKLPGLLELEYEGFYVRGFFVTKKKY-NHNH2. (SEQ ID NO: 17) Figure 14A D-9 was synthesized on a Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) following the conditions described in the experimental method. o NC-3@56-mer. The product was observed to be of the expected quality, such as... Figure 14B Provided in [the document / source].

[0676] D-9°NC-3@35-mer:AKEFLKYINP KLPGLLELEY EGFYVRGFFV TKKKY-NHNH2. (SEQ IDNO: 18)( Figure 15A D-9 was synthesized on a Liberty Blue 2.0 automated microwave peptide synthesizer (CEM) following the conditions described in the experimental method. o NC-3@35-mer. The product was observed to be of the expected quality, such as... Figure 15B Provided in [the document / source].

[0677] D-9 o Synthesis of NN-6@16-mer: Tfa-Thz-AVYECVFGKPKEKVY-NHNH2 (SEQ ID) NO:25). Used in the preparation of D-9. o The NN-6@16-mer method in Figure 16B It is shown in the figure, and D-9 is included. o HPLC chromatographic analysis of NN-6@16-mer is provided Figure 16C middle.

[0678] Synthesis of D-9oN-C-7@72-mer: MKATVDPLEKKLLDYRQRLIKILANSFYGYYGYCKARWY-C(Acm)-KE-C(Acm) AESVTAWGRE YIEMVIRELE EKFGFKVLY-NHNH2 (SEQ ID NO: 23). The method used to prepare D-9oN-C-7@72-mer is described in... Figure 17B As shown in the image. Figure 17C This is an analytical HPLC chromatogram (λ=214 nm) showing the peak transition during the NCL reaction. Figure 17D It is an ESI-MS with MPAA attachment, specifically D-9°NC-7@72-mer, and Figure 17E The connection of the MPAA-attachment is shown.

[0679] The following peptides were produced via solid-phase peptide synthesis on a PurePep® Chorus automated peptide synthesizer, following the conditions described in the experimental method. The products were analyzed by HPLC chromatographic analysis to confirm peptide production.

[0680]

[0681]

[0682]

[0683] Natural Chemical Linkage (NCL): Reagent Preparation: NaOH, 1 M and 6 M. Dissolve 40 mg or 240 mg of NaOH in 1 ml of deionized H₂O. HCl, 6 M. Mix 1 ml of 12 M HCl with 1 ml of deionized H₂O. Two different phosphate solutions (0.1 M) containing 6 M Gn·HCl (pH 3.0–3.1 and pH 5.7–6.0, respectively). For a 10-ml solution, mix 156 mg of NaH₂PO₄·2H₂O and 5.74 g of Gn·HCl in a 10-ml volumetric flask and adjust to the final volume with deionized H₂O. Adjust the pH to 3.0–3.1 with 6 M NaOH and 6 M HCl. Filter the solution using a 13 mm × 0.22 µm microporous membrane filter. The filtered solution can be stored at 4°C for at least 1 month. Na₂HPO₄·12H₂O (0.1 M) (pH 5.7–6.0) containing 6 M Gn·HCl was also prepared in the same manner. NaNO₂, 0.5 M: 17 mg of NaNO₂ was dissolved in 0.5 ml of deionized H₂O. TCEP, 0.1 M: 58 mg of TCEP·HCl was dissolved in 1 ml of 0.2 M phosphate solution (pH 3.0) containing 6 M guanidine hydrochloride (Gn·HCl). The pH was adjusted to 6.0–7.0, and the solution was filtered using a 13 mm × 0.22 µm microporous membrane filter. VA-044, 0.1 M: 9.7 mg of VA-044 was weighed into a 2-ml Eppendorf reaction tube, and 0.3 ml of 0.2 M phosphate solution (pH 6.9–7.0) containing 6 M Gn·HCl was added. VA-044 was completely dissolved using a vortex and ultrasonic cleaning bath.

[0684] Synthesis of D-9oN-N-10@(His)6+101-mer:

[0685] (His)6-MILDTDYITENGKPVIRVFKKENGEFKIEYDRTFEPYFYCLLKDDSAIEDVKKVTAKRHGTVVKVKRAEKVQKKFLGRPVEVWKLYFNHPQDVPAIRDRIR-NHNH2 (SEQ ID NO: 52). ( Figure 18A D-9 was synthesized using natural chemical linking method A. o NN-10@(His)6+101-mer. Used for the preparation of D-9. o The method NN-10@(His)6+101-mer in Figure 18B It is shown in the figure, and D-9 is included.o HPLC chromatographic analysis of NN-10@(His)6+101-mer is provided. Figure 18C Purified D-9 o The deconvolutional MS (ESI-MS) spectrum of NN-10@(His)6+101-mer was calculated to be 12875.8 and measured to be 12876.8. For example... Figure 18B As shown, D-9 o NN-1@(His)6+39-mer (2 mg, 1 equivalent) was dissolved in 75 µL of acidified ligation buffer (aqueous solution of 6 M Gn·HCl and 0.1 M NaH2PO4, pH 3.0). The mixture was cooled in an ice-salt bath (-15 °C), and 7.2 μL of 0.5 M NaNO2 (in acidified ligation buffer) was added. The reaction was maintained in an ice-salt bath with stirring for 20 min, after which 70 µL of 0.2 M MPAA (in 6 M Gn·HCl and 0.1 M Na2HPO4, pH 6.5) was added with D-9. o NN-2@62-mer (2.58 mg, 1 equivalent) was used, and the pH of the reaction mixture was adjusted to 6.6–6.8 at room temperature using NaOH solution. After 14 h, the reaction mixture was reduced by 0.15 M TCEP and purified by HPLC (purification conditions: 5%–95% CH3CN (0.1% TFA) gradient in H2O (with 0.1% TFA) over 30 min on a Welch C4 column).

[0686] Synthesis of D-9oN-N-13@108-mer:

[0687] D-9 was synthesized using natural chemical linking method B. o NN-13@108-mer. Used for the preparation of D-9. o The NN-13@108-mer method in Figure 19B As shown in the figure, and D-9 o HPLC chromatographic analysis of NN-13@108-mer is provided Figure 19C In the middle. For example Figure 19B As shown, fragment D-9 was placed in a 1.5 mL Eppendorf tube. o NN-7@52-mer (2.2 mg) was dissolved in 300 µL of 6 M Gn.HCl containing 200 mM MPAA. Acac was dissolved in water to a final concentration of 150 mM and 2.5 equivalents were added to D-9. oIn a solution of NN-7@52-mer, add a small stir bar to the Eppendorf tube and allow the reaction to stir for 4 hours. Add fragment D-9. o NN-8@56-mer (2.29 mg, 1 equivalent) was dissolved in 300 µL of 6 M Gn.HCl containing 200 mM Na2HPO4·12H2O at pH 8.5. This solution was then added to the thioesterification solution, resulting in a 600 µL solution at pH 5.3. The pH of this solution was adjusted to 7.0 by adding 1 M NaOH and the reaction was stirred overnight. After 18 hours, 50 mM TCEP was added and the mixture was analyzed by HPLC after stirring for 30 min. c) Analytical HPLC chromatogram of peak transition during the NCL reaction (λ = 214 nm). Column: Welch C4. Gradient: 5%-95% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) for 30 min [√ = confirmed peak]. Figure 19D Purified D-9 is shown. o Deconvolutional MS (ESI-MS) spectrum of NN-13@108-mer [calculated value: 12467.52, observed value: 12467.39].

[0688] Using this method, the following peptides were synthesized.

[0689]

[0690] Using similar methods, such as Figure 3B The example shown is the generation of D-9°NC-11. Furthermore, D-9°NC-12 is generated through Cys at one or more locations. 500 Cys 539 Cys 595 Cys 651 and Cys 714 D-9°NC-11 is generated by desulfurizing one or more cysteine ​​residues to alanine residues. D-9°NC-12 is produced by desulfurizing D-9°NC-11 via Cys... 506 and Cys 509 It is generated by deprotecting Acm.

[0691] 6.14. Example 14. Chemical synthesis of (D)-terminal deoxynucleotidyl transferase via solid-phase peptide synthesis and natural chemical linkage (Method 1)

[0692] Figure 20A provides the amino acid sequence of a (D)-terminal deoxynucleotidyl transferase, in which all amino acids are D-type amino acids. The (D)-terminal deoxynucleotidyl transferase is a 381-aa protein. Figure 20 B provides a synthetic chemical linkage pathway for terminal deoxynucleotidyl transferases. The synthesis of terminal deoxynucleotidyl transferases is designed to involve the synthesis of seven synthetic peptides (D-TdT-WT-1 to D-TdT-WT-7) via solid-phase peptide synthesis, and these seven synthetic peptides are linked at specific cysteine ​​residues, as shown.

[0693] To synthesize these seven synthetic peptides, the experimental methods related to D-polymerase described above were used. For example, Figure 21A The following diagram illustrates D-TdT-WT-1@(His)6+46-mer: (His)6NSSPSPVPGSQNVPAPAVKKISQYAC(Acm)QRRTTLNNYNQLFTDALDIL-NHNH2 (SEQ ID NO: 57). D-TdT-WT-1@His6+46-mer was synthesized on a PurePep® Chorus automated peptide synthesizer following the conditions described in the experimental method. The D-TdT-WT-1@His6+46-mer synthesized by this method was analyzed by HPLC chromatogram, and the results are provided. Figure 21B middle.

[0694] Figure 21A It is the peptide sequence of D-TdT-WT-1@(His)6+46-mer. Figure 21B This is the analytical HPLC chromatogram of crude D-TdT-WT-1@(His)6+46-mer (λ=214 nm). Column: Welch C4. Gradient: 1-20 min in ACN (0.1% TFA) 100%-80% water (0.1% TFA), 20-40 min in ACN (0.1% TFA) 80%-50% water (0.1% TFA), 40-60 min in ACN (0.1% TFA) 50%-20% water (0.1% TFA), and 60-70 min in ACN (0.1% TFA) 20%-0% water (0.1% TFA) [√=confirmation peak]. Figure 21CThe image shows the deconvolution MS (ESI-MS) spectrum of purified D-TdT-WT-1@His6+46-mer [calculated value: 5922.97, observed value: 5922.97]. Purification conditions: 1-20 min in 100%-80% water (0.1% TFA) in ACN (0.1% TFA), 20-40 min in 80%-50% water (0.1% TFA) in ACN (0.1% TFA), 40-50 min in 50%-20% water (0.1% TFA), and 50-60 min in 20%-0% water (0.1% TFA) in ACN (0.1% TFA) (C18 semi-preparative column).

[0695] Furthermore, the following peptides were produced via solid-phase peptide synthesis on the PurePep® Chorus automated peptide synthesizer, following the conditions described in the experimental method. The products were analyzed by HPLC chromatographic analysis, confirming the peptide production.

[0696]

[0697] Following the protocol described above, these seven synthetic peptides were linked at specific cysteine ​​residues. For example, in a 1.5 mL Eppendorf tube, fragment D-TdT-WT-1@(His)6+46-mer (3.0 mg) was dissolved in 300 µL of 6 M Gn.HCl containing 200 mM M PAA. Acac was dissolved in water to 150 mM and 2.5 equivalents were added to the solution of D-TdT-WT-1@(His)6+46-mer. A small stir bar was added to the Eppendorf tube and the reaction was allowed to stir for 4 hours. Fragment D-TdT-WT-2@61-mer (2.74 mg, 0.8 equivalents) was dissolved in 300 µL of 6 M Gn.HCl containing 200 mM Na2HPO4·12H2O at pH 8.5. Figure 22A This solution was then added to the thioesterification solution, yielding a 600 µL solution at pH 5.3. The pH of this solution was adjusted to pH 7.0 by adding 1 M NaOH, and the reaction was stirred at room temperature. After 48 hours, 50 mM TCEP was added, and the mixture was analyzed by HPLC after stirring for 30 min. Figure 22B D-TdT-WT-8@(His)6+107-mer was purified by preparative HPLC (retention time: 13.8 min). Figure 22CColumn: Welch C4. Gradient: 70%–30% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) for 30 min [√ = confirm peak]. The observed MS for D-TdT-WT-8@(His)6+107-mer was 12663.23 (calculated value 12663.21). Figure 22D )

[0698] Using this method, the following peptides were synthesized.

[0699]

[0700]

[0701]

[0702] Preparation of D-TdT-WT-14@(His)6+381-mer:

[0703] D-TdT-WT-13@(His)6+381-mer was dissolved in 200 mM TCEP solution (6M Gn.HCl and 0.1 M Na2HPO4, pH 7.0) containing 20 mM VA-044 and 40 mM reduced L-glutathione. The reaction was stirred overnight at 37 °C. The desulfurization product D-TdT-WT-14@(His)6+381-mer was purified by semi-preparative HPLC (retention time: 17.2 min). The observed MS value of D-TdT-WT-14@(His)6+381-mer was 44854 (calculated value 44855.2). Column: Welch C4. Gradient: 20% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) within 10 min, then 20%-70% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) within 20 min. This reaction results in Cys at one or more sites. 47 Cys 108 Cys 162 Cys 224 Cys 268 and Cys 317 One or more cysteine ​​residues in D-TdT-WT-13 are desulfurized to alanine residues.

[0704] Synthesis of D-TdT-WT-15@(His)6+381-mer:

[0705] D-TdT-WT-14@(His)6+381-mer was dissolved in an aqueous solution of 6 M Gn.HCl and 0.1 M Na2HPO4, 40 mM TCEP, pH 7.0. PdCl2 was dissolved in an aqueous solution of 6 M Gn.HCl and added to the peptide solution. The reaction was stirred at 30 °C. After 2 hours, 2 M DTT (in an aqueous solution of 6 M Gn.HCl) was added. The reaction mixture was stirred for 30 min and purified by HPLC to obtain D-TdT-15@(His)6+381-mer (retention time: 17.5 min). The observed MS value of D-TdT-WT-15@(His)6+381-mer was 44365 (calculated value 44367.2). Column: Welch C4. Gradient: 20% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) over 10 min, then 20%-70% CH3CN (with 0.1% TFA) in H2O (with 0.1% TFA) over 20 min. This reaction leads to Cys 26 Cys 59 Cys 87 Cys 173 Cys 249 Cys 275 and Cys 309 The Acm protection at the location is removed.

[0706] 6.15. Example 15: Synthesis of L-polynucleotides using D-terminal deoxynucleotidyltransferase.

[0707] Mix D-terminal deoxynucleotidyl transferase with reaction buffer, L-DNA fragment, L-dNTPs (e.g., L-dATP, L-dTTP, L-dGTP, or L-dCTP), and water. Incubate the mixture at 22°C–37°C for 15–30 minutes. Then stop the reaction by heating at 70°C for 10 minutes or by adding 2 µL of 0.5 M EDTA.

[0708] Mix D-terminal deoxynucleotidyl transferase with reaction buffer, L-DNA fragment, L-3'-OR-dNTP (N=A, C, G, or T), and water. Incubate the mixture at 22°C–37°C for 15–30 minutes. Remove the 3'-OR group using a deprotection reaction (i.e., if R = azidomethyl, incubate at 37°C–55°C in 50 mM TCEP buffer for 5–15 minutes). The next nucleotide addition can be performed by repeating the reaction above.

[0709] The L-polynucleotides generated by this method were sequenced, and the incorporation of L-dNTPs into the 3' end of the DNA fragments was confirmed. The synthesized L-polynucleotides can be used for information storage.

[0710] D-terminal deoxynucleotidyl transferase is also used for 3' end labeling of DNA and oligonucleotides. The enzyme is mixed with radiolabeled L-ddNTPs, buffer, linear DNA, and water. The mixture is incubated at 37°C for 15 minutes. The reaction is then stopped by heating at 70°C for 10 minutes or by adding 2 µL of 0.5 M EDTA.

[0711] The L-polynucleotides generated by this method were sequenced, and the L-ddNTPs were confirmed to be incorporated into the 3' end of the linear DNA.

[0712] 6.16. Example 16: Synthesis of (L) 3'-O-azidomethyl-7-denitro-dNTP-labeled intermediate

[0713] Material

[0714] Unless otherwise stated, all solvents and reagents are reagent-grade, commercially available, and used without further purification. All chemicals were purchased from Sigma-Aldrich, Fisher Scientific, TCI, etc. 1 H NMR from Bruker Ascend, Chapman University TM Recorded on a (400 MHz) spectrometer and reported in parts per million (ppm) of CDCl3 (7.26 ppm) or D2O. Data are reported as follows: (s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, dd = doublet, J = coupling constant, in Hz, integral). Proton decoupling... 31 P NMR spectra from Bruker Ascend, Chapman University TMRecorded on a 121.4 MHz spectrometer. High-resolution mass spectrometry (HRMS) was obtained from the Analytical Chemistry Instrumentation Facility of Chapman University School of Pharmacy and University of California, Riverside. Starting materials β-L-deoxythymidine, β-L-deoxycytidine, β-L-deoxyadenosine, and β-L-deoxyguanosine were purchased from Chemgenes. Analytical (Polaris 180A C18-A, 4.6 x 250 mm, 5 μm) and semi-preparative (Polaris 180A C18-A, 4.6 x 250 mm, 5 μm) HPLC columns were purchased from Agilent. 3'-O-modified nucleotides were purified by reversed-phase HPLC on a 4.6 x 250 mm C18 column (Polaris) using the following mobile phases: A, 25 mM TEAB buffer in water; B, 25 mM TEAB buffer in acetonitrile. Elution was performed under isocratic conditions as described in each procedure.

[0715] 6.16.1. Scheme 29: Synthesis of NH2-L-3'-O-azidomethyl-7-denitro-dATP:

[0716]

[0717]

[0718]

[0719] 6.16.1.1 Experimental Procedure:

[0720]

[0721] Synthesis of 7dNATP-FL-1: At room temperature, powdered KOH (1.8 g, 7.57 mmol) and TDA-1 (0.3 ml, 0.86 mmol) were added to a solution of 4-chloro-5-iodo-7H-pyrrolo[2,3-d]pyrimidine (4.0 g, 14.4 mmol) in MeCN (200 ml). After stirring for 30 min, 1-Cl-2-deoxy sugar (8.6 g, 18.7 mmol) was introduced and stirring was continued for another 30 min. The insoluble material was filtered off and washed several times with hot acetone. The combined filtrates were evaporated to dryness. The residue was subjected to purification by chromatography on silica (EtOAc:hexane 1:1 to 100% EtOAc) to give 7-dNATP-FL-1 (4.22 g, 89%) as a yellow solid. 1HNMR (400 MHz, CD3OD) δ 8.44 (s, 1H), 7.89 (d, 2H), 7.78 (d, 2H), 7.73 (s, 1H), 6.66 (dt, J = 1.61Hz, J = 7.85Hz, 1H), 4.51-4.49 (m, 1H), 3.56 (s, 1H), 2.98-2.93 (m, 1H), 2.75–2.70 (m, 1H), 2.34 (s, 1H) 2.32 (s, 1H); HRMS (ESI): m / z [M] calculated as C 27 H 23 ClIN3O5 631.0371; Measured value [M+ H]+ 632.0456

[0722]

[0723] Synthesis of 7dNATP-FL-2: A suspension of 7dNATP-FL-1 (1.2 g, 1.89 mmol) in a mixture of 28% NH3 aqueous solution and dioxane (1:1, 100 ml) was stirred under reflux for 17 h. The residue was subjected to purification by chromatography on silica (EtOAc:hexane:CH3OH 1:1:0.1) to give 7-dNATP-FL-2 (296 mg, 41.4%) as a yellow solid. 1 HNMR (400 MHz, CD3OD) δ 8.11 (s, 1H), 7.61 (s, 1H), 7.76 (s,1H), 6.52 (dt, J = 2.1Hz, J = 6.0Hz,1H), 4.60 (s, 1H), 4.53-4.51 (m, 1H), 4.02-4.00 (m, 1H), 3.82–3.79(dd, J = 3.3Hz, J = 11.9Hz, 1H), 3.75-3.71(dd, J = 3.5Hz, J = 12Hz, 1H), 2.66–2.59 (m, 1H), 2.36–2.30 (m, 1H); HRMS (ESI): m / z [M] calculated as C 11 H 13 IN4O3 376.0039; Measured value [M + H] +377.0113

[0724]

[0725] Synthesis of 7dNATP-FL-3: CuI (24 mg, 0.1260 mmol) and triethylamine (250 μL) were added to a solution of 7dNATP-FL-2 (236 mg, 0.6298 mmol) in N,N-dimethylformamide (5 mL), and the mixture was stirred at room temperature for several minutes. Then, 2,2,2-trifluoro-N-(prop-2-ynyl)acetamide (285 mg, 1.89 mmol) and tetrakis(triphenylphosphine)palladium (0) (72.78 mg, 0.06 mmol) were added. The reaction was allowed to run at room temperature for 24 h and concentrated. The resulting residue was purified by column chromatography (1:10:10 to 1:4:4, CH3OH:CH2Cl2:EtOAc) to give 7dNATP-FL-3 (164.2 mg, 65.3%) as a yellow solid. 1 HNMR (400 MHz, CD3OD) δ 10.10 (s, 1H), 8.12 (s, 1H), 7.76 (s, 1H), 6.53-6.46 (m, 1H), 5.26 (d, 1H), 5.08 (t, J = 5.6 Hz, 1H), 4.35– 4.26 (m, 2H), 3.84–3.81(m, 1H), 2.59(dd, J = 4.8Hz, J = 0.8Hz, 1H), 2.49–2.43 (m, 1H), 2.21–2.16 (m, 1H); HRMS (ESI): m / z for C 16 H 16 Calculated value of F3N5O4 [M]: 399.1154; Measured value [M+H]: + 400.1234

[0726]

[0727] Synthesis of 7dNATP-FL-4: Imidazole (39.8 mg, 0.586 mmol) and tert-butyl(chloro)diphenylsilane (118.12 mg, 3.25 mmol) were added to a solution of 7dNATP-FL-3 (156 mg, 0.3907 mmol) in N,N-dimethylformamide (4 mL) at 0 °C, and then the temperature was raised to room temperature. After stirring at room temperature for 10 h, the solution was added to ice water and extracted with ethyl acetate × 2. The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (1:2 to 100%, hexane:EtOAc) to give 7dNATP-FL-4 (128 mg, 63.8%) as a yellow oil. 1 H NMR (400 MHz, CD3OD) δ 8.11 (s, 1H), 7.66 (d, 4H), 7.51 (s, 1H),7.42-7.33 (m, 6H), 6.56 (t, J = 6.5 Hz, 1H), 4.63–4.59 (m, 1H), 4.28 (s, 2H), 4.01–4.00 (m, 1H), 3.90 (dd, J = 11.5 Hz, J = 3.15 Hz, 1H), 3.81 (dd, J =11.4 Hz, J = 3.82 Hz, 1H), 2.57–2.38 (m, 2H), 1.05 (s, 9H); HRMS (ESI): m / z for C 32 H 34 The calculated value of F3N5O4Si [M] is 637.2362; the measured value is [M + H]. + 638.2438

[0728]

[0729] Synthesis of 7dNATP-FL-5: A solution of 7dNATP-FL-4 (128 mg, 0.2492 mmol) was dissolved in a mixture of CH3OH:N,N-dimethylacetal (10:1) and stirred at 40 °C. The reaction, monitored by TLC, was completed after 2 h. The solvent was removed under vacuum. Purification by chromatography on silica (EtOAc: CH3OH = 15:1 to 10:1) yielded 7dNATP-FL-5 (99.5 mg, 70.2%) as a clear yellow oil. HRMS (ESI): m / z against C 35 H39 The calculated value of F3N6O4Si [M] is 692.2788; the measured value is [M + H]. + 693.2865

[0730]

[0731] Synthesis of 7dNATP-FL-6: At room temperature, AcOH (800 uL) and acetic anhydride (800 uL) were added to a solution of 7dNATP-FL-5 (99.5 mg, 0.175 mmol) in DMSO (4 mL). After stirring at room temperature for 18 hours, ice-cold water and solid sodium bicarbonate were added to the solution. The aqueous layer was extracted twice with ethyl acetate. The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (hexane:EtOAc = 3:1 to 1:1) to give 7dNATP-FL-6 (48 mg, 40%) as a white solid. HRMS (ESI): m / z against C 35 H 39 The calculated value of F3N6O4Si [M] is 752.2821; the measured value is [M + H]. + 753.2897.

[0732]

[0733] Synthesis of 7dNATP-FL-7: Cyclohexene (0.03 ml, 0.16 mmol) was added to a solution of 7dNATP-FL-6 (20 mg, 0.032 mmol) in dry CH2Cl2 at 4 °C, followed by dropwise addition of sulfonyl chloride (1 M CH2Cl2 solution, 0.03 ml, 0.031 mmol) under N2. After 40 min, TLC indicated complete consumption of 7dNATP-FL-6, the solvent was evaporated, and the residue was subjected to high vacuum for 20 min. It was then redissolved in dry DMF (1 ml) and treated with NaN3 (30 mg, 0.45 mmol). The resulting suspension was stirred at room temperature for 2 h. The reaction was quenched with CH2Cl2, and the organic layer was washed with a saturated aqueous NaCl solution. After solvent removal, the resulting yellow gel was redissolved in 2 M HCl:acetonitrile (2:1) and stirred at room temperature for 30 min. The solvent was removed, and the reaction was quenched with CH2Cl2. l2 The aqueous layer was post-treated with a saturated NaHCO3 aqueous solution. The aqueous layer was extracted three times with CH2Cl2. Purification was performed on silica by chromatography (EtOAc:hexane 1:1 to 100% EtOAc) to give 7dNATP-FL-7 (15 mg, 73%) as a pale yellow foam.1 HNMR (400 MHz, CD3OD) δ 8.86 (s), 8.09(s), 6.44 (app dt, J =2.5 Hz, J = 6.0 Hz, 1H) 4.86-4.80 (m, 2H), 4.57-4.54(m, 1H),4.19–4.16 (m, 1H), 3.81 (dd, J = 3.6 Hz, 12 Hz, 1H), 3.75 (dd, J =12.1 Hz, J = 3.3 Hz, 1H), 2.73–2.66 (m, 1H), 2.53 (dd, J = 2.3 Hz, J = 5.7Hz, 1H), 2.49 (dd, J = 2.3 Hz, J = 5.9 Hz, 1H); HRMS (ESI): m / z for C 17 H 17 Calculated value of F3N8O4 [M]: 454.1341; Measured value [M + H]: + 455.1415.

[0734]

[0735] Synthesis of 7dNATP-FL-8: 7-dNATP-FL-7 (40 mg, 0.088 mmol) was dried overnight in a vacuum desiccator using P2O5. POCl3 (20.5 μL, 0.22 mmol) was added dropwise to a solution of 7dNATP-FL-7 in trimethyl phosphate (1 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h, and then a thoroughly vortexed mixture of tributylammonium pyrophosphate (110 mg) and tributylamine (157 μL, 0.620 mmol) in anhydrous DMF (0.5 mL) was added. The mixture was stirred at room temperature for 1 h, and then 0.1 M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.5, 20 mL) was added, and the mixture was stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure, and the residue was diluted with 10 mL of water. The crude mixture was extracted with CH2Cl2 (2 × 10 mL), and the aqueous layer was concentrated under reduced pressure. The residue was then purified by anion exchange chromatography on a DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.5; 0.1–0.8 M). Fractions containing the product (0.3 M–0.4 M) were collected and concentrated under reduced pressure.

[0736] HRMS (ESI): m / z for C 17 H 17 Calculated value of F3N8O4 [M]: 598.0486; Measured value [M – H]: – 597.0413.

[0737] The 7dNATP-FL-8 marker is made according to the synthesis described in Scheme 25.

[0738] 6.16.2. Scheme 30: NH2-L-3'- O Synthesis of -azidomethyl-7-denitro-dGTP:

[0739]

[0740]

[0741]

[0742]

[0743] 6.16.2.1 Experimental Procedure:

[0744]

[0745] Synthesis of denitrified-dGTP-FL-1: At room temperature, 2-amino-4-chloro-7H-pyrrolo[2,3-d]pyrimidine (421 mg, 2.50 mmol) was added to a suspension of KOH (0.68 g, 8.71 mmol) and TDA-1 (0.1 mL, 0.32 mmol) in CH3CN (30 mL). After stirring for 5 min, 1-Cl sugar (1.27 g, 3.25 mmol) was added, and stirring was continued for 30 min. The insoluble material was filtered off, the precipitate was washed with CH3CN, and the filtrate was evaporated to dryness. The residue was purified by column chromatography (hexane–EtOAc, 4:1 to 1:1) to obtain a white foamy product (1.10 g, 82%). At room temperature, a solution of NaOCH3 in CH3OH (0.5 M, 30 mL) was added to the starting material (1.10 g, 2.00 mmol). The solution was heated to reflux and stirred for 3 hours. The mixture was cooled to room temperature and neutralized by adding AcOH. The mixture was then evaporated to dryness. The residue was purified by column chromatography (hexane–EtOAc, 1:1 to 100% EtOAc) to obtain denitrified-dGTP-FL-1 (420 mg, 75%) as a pale yellow foam. R f 0.21 (100% EtOAc), 1 HNMR (400 MHz, CD3OD) δ 7.00(d, J = 3.7 Hz, 1H), 6.37 (dd, J 1',2'b = 5.9 Hz, J 1',2'a = 8.6 Hz, 1H, H-1'), 6.32 (d, J = 3.7 Hz, 1H), 4.51 (app dt, J 3',2'a = 5.8 Hz, J 3',2'b = J 3',4' = 2.3Hz, 1H, H-3'), 4.05–3.97 (m, 4H, OCH3, H-4'), 3.81 (dd, J 5'a,4' = 3.4 Hz, J gem =12.1 Hz, 1H, H-5'a), 3.72 (dd, J 5'b,4' = 3.5 Hz,J gem = 12.1 Hz, 1H, H-5'b),2.70 (ddd, J 2'a,3' = 5.9 Hz, J 2'a,1' = 8.6 Hz, J gem = 14.5 Hz, 1H, H-2'a), 2.24(ddd, J 2'b,3' = 2.3 Hz, J 2'b,1' = 5.9 Hz, J gem = 14.5 Hz, 1H, H-2'b).

[0746]

[0747] Synthesis of denitrified-dGTP-FL-2: At room temperature, butyric anhydride (1.22 mL, 7.50 mmol) was added to a solution of denitrified-dGTP-FL-1 (420 mg, 1.50 mmol) in pyridine (5 mL). After stirring for 24 h, the reaction mixture was quenched with CH3OH. The mixture was then evaporated and extracted with EtOAc, washed with 2N HCl and saturated NaHCO3 aqueous solution. The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (1:1, hexane–EtOAc) to obtain an intermediate (412 mg, 81%). Benzoyl chloride (705 μL, 6.07 mmol) was added to a solution of the intermediate (412 mg, 1.21 mmol) in pyridine at room temperature. After stirring for 2 h, the mixture was quenched with CH3OH and evaporated. The residue was diluted with EtOAc and washed with 2N HCl and saturated NaHCO3 aqueous solution. The organic layer was dried over Na2SO4, then filtered and concentrated. The residue was purified by column chromatography (3:1, hexane–EtOAc) to obtain denitrified-dGTP-FL-2 (425 mg, 67%) as a white foam. R f 0.53 (3:1, hexane–EtOAc), R f 0.52 (3:1 hexane–EtOAc), 1H NMR (400 MHz, CDCl3) δ7.89–7.82 (m, 4H, ArH), 7.54–7.46 (m, 2H, ArH), 7.41–7.35 (m, 4H, ArH), 7.17 (d, J = 3.7 Hz, 1H), 6.50 (d, J = 3.7 Hz, 1H), 6.39 (dd, J 1',2'b = 6.8 Hz, J 1',2'a = 8.5 Hz, 1H, H-1'), 5.25 (app dt, J 3',2'a = 6.2 Hz, J 3',2'b = J 3',4' = 2.2 Hz, 1H, H-3'), 4.35–4.29 (m, 2H, H-5'a, H-5'b), 4.24–4.19(m, 1H, H-4'), 3.79 (s, 3H, OCH3), 2.67–2.53 (m, 2H, (CH3)2C H ), 2.48–2.39 (m,1H, H-2'a), 2.33–2.24 (m, 1H, H-2'b), 1.25–1.16 (m, 12H, (C H 3 )2CH).

[0748]

[0749] Synthesis of denitrification-dGTP-FL-3: At room temperature, NIS (200 mg, 0.892 mmol) was added to a solution of denitrification-dGTP-FL-2 (425 mg, 0.81 mmol) in DMF (8.0 mL). After stirring overnight, the mixture was diluted with EtOAc and saturated with NaHCO3. 3(水溶液) Wash. Dry the organic layer with Na2SO4, filter, and concentrate. Purify the residue by column chromatography (hexane–EtOAc, 10:1 to 3:1) to obtain 7-dN-dGTP-FL-3 (330 mg, 63%) as a pale yellow foam. R f 0.42 (3:1, hexane–EtOAc), 1HNMR (400 MHz, CDCl3) δ 7.84–7.72 (m, 4H, ArH), 7.52–7.43 (m, 2H, ArH), 7.41–7.31 (m, 4H, ArH), 7.28 (s, 1H), 6.31 (dd, J =6.2 Hz, 8.0 Hz, 1H, H-1'), 5.24–5.19 (m, 1H, H-3'), 4.33–4.27 (m, 2H, H-5'a,H-5'b), 4.22–4.17 (m, 1H, H-4'), 3.77 (s, 3H, OCH3), 2.59 (septet, J = 6.9Hz, 2H, (CH3)2C H ), 2.35–2.19 (m, 2H, H-2'a, H-2'b), 1.21–1.19 (m, 6H, (C H 3 )2CH), 1.19–1.16 (m, 6H, (C H 3 )2CH); HRMS (ESI) m / z for C 34 H 35 The calculated value of IN4O8 [M] is 754.1518; the measured value is [M + H]. + 755.1591.

[0750]

[0751] Synthesis of denitrified-dGTP-FL-4: At room temperature, a 0.5 M CH3O3 solution (2 mL) of CH3OH was added to a solution of denitrified-dGTP-FL-3 (330 mg, 0.508 mmol) in CH3OH (8.0 mL). After stirring for 1.5 h, the mixture was neutralized by adding AcOH (100 uL) and then concentrated. The residue was purified by column chromatography (hexane–EtOAc, 3:1 to 100% EtOAc) to obtain denitrified-dGTP-FL-4 (240 mg, 75%) as a white solid. R f 0.23 (100% EtOAc); 1 HNMR (400 MHz, CD3OD) δ 8.01–7.95 (m, 2H, ArH), 7.66–7.59 (m, 2H, ArH,H-6), 7.57–7.50 (m, 2H, ArH), 6.66 (dd,J = 6.7, 6.6 Hz, 1H, H-1'), 4.59–4.51(m, 1H, H-3'), 4.10 (s, 3H, OC H 3 ), 3.99–3.94 (m, 1H, H-4'), 3.79 (dd, J =3.8, 12.0 Hz, 1H, H-5'a), 3.73(dd, J = 4.4, 12.0 Hz, H-5'b), 2.68–2.57 (m,1H, H-2'a), 2.38(ddd, J = 3.4, 6.1, 13.4 Hz, H-2'b); HRMS (ESI) m / z for C 19 H 19 Calculated value of IN4O5 [M]: 510.0409; Measured value [M + H]: + 511.0482.

[0752]

[0753] Synthesis of denitrified-dGTP-FL-5: CuI (15 mg, 0.095 mmol) and Et3N (0.5 mL) were added to a solution of denitrified-dGTP-FL-4 (200 mg, 0.392 mmol) in DMF (5.0 mL) at room temperature. After stirring under N2 for 5 min at room temperature, alkyne (177.5 mg, 1.18 mmol) and Pd(PPh3)4 (45 mg, 0.039 mmol) were added to the mixture at room temperature. After stirring for 24 h, the mixture was concentrated and the residue was purified by column chromatography (hexane–EtOAc, 1:1 to hexane–EtOAc–CH3OH, 2:2:1) to obtain denitrified-dGTP-FL-5 (185 mg, 88%) as a pale yellow oil. R f 0.11 (2:2:1, hexane–EtOAc–CH3OH); 1 HNMR (400 MHz, CD3OD) δ 8.03–7.91 (m,2H, ArH), 7.71–7.50 (m, 4H, ArH, H-6), 6.64 (dd, J = 6.7, 6.6 Hz, 1H, H-1'), 4.58–4.52 (m, 1H, H-3'), 4.33 (s, 2H, C H2 N), 4.10 (s, 3H, OC H 3 ), 3.99–3.94(m, 1H, H-4'), 3.79 (dd, J = 3.8, 12.0 Hz, 1H, H-5'a), 3.73 (dd, J = 4.4,12.0 Hz, H-5'b), 2.67–2.57 (m, 1H, H-2'a), 2.39 (ddd, J = 3.4, 6.1, 13.4 Hz, H-2'b); HRMS (ESI) m / z for C 24 H 22 Calculated value of F3N5O6 [M]: 533.1534; Measured value [M + Na]: + 556.1428.

[0754]

[0755] Synthesis of denitrified-dGTP-FL-6: TBDPSCl (118 uL, 0.454 mmol) was added to a solution of denitrified-GTP-FL-5 (220 mg, 0.412 mmol) in pyridine (2.0 mL) at 0 °C, and the mixture was slowly heated to room temperature. After stirring for 72 h, the mixture was quenched by adding CH3OH (5 mL) and then concentrated. The residue was diluted with EtOAc and washed with 2N HCl and saturated NaHCO3. The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (hexane–EtOAc, 4:1 to 1:1) to obtain denitrified-dGTP-FL-6 (235 mg, 74%) as a white foam. R f 0.34 (1:1, hexane – EtOAc); 1 HNMR (400 MHz, CD3OD) δ 7.98–7.90 (m,2H, ArH), 7.74–7.31 (m, 14H, ArH, H-6), 6.65 (dd, J = 6.7, 6.6 Hz, 1H, H-1'), 4.71–4.64 (m, 1H, H-3'), 4.33 (s, 2H, C H 2 N), 4.10 (s, 3H, OC H 3), 4.05–3.98(m, 1H, H-4'), 3.96–3.83 (m, 2H, H-5'a, H-5'b), 2.70–2.54 (m, 1H, H-2'a), 2.49–2.37 (m, 1H, H-2'b), 1.06 (s, 9H, (C H 3 )3C); HRMS (ESI) m / z for C 40 H 40 The calculated value of F3N5O6Si [M] is 771.2713; the measured value is [M + H]. + 772.2789.

[0756]

[0757] Synthesis of denitrification-dGTP-FL-7: At room temperature, AcOH (1.5 mL) and Ac2O (3 mL) were added to a solution of denitrification-dGTP-6 (180 mg, 0.233 mmol) in DMSO (6.0 mL). After stirring for 24 hours, the mixture was transferred to a solution containing saturated NaHCO3. 3(溶液) Quenching. The mixture was stirred for 30 min and diluted with EtOAc, then washed with saturated NaHCO3. The organic layer was dried over Na2SO4, then filtered and concentrated. The residue was purified by column chromatography (hexane–EtOAc, 4:1 to 1:1) to obtain denitrified-dGTP-FL-7 (154 mg, 79%) as a white foam. R f 0.67 (1:1, hexane–EtOAc); 1 HNMR (400 MHz, CD3OD) δ 7.97–7.90 (m, 2H, ArH), 7.70–7.32 (m,14H, ArH, H-6), 6.58 (dd, J = 6.2, 7.3 Hz, 1H, H-1'), 4.83–4.78 (m, 1H, H-3'), 4.77–4.67 (m, 2H, C H 2 S), 4.30 (s, 2H, C H 2 N), 4.12–4.04 (m, 4H, H-4',OC H 3 ), 3.88 (dd, J= 4.2, 11.9 Hz, 1H, H-5'a), 3.81 (dd, J = 4.2, 11.9 Hz,1H, H-5'b), 2.69–2.58 (m, 1H, H-2'a), 2.50 (ddd, J = 3.1, 6.2, 13.7 Hz, 1H,H-2'b), 2.12 (s, 3H, SC H 3 ), 1.06 (s, 9H, (C H 3 )3C); HRMS (ESI) m / z for C 42 H 44 Calculated value of F3N5O6Ssi [M]: 831.2751; Measured value [M + H]: + 832.2826.

[0758]

[0759] Synthesis of denitrogenated-dGTP-FL-8: At 0 °C, cyclohexene (500 μL) and 1 M sulfonyl chloride (555 μL, 0.555 mmol) were added to a solution of denitrogenated-dGTP-FL-7 (154 mg, 0.185 mmol) in CH2Cl2 (2.0 mL). After stirring for 30 min, the mixture was concentrated. The residue was then dissolved in DMF (2.0 mL), and NaN3 (120 mg, 1.85 mmol) was added to the mixture at room temperature. After stirring for 1 h, the mixture was diluted with EtOAc and washed with H2O. The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (hexane–EtOAc, 4:1 to 1:1) to obtain denitrogenated-dGTP-FL-8 (142 mg, 93%) as a colorless oil. R f 0.67 (1:1, hexane–EtOAc); 1 HNMR (400 MHz, CD3OD) δ 7.96–7.89 (m, 2H, ArH), 7.69–7.29 (m, 14H, ArH, H-6), 6.55 (app t, J = 6.7 Hz, 1H, H-1'), 4.83 (d, J = 9.0 Hz, 1H,C H 2 N3), 4.78–4.73 (m, 2H, H-3', CH 2 N3), 4.30 (s, 2H, C H 2 N), 4.15–4.06 (m, 4H,H-4', OC H 3 ), 3.90 (dd, J = 4.2, 11.3 Hz, 1H, H-5'a), 3.84 (dd, J = 4.4, 11.3Hz, 1H, H-5'b), 2.79–2.69 (m, 1H, H-2'a), 2.55 (ddd, J = 3.7, 6.3, 13.6 Hz,1H, H-2'b), 1.06 (s, 9H, (C H 3 )3C); HRMS (ESI) m / z for C 41 H 41 The calculated value of F3N8O6Si is [M] 826.2897; the measured value is [M + H]. + 827.2973.

[0760]

[0761] Synthesis of denitrified-dGTP-FL-9: At room temperature, NH4F (13.4 mg, 0.363 mmol) was added to a solution of denitrified-dGTP-FL-8 (98 mg, 0.121 mmol) in MeOH (2.0 mL). After stirring for 24 hours, the mixture was concentrated and the residue was diluted with EtOAc and then washed with H2O. The organic layer was dried over Na2SO4, filtered, and concentrated. The residue was purified by column chromatography (hexane–EtOAc, 4:1 to 1:1) to obtain denitrified-dGTP-FL-9 (46 mg, 67%) as a colorless oil. R f 0.27 (1:1, hexane – EtOAc); 1 HNMR (400 MHz, CD3OD) δ 8.00–7.94 (m, 2H, ArH), 7.67 (s, 1H, H-6), 7.65–7.60 (m, 1H, ArH), 7.57–7.51 (m,2H, ArH), 6.58 (dd, J = 6.2, 7.4 Hz, 1H, H-1'), 4.86–4.79 (m, 2H, C H2 N3),4.70–4.64 (m, 1H, H-3'), 4.33 (s, 2H, C H 2 N), 4.15–4.04 (m, 4H, H-4', OC H 3 ),3.83–3.71 (m, 2H, H-5'a, H-5'b), 2.77–2.68 (m, 1H, H-2'a), 2.55 (ddd, J =3.0, 6.3, 13.6 Hz, 1H, H-2'b); HRMS (ESI) m / z for C 25 H 23 Calculated value of F3N8O6 [M]: 588.1705; Measured value [M + H]: + 589.1776.

[0762] 6.17. Scheme 30a: NH2-L-3'- O Synthesis of -azidomethyl-7-denitro-dGTP:

[0763]

[0764]

[0765] Experimental procedure:

[0766]

[0767] Synthesis of 7-dN-dGTP-FL-2: At room temperature, N-(4-chloro-5-iodo-7h-pyrrolo[2,3-d]pyrimidin-2-yl)-2,2-dimethylpropionamide (756 mg, 2.00 mmol) was added to a suspension of KOH (0.68 g, 8.71 mmol) and TDA-1 (0.1 mL, 0.32 mmol) in CH3CN (30 mL). After stirring for 10 min, 1-Cl sugar (1.02 g, 2.6 mmol) was added after 30 min, and stirring was continued for another 30 min. The insoluble material was filtered off, the precipitate was washed with 15 mL CH3CN, and the filtrate was evaporated to dryness. The residue was purified by column chromatography (dichloromethane) to obtain 7-dN-dGTP-FL-2 (1.15 g, 80%) as a brown foam. 1HNMR (400 MHz, CD3OD) δ 8.05 (s, 1H), 7.79 (s,1H,), 7.33 (s, 1H), 7.92 ~ 7.80 (m, 4H),7.22 ~ 7.16 (m, 4H), 6.64 (dd, J = 1.4 Hz, J = 7.6 Hz, 1H), 5.70 (dt, 1H), 4.66 (dt, J = 5.8 Hz, J = 2.3 Hz,1H, H-3'), 4.56 (dd, J 5'a,4' = 3.4 Hz, J gem = 12.1 Hz, 1H, H-5'a), 4.48 (dd, J =3.5 Hz, J = 12.1 Hz, 1H), 2.85 (ddd, J 2'a,3' = 5.9 Hz, J 2'a,1' = 8.6 Hz, J gem =14.5 Hz, 1H, H-2'a), 2.71 (ddd, J = 2.3 Hz, J = 5.9 Hz, J gem = 14.5 Hz, 1H),2.36 (s, 3H,), 2.34 (s, 3H,) 1.26 (s, 9H)。

[0768]

[0769] At room temperature, an aqueous solution of NaOH (2.0 M, 3.0 mL) was added to 7dN-dGTP-FL-2 (0.73 g, 1.00 mmol). The solution was heated to reflux and stirred for 3 hours. TLC showed that all starting material was consumed. The mixture was cooled to room temperature and neutralized by adding 2.0 M HCl. The mixture was then evaporated to dryness. The residue was purified by column chromatography (DCM; DCM:MeOH, 95:5; DCM:MeOH, 90:10; DCM:MeOH, 85:15) to obtain 7-dN-dGTP-FL-3 (220 mg, 55%) as a pale yellow foam. 1 HNMR (400 MHz, CD3OD) δ 7.81 (s, 1H), 7.79 (s, 1H), 7.16 (d, J = 9.0 Hz, 1H), 6.26 (d, J = 3.7 Hz, 1H), 4.35 (dt, J = 5.8 Hz, J = 2.3 Hz, 1H, H-3'), 3.82 (dd, J 5'a,4' = 3.4 Hz, J gem = 12.1 Hz, 1H, H-5'a),3.62 (dd, J 5'b,4' = 3.5 Hz, J gem = 12.1 Hz, 1H, H-5'b), 2.38 (ddd, J 2'a,3' = 5.9Hz, J 2'a,1' = 8.6 Hz, J gem = 14.5 Hz, 1H, H-2'a), 2.14 (ddd, J 2'b,3' = 2.3 Hz, J 2'b,1' = 5.9 Hz, J gem = 14.5 Hz, 1H, H-2'b). LRMS (ESI) m / z for C 11 H 13I Calculated value of N4O4 [M]: 392.14; Measured value [M + NH]: + 393.15.

[0770]

[0771] Synthesis of 7dN-dGTP-FL-4: CuI (15 mg, 0.095 mmol) and Et3N (0.5 mL) were added to a solution of 7dN-dGTP-FL-3 (186 mg, 0.5 mmol) in DMF (4.0 mL) at room temperature. After stirring under N2 for 5 min at room temperature, alkyne (220.5 mg, 1.5 mmol) and Pd(PPh3)4 (45 mg, 0.039 mmol) were added to the mixture at room temperature. After heating at 55 °C for 13 h, HPLC showed that the starting nucleosides were consumed. The mixture was concentrated and the residue was purified by column chromatography (DCM; DCM:MeOH, 90:10; DCM:MeOH, 85:1) to obtain 7dN-dGTP-FL-4 (170 mg, 82%) as a brown foam. MS (ESI) m / z against C 16 H 16 Calculated value of F3N5O5 [M]: 415.11; Measured value [M + H]: + 416.15.

[0772]

[0773] Synthesis of 7dN-dGTP-FL-5: At room temperature, 100 mg of N,N-dimethylformamide dimethyl acetal was added to a solution of 7dN-dGTP-FL-4 (108 mg, 0.25 mmol) in ACN (5.0 mL). After stirring at room temperature under N2 for 45 min, HPLC showed that the starting nucleoside was consumed. The mixture was concentrated and the residue was purified by column chromatography (DCM; DCM:MeOH, 90:10; DCM:MeOH, 85:1) to give brown foam.

[0774] 6.18. Example 17: Synthesis of NH2-L-3'-O-N3-dNTP via an alternative pathway

[0775] 6.18.1. Scheme 32: Synthesis of NH2-L-3'-O-N3-dUTP

[0776]

[0777]

[0778]

[0779] 6.18.1.1 Experimental Procedure:

[0780]

[0781] Synthesis of dUTP-FL-1: Sodium azide (854 mg, 13.14 mmol) and N-iodosuccinimide (1.48 g, 6.57 mmol) were added to a solution of β-L-deoxyuridine (1 g, 4.38 mmol) in H2O (10 mL), and the mixture was stirred at room temperature for 19 hours. After complete reaction, the reaction mixture was filtered off and washed with water. The remaining filtrate was extracted with DCM×2 and the combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (1:10 to 1:3, CH3OH:CH2Cl2) to give dUTP-FL-1 (1.27 g, 80%) as a white solid. 1 HNMR (400 MHz, CD3OD) δ 8.54 (s, 1H), 6.24 (app t, J = 6.5 Hz, 1H), 4.42 (app dt, J =3.4 Hz, J =6.1 Hz, 1H), 3.97–3.93 (m, 1H), 3.84 (dd, J = 12.0 Hz, J = 3.0 Hz, 1H), 3.75 (dd, J = 12 Hz, J = 3.3 Hz, 1H), 2.37–2.18 (m, 2H). HRMS (ESI): m / z for C9H 11 Calculated value of IN₂O₅ [M]: 353.9717; Measured value [M – H]: – 352.9644

[0782]

[0783] Synthesis of dUTP-FL-2: CuI (6.5 mg, 0.34 mmol) and triethylamine (50 μL) were added to a solution of dUTP-FL-1 (60 mg, 0.17 mmol) in N,N-dimethylformamide (3 mL), and the mixture was stirred at room temperature for several minutes to activate the CuI. Then, 2,2,2-trifluoro-N-(prop-2-ynyl)acetamide (76.8 mg, 0.51 mmol) and tetrakis(triphenylphosphine)palladium (0) (19.6 mg, 0.0017 mmol) were added. The reaction was allowed to run at room temperature for 24 h and concentrated. The resulting residue was purified by column chromatography (1:10:10 to 1:4:4, CH3OH: CH2Cl2: EtOAc) to give dUTP-FL-2 (32 mg, 50%) as a yellow oil. 1 HNMR (400 MHz, CD3OD ) δ 8.34 (s, 1H), 6.25 (app t, J =6.5 Hz, 1H), 4.6 (s, 1H), 4.43-4.39 (m, 1H), 4.29 (s, 2H), 4.42 (app dt, J =7.0 Hz, J = 7.1 Hz, 1 H), 3.97–3.94 (m, 1H), 3.82 (dd, J = 12.0 Hz, J = 3.0Hz, 1H), 3.75 (dd, J = 12 Hz, J = 3.5 Hz, 1H); HRMS (ESI): m / z for C 14 H 14 Calculated value of F3N3O6 [M]: 377.0825; Measured value [M + H]: + 378.0893

[0784]

[0785] Synthesis of dUTP-FL-3: Imidazole (160.32 mg, 2.35 mmol) and tert-butyldimethyl chloride (260.29 mg, 1.73 mmol) were added to a solution of dUTP-FL-2 (590 mg, 1.57 mmol) in N,N-dimethylformamide (10 mL) at 0 °C, and then the temperature was raised to room temperature. After stirring at room temperature for 10 h, ice water was added to the solution and the mixture was extracted with ethyl acetate × 2. The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (1:3 to 1:1, hexane:EtOAc) to give dUTP-FL-3 (210 mg, 43%) as a yellow oil. 1 HNMR (400MHz, CDCl3) δ 8.69 (s,1H), 8.11 (s, 1H), 6.19–6.24 (m, 1H), 4.64 (app dt, J =11.9 Hz, J =14.6 Hz, 1H), 4.47 (d, 1H), 4.16–4.05 (m, 1H), 3.92 (dd, J = 2.5Hz, J = 11.4 Hz, 1H), 3.80 (dd, J = 2.4 Hz, J = 11.5 Hz, 1H), 2.15 (s, 3H), 0.95 (s, 9H), 0.17 (d, 6H); HRMS (ESI): m / z for C 20 H 28 The calculated value of F3N3O6Si [M] is 491.1699; the measured value is [M + H]. + 492.1778

[0786]

[0787] Synthesis of dUTP-FL-4: At room temperature, AcOH (200 uL) and acetic anhydride (200 uL) were added to a solution of dUTP-FL-3 (164 mg, 0.46 mmol) in DMSO (1 mL). After stirring at room temperature for 24 hours, ice water and solid sodium bicarbonate were added to the solution. The aqueous layer was extracted twice with ethyl acetate. The combined organic layers were dried over Na2SO4, filtered, and concentrated. The resulting residue was purified by column chromatography (3:1 to 1:1, hexane:EtOAc) to give dUTP-FL-4 (70 mg, 27%) as a colorless oil. 1HNMR (400 MHz, CDCl3) δ 9.35(s,1H), 7.96 (s, 1H), 7.49 (s,1H), 6.10 (dd, J = 5.7 Hz, 1H), 4.33–3.32 (m, 1H), 4.24–4.13 (m, 2H), 4.02(d, 1H), 3.83 (dd, J = 2.4 Hz, J = 11.5 Hz, 1H), 3.75 (dd, J = 2.0 Hz, J =11.4 Hz, 1H), 2.38–2.33 (m, 1H), 2.01 (s, 3H), 0.78 (s, 9H), 0.01 (d, 6H); HRMS (ESI): m / z for C 22 H 32 Calculated value of F3N3O6SSi [M]: 551.1733; Measured value [M + H]: + 552.1825

[0788]

[0789] Synthesis of dUTP-FL-5: Cyclohexene (30 μL, 0.3 mmol) was added to a solution of dUTP-FL-4 (50 mg, 0.63 mmol) in dry CH2Cl2 at 4 °C, followed by dropwise addition of sulfonyl chloride (1 M CH2Cl2 solution, 0.11 mL, 0.11 mmol) under N2. After 40 min, TLC indicated complete consumption of dUTP-FL-4, the solvent was evaporated, and the residue was subjected to high vacuum for 20 min. It was then redissolved in dry DMF (1 mL) and treated with NaN3 (30 mg, 0.45 mmol). The resulting suspension was stirred at room temperature for 2 h. The reaction was quenched with CH2Cl2, and the organic layer was washed with a saturated aqueous NaCl solution. After solvent removal, the resulting yellow gel was redissolved in 2 M HCl:acetonitrile (2:1) and stirred at room temperature for 30 min. The solvent was removed, and the reaction was post-treated with CH2Cl2 and a saturated aqueous NaHCO3 solution. The aqueous layer was extracted three times with CH2Cl2. The solution was purified by chromatography on silica (EtOAc:hexane 1:1 to 100% EtOAc) to obtain dUTP-FL-5, which was a pale yellow foam.

[0790] HRMS (ESI): m / z for C 15 H 15Calculated value of F3N6O6 [M]: 432.1005; Measured value [M + Na]: + 455.0887

[0791] 6.18.2. Scheme 33: Synthesis of NH2-L-3'-O-azidomethyl-dCTP

[0792]

[0793] 6.18.2.1 Experimental Procedure:

[0794] Synthesis of dCTP-FL-1: Add the following to a dry round-bottom flask: -L-deoxycytidine (1 g, 4.40 mmol), iodine (1.67 g, 60 mmol), and mCPBA (0.75 g, 4.40 mmol) were dissolved in 15 mL of DMF. The reaction was stirred at room temperature for 2 h, and then evaporated to dryness under reduced pressure. The resulting crude product was purified by column chromatography (1:9, DCM-MeOH) to give dCTP-FL-1 (0.83 g, 53% yield) as an orange solid. 1 H NMR (500 MHz, CD3OD) δ 8.49 (s,1H), 6.17 (t, J = 6.3 Hz, 1H), 4.36 (dt, J = 6.3, 4.1 Hz, 1H), 3.92 (dd, J =6.9, 3.4 Hz, 1H), 3.80 (dt, J = 15.6, 4.9 Hz, 1H), 3.71 (dd, J = 12.0, 3.4Hz, 1H), 2.34 (ddd, J = 13.6, 6.2, 4.2 Hz, 1H), 2.20–2.07 (m, 1H). HRMS (ESI): m / z [M] calculated as C9H 12 IN3O4 352.9900; Measured value [M + Na] + 375.9765.

[0795] Synthesis of dCTP-FL-2: dCTP-FL-1 (650 mg, 1.84 mmol) was added to a light-protected round-bottom flask and dissolved in DMF (10 ml). Subsequently, CuI (70 mg, 0.36 mmol), triethylamine (0.5 ml), 2,2,2-trifluoro-N-prop-2-ynylacetamide (833.7 mg, 5.52 mmol), and finally Pd(PPh3)4 (212.5 mg, 0.184 mmol) were added to the mixture, and the mixture was stirred at room temperature under an argon atmosphere for 18 h. Bicarbonate resin (100 mg) was added, and the mixture was stirred for another 1 h. The reaction mixture was filtered through diatomaceous earth, and the filtrate was evaporated under reduced pressure to obtain the residue. The residue was purified by silica gel chromatography (CH2Cl2: EtOAc: MeOH 4.5:4.5:1) to give the desired product (490 mg, 71% yield) as a beige solid. 1 H NMR (500 MHz, D2O) δ 8.04 (s, 1H), 6.08 (t, J = 6.5Hz, 1H), 4.37 – 4.23 (m, 1H), 4.23 (s, 2H), 3.94 (d, J = 4.3 Hz, 1H), 3.73(dd, J = 12.6, 3.0 Hz, 1H), 3.64 (dd, J = 12.3, 5.0 Hz, 1H), 2.39–2.28 (m, 1H), 2.19–2.13 (m, 1H). HRMS (ESI): m / z for C 14 H 15 Calculated value of F3N4O5 [M]: 376.1000; Measured value [M + H]: + 377.1066.

[0796] Synthesis of dCTP-FL-3: Imidazole (82.12 mg, 1.195 mmol) and tert-butyldimethylsilyl chloride (144.14 mg, 0.956 mmol) were added to a solution of dCTP-FL-2 (300 mg, 0.797 mmol) in anhydrous DMF (5.0 mL) at 0 °C. The solution was stirred at 0 °C under nitrogen for 2 h. After the reaction was complete (monitored by TLC), cold water (10 mL) was added and the mixture was extracted with EtOAc (3 × 30 mL). The combined organic layers were dried over Na2SO4, filtered, and evaporated under reduced pressure. The resulting residue was purified by column chromatography (100% EtOAc to EtOAc:MeOH 9.5:0.5) to give dCTP-FL-3 (210 mg, 54%) as a beige solid. 1 H NMR (500 MHz, DMSO-d6) δ 9.92 (t, J = 5.3 Hz,1H), 7.97 (s, 1H), 7.83 (s, 1H), 6.87 (d, J = 22.9 Hz, 1H), 6.11 (dd, J =7.2, 6.1 Hz, 1H), 5.27 (d, J = 4.1 Hz, 1H), 4.24 (d, J = 5.3 Hz, 2H), 4.17(td, J = 6.0, 3.1 Hz, 1H), 3.88 (q, J = 2.8 Hz, 1H), 3.81 (dd, J = 11.5, 2.6Hz, 1H), 3.72 (dd, J = 11.6, 3.1 Hz, 1H), 2.19–2.17 (m, 1H), 1.96–1.88 (m, 1H), 0.86 (s, 9H), 0.07 (s, 6H). HRMS (ESI): m / z for C 20 H 29 The calculated value of F3N4O5Si [M] is 490.1900; the measured value is [M + Na]. + 513.1751.

[0797] Synthesis of dCTP-FL-4: Acetic acid (2 ml) and acetic anhydride (2 ml) were added to a stirred solution of dCTP-FL-3 (210 mg, 0.428 mmol) in DMSO (6 ml) and the mixture was stirred at room temperature for 48 h. After the reaction was complete, a saturated NaHCO3 solution was added at 0 °C and the mixture was stirred for 30 min. The aqueous layer was extracted with EtOAc (3 × 30 ml). The combined organic extracts were dried over Na2SO4, filtered, and evaporated under reduced pressure. The crude product was purified by column chromatography (1:1, hexane–EtOAc) to give dCTP-FL-4 (130 mg, 52%) as a colorless oil. 1 H NMR (500 MHz, CDCl3) δ 8.32(s, 1H), 8.14 (s, 1H), 6.15 (t, J = 6.4 Hz, 1H), 4.62 (dd, J = 32.9, 11.7 Hz,2H), 4.42 (dt, J = 5.7, 2.8 Hz, 1H), 4.37 (t, J = 4.2 Hz, 2H), 4.18 (dd, J =4.8, 2.3 Hz, 1H), 3.94 (dd, J = 11.5, 2.6 Hz, 1H), 3.79 (dd, J = 11.5, 2.2Hz, 1H), 2.66 (ddd, J = 13.7, 5.9, 2.9 Hz, 1H), 2.57 (s, 3H), 2.13 (s, 3H), 2.02 (s, 1H), 0.88 (s, 9H), 0.10 (s, 6H). HRMS (ESI): m / z for C 24 H 35 Calculated value of F3N4O6SSi [M]: 592.2000; Measured value [M + H]: + 593.2071.

[0798] Synthesis of dCTP-FL-5: Cyclohexene (1 mL) was added to a stirred solution of dCTP-FL-4 (130 mg, 0.67 mmol) in anhydrous CH2Cl2 (6 mL), and a CH2Cl2 solution of SO2Cl2 (1.0 M, 0.4 mL) was added. After stirring at 0 °C for 1 h, the volatiles were removed under reduced pressure. Dry DMF (3 mL) and NaN3 (169 mg, 2.61 mmol) were added to the residue, and the mixture was stirred at room temperature for 6 h. The reaction mixture was dispersed in cold distilled water (30 mL) and extracted with EtOAc (2 × 30 mL). The combined organic extracts were dried over Na2SO4 and concentrated under reduced pressure. Acetonitrile (10 mL) and 2M HCl (3 drops) were added to the resulting residue, and the reaction was stirred at room temperature for 1–2 h. The mixture was neutralized with saturated NaHCO3 solution and diluted with EtOAc. The organic layer was dried over Na2SO4 and concentrated under reduced pressure. The residue was purified by column chromatography (hexane–EtOAc (1:1) to 100% EtOAc to EtOAc-MeOH (19:1)) to give dCTP-FL-5 (25 mg, 28%) in orange oil. 1 H NMR (500 MHz, DMSO-d6) δ 9.89 (t, J = 4.8 Hz, 1H), 8.06 (s,1H), 7.80 (s, 1H), 6.84 (s, 1H), 6.00 (dd, J = 7.3, 6.0 Hz, 1H), 5.11 (s,1H), 4.76 (s, 2H), 4.25 (dt, J = 6.0, 3.0 Hz, 1H), 4.20 (d, J = 5.1 Hz, 2H), 3.93–3.88 (m, 1H), 3.59–3.47 (m, 1H), 2.32–2.23 (m, 1H), 2.14–2.01 (m, 1H). HRMS (ESI): m / z for C 15 H 16 Calculated value of F3N7O5 [M]: 431.1200; Measured value [M + H]: + 432.1237.

[0799] Synthesis of dCTP-FL-6: dCTP-FL-5 (68 mg, 0.157 mmol) and proton sponge (40 mg, 0.189 mmol) were dried overnight in a vacuum desiccator using P2O5. POCl3 (30 μL, 0.375 mmol) was added dropwise to a solution of dCTP-FL-5 and proton sponge in trimethyl phosphate (1 mL) at 0 °C. The mixture was stirred at 0 °C for 2 h, and then a thoroughly vortexed mixture of tributylammonium pyrophosphate (148 mg) and tributylamine (233.8 μL, 1.26 mmol) in anhydrous DMF (2 mL) was added. The mixture was stirred at room temperature for 1.5 h, and then 0.1 M triethylammonium bicarbonate buffer (TEAB buffer, pH 8.5, 0.1 M, 5 mL) was added, and the resulting mixture was stirred at room temperature for 1 h. Then, concentrated ammonium hydroxide (5 mL) was added to the mixture and stirred overnight at room temperature. The resulting mixture was concentrated under reduced pressure, and the residue was diluted with 20 mL of water. The crude mixture was extracted with CH2Cl2 (2 × 20 mL), and the aqueous layer was concentrated under reduced pressure. The residue was then purified by anion exchange chromatography on a DEAE-Sephadex A-25 using a gradient of TEAB (pH 8.5; 0.1–0.8 M). Fractions containing the product (0.3 M – 0.6 M) were collected and concentrated under reduced pressure. The residue was diluted with ddH2O and subjected to C18 HPLC (for 60 min, 100% to 0% A in B, where A = 25 mM TEAB in water, B = 25 mM TEAB in ACN). The product (retention time: 11.3 min) was collected and concentrated under reduced pressure to give dCTP-FL-6 (6.7 mg, yield 7.4%) as a syrup. 1 H NMR (500 MHz, D2O) δ 7.64 (s,1H), 6.22 (dd, J = 8.5, 5.9 Hz, 1H), 4.78 – 4.72 (m, 2H), 4.70 (s, 2H), 4.55– 4.51 (m, 1H), 4.25 (t, J = 6.6 Hz, 1H), 4.14 – 4.04 (m, 2H), 2.44 – 2.37 (m, 1H), 2.31 – 2.22 (m, 1H). 31 P NMR (202 MHz, D2O): δ –10.47 (bs, 1P), –11.23(d, J= 20.0 Hz, 1P), –22.96 (bs, 1P)

[0800] 6.18.3. Scheme 34: Synthesis of NH2-L-3'-O-azidomethyl-dGTP

[0801]

[0802] 6.18.3.1 Experimental Procedure:

[0803] Synthesis of dGTP-FL-1:

[0804] N-bromosuccinimide (1.33 g, 7.2 mmol, 1 equivalent) was added in portions to a suspension of 2'-deoxyguanosine (2.0 g) in a mixture of acetonitrile (17.5 ml) and water (5 ml). The reaction mixture was stirred at room temperature for 30 min. The solvent was removed, and the precipitate was suspended in acetone (15 ml), stirred at room temperature for 4 h, and cooled at -20 °C for 48 h. The precipitate was collected by filtration, washed extensively with cold acetone, and dried under vacuum to give 2.1 g (82%) of a slightly yellow powder. NMR (500 MHz, δ 10.84 (s, 1H), 6.29 (s, 1H), 6.24 (dd, ) J =8.2, 6.6 Hz, 1H), 4.53 – 4.48 (m, 1H), 3.95 (dd, J = 6.2, 3.7 Hz, 1H), 3.75 (dd, J = 11.9, 3.9 Hz, 1H), 3.62 (dd, J = 12.0, 4.2 Hz, 1H), 3.10 – 3.03 (m,1H), 2.15 (dd, J = 13.2, 6.4 Hz, 1H).

[0805] Synthesis of dGTP-FL-2:

[0806] dGTP-FL-1 (1.0 g, 2.891 mmol) was dissolved in dry DMF (15 ml). 2,2,2-trifluoro-N-prop-2-ynylacetamide (1.56 g, 10.34 mmol), Pd(PPh3)4 (0.33 g, 0.289 mmol), CuI (0.29 g, 1.514 mmol), and Et3N (0.6 ml, 4.33 mmol) were added to the reaction mixture and stirred under argon at 55 °C. After stirring for 3.5 h, the reaction mixture was evaporated and purified by silica gel column chromatography (CH2Cl2 / ethyl acetate / MeOH; 4.5:4.5:1) to give a pure product (730 mg, 60%) as a beige solid. NMR (500 MHz, δ 10.96 (s, 1H), 10.22 (t, δ 1H) J = 5.4 Hz, 1H), 6.70 (s, 2H), 6.28 – 6.11 (dd, 1H), 4.89 (s, 1H), 4.39 – 4.34 (m, 3H), 3.78 (td, J = 5.6, 3.2 Hz, 2H), 3.63 – 3.60 (m,1H), 3.49 (d=m, J = 5.7 Hz,1H), 3.05 – 2.99 (m, 1H), 2.09 (ddd, J = 13.1, 6.6, 3.0 Hz, 1H).

[0807] 6.19. Example 18. Synthesis of L-3'-O-nitrobenzyl-dNTP

[0808] 6.19.1. Synthesis of L-3'-O-nitrobenzyl-dATP

[0809]

[0810]

[0811]

[0812] Experimental procedure:

[0813]

[0814] Synthesis of dATP-NB-1: Imidazole (4.5 g, 66 mmol) and TBSCl (4.82 g, 32 mmol) were added to a solution of β-L-2'-deoxyadenosine (2.5 g, 10.0 mmol) in DMF (25 mL) at room temperature. The mixture was stirred overnight at room temperature and quenched by adding CH3OH (20 mL). The resulting mixture was concentrated under vacuum. The residue was diluted with CH2Cl2 and quenched with saturated NaHCO3. 3(水溶液) The residue was washed, then dried over Na₂SO₄, filtered, and concentrated under vacuum. At room temperature, (Boc)₂O (6.55 g, 30 mmol) and DMAP (3.66 g, 30 mmol) were added to a solution of the crude residue in DMF (15 mL). The mixture was stirred overnight at room temperature. The mixture was concentrated under reduced pressure, and the residue was diluted with CH₂Cl₂ and washed with saturated NaHCO₃ (aqueous solution). The organic layer was dried over Na₂SO₄, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give dATP-NB-1 (5.6 g, 82%) as a white foam. R f 0.72 (100% EtOAc); 1 HNMR (400 MHz, CDCl3) δ 8.85 (s, 1H, H-8), 8.43 (s, 1H, H-2), 6.53 (app t, 1H, J = 6.3 Hz, H-1'), 4.62 (app dt, 1H, J = 5.6, 3.7 Hz, H-3'), 4.05–4.01 (m, 1H, H-4'), 3.89 (dd, 1H, J = 4.2, 11.5 Hz, H-5'a), 3.79 (dd,1H, J = 3.1, 11.5 Hz, H-5'b), 2.67–2.58 (m, 1H, H-2'a), 2.47 (ddd, 1H, J =4.1, 6.3, 13.1 Hz, H-2'b), 1.44 (s, 18H, Boc 2 x t Bu), 0.92 (s, 18H, TBS 2x) t Bu), 0.11 (s, 6H, TBS 2 x CH3), 0.09 (s, 6H, TBS 2 x CH3); HRMS (ESI) m / z for C32 H 57 Calculated value of N5O7Si2 [M]: 679.3797; Measured value [M + H]: + 680.3812.

[0815]

[0816] Synthesis of dATP-NB-2: 1 M TBAF (24.7 mL) was added to a solution of dATP-NB-1 (5.6 g, 8.24 mmol) in THF (30 mL) at room temperature. The mixture was stirred at room temperature for 30 min and concentrated under vacuum. The residue was purified by silica gel column chromatography to give dATP-NB-2-diol (2.6 g, 70%) as a white foam. R f 0.35 (100% EtOAc); 1 HNMR (400 MHz, CD3OD) δ 8.79 (s, 1H, H-8), 8.73 (s, 1H, H-2), 6.54 (app t, 1H, J = 6.7 Hz, H-1'), 4.65 (app dt, 1H, J = 5.9, 3.3 Hz, H-3'), 4.03–3.98 (m, 1H, H-4'), 3.78 (dd, 1H, J = 3.7, 12.0 Hz, H-5'a), 3.66 (dd,1H, J = 4.0, 12.0 Hz, H-5'b), 2.83–2.75 (m, 1H, H-2'a), 2.45 (ddd, 1H, J =3.4, 6.3, 13.5 Hz, H-2'b), 1.33 (s, 18H, Boc 2 x t Bu); At 0 °C, imidazole (0.78 g, 11.5 mmol) and TBSCl (1.13 g, 7.49 mmol) were added to a solution of dATP-NB-2-diol (2.6 g, 4.65 mmol) in DMF (15 mL). The mixture was gradually heated to room temperature and stirred for two days. The reaction was quenched with water, and the mixture was extracted three times with EtOAc. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography to give dATP-NB-2 (3.4 g, 73%) as a white foam. R f0.27 (1:1, hexane – EtOAc); 1 HNMR (400 MHz, CDCl3) δ 8.85 (s, 1H, H-8), 8.40 (s, 1H, H-2), 6.56(app t, 1H, J = 6.4 Hz, H-1'), 4.74–4.67 (m, 1H, H-3'), 4.11–4.07 (m, 1H, H-4'), 3.90–3.86 (m, 2H, H-5'a, H-5'b), 2.79–2.68 (m, 1H, H-2'a), 2.57 (ddd,1H, J = 4.3, 6.4, 13.6 Hz, H-2'b), 1.45 (s, 18H, Boc 2 x t Bu), 0.91 (s, 9H, TBS) t Bu), 0.11 (s, 3H, TBS of C H 3 ), 0.10 (s, 3H, TBS of C H 3 ); HRMS (ESI) m / z for C 26 H 43 Calculated value of N5O7Si [M]: 565.2932; Measured value [M + H]: + 566.3025.

[0817]

[0818] Synthesis of dATP-NB-3: A solution of TBAOH (0.86 mL, 4.24 mmol, 55% aqueous solution) and NaI (20 mg) in NaOH (1M, 5 mL) was added to a solution of dATP-NB-2 (1.2 g, 2.21 mmol) in CH2Cl2 (5 mL). A solution of 2-nitrobenzyl bromide (2.3 g, 10.6 mmol) in CH2Cl2 (3 mL) was added dropwise to the mixture, and the reaction mixture was stirred in the dark at room temperature for two hours. The organic layers were separated, and the aqueous layer was washed twice with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (4:1 to 1:1, hexane–EtOAc) to give dATP-NB-3 (1.43 g, 96%) as a white foam. R f 0.55 (1:1, hexane–EtOAc).

[0819]

[0820] Synthesis of dATP-NB-4: Silica gel 60 (10 g, 100–200 mesh) was added to a solution of dATP-NB-3 (245 mg, 0.35 mmol) in CH2Cl2 (20 mL) at room temperature. The mixture was evaporated to dryness under reduced pressure. The residue was heated to 70–80 °C under vacuum using an oil pump for 5 hours, and then purified by silica gel column chromatography to give dATP-NB-4 (132 mg, 74%) as a colorless syrup. R f 0.46 (100% EtOAc); 1 H NMR (400 MHz, CDCl3) δ8.36 (s, 1H, H-8), 8.15 (s, 1H, H-2), 8.09 (dd, 1H, J = 1.2, 8.7 Hz, ArH),7.82 (d, 1H, J = 8.0 Hz, ArH), 7.68 (dt, 1H, J = 1.2, 7.7 Hz, ArH), 7.50–7.45(m, 1H, ArH), 6.50 (app t, 1H, J = 7.1 Hz, H-1'), 6.50 (bs, 2H, NH2), 5.01–4.91 (m, 2H, C H 2 Ar), 4.45–4.41 (m, 1H, H-3'), 4.32–4.28 (m, 1H, H-4'), 3.91 (dd, 1H, J = 4.6, 11.3 Hz, H-5'a), 3.84 (dd, 1H, J = 3.4, 11.3 Hz, H-5'b), 2.74–2.68 (m, 2H, H-2'a, H-2'b), 0.92 (s, 9H, TBS t Bu), 0.107 (s, 3H, TBS of C H 3 ), 0.103 (s, 3H, TBS of C H 3 ); HRMS (ESI) m / z for C 23 H 33Calculated value of N6O5Si [M]: 500.2203; Measured value [M + H]: + 501.2631.

[0821]

[0822] Synthesis of dATP-NB-5: At room temperature, benzoyl chloride (43.8 mmol) was added to a solution of dATP-NB-4 (145 mg, 0.28 mmol) in pyridine (2 mL). L, 0.38 mmol). The mixture was stirred for 2 hours. The mixture was diluted with EtOAc and washed with 1N HCl, saturated NaHCO3 solution and water. The organic layer was dried over Na2SO4, filtered and concentrated. The residue was purified by silica gel column chromatography to give dATP-NB-5 (131 mg, 75%) as a colorless syrup. R f 0.07 (1:2, hexane – EtOAc); 1 HNMR (400 MHz, CDCl3) δ 9.03 (bs, 1H, N H Bz), 8.81 (s, 1H, H-8), 8.35 (s, 1H, H-2), 8.10 (dd, 1H, J = 1.2, 8.3 Hz, ArH), 8.05–8.00 (m, 2H, ArH), 7.82 (dd, 1H, J = 0.9, 8.0 Hz, ArH), 7.69 (dt, 1H, J = 1.2, 7.5 Hz, ArH), 7.64–7.58 (m, 1H, ArH), 7.55–7.46 (m, 3H, ArH), 6.57 (app t, 1H, J =6.4 Hz, H-1'), 5.04–4.90 (m, 2H, C H 2 Ar), 4.47–4.42 (m, 1H, H-3'), 4.35–4.31(m, 1H, H-4'), 3.93 (dd, 1H, J = 4.3, 11.6 Hz, H-5'a), 3.85 (dd, 1H, J = 3.3,11.6 Hz, H-5'b), 2.77–2.72 (m, 2H, H-2'a, H-2'b), 0.91 (s, 9H, TBSt Bu), 0.10 (s, 6H, TBS of 2x C H 3 HRMS (ESI) m / z for C 30 H 36 Calculated value of N6O6Si [M]: 604.2466; Measured value [M + H]: + 604.2531.

[0823]

[0824] Synthesis of dATP-NB-6: At room temperature, 1 M TBAF (1 mL, 1.0 mmol) was added to a solution of dATP-NB-5 (479 mg, 0.79 mmol) in THF (5 mL). The mixture was stirred for 2 hours. The mixture was concentrated and the residue was purified by silica gel column chromatography to give dATP-NB-6 (350 mg, 87%) as a colorless syrup. R f 0.54 (1:1, hexane–EtOAc); 1 HNMR (400 MHz, CDCl3) δ 9.05 (bs, 1H, N H Bz), 8.83 (s, 1H, H-8), 8.35 (s, 1H, H-2), 8.12–8.07 (m, 2H, H-2, ArH), 8.07–8.03 (m, 2H, ArH), 7.79–7.75 (m, 1H, ArH), 7.70 (dt, 1H, J = 1.3, 7.4 Hz, ArH), 7.68–7.62 (m, 1H,ArH), 7.60–7.50 (m, 3H, ArH), 6.37 (dd, 1H, J = 6.6, 9.8 Hz, H-1'), 5.93, dd,1H, J = 1.9, 11.9 Hz), 5.04 (d, 1H, J = 13.8 Hz, C H 2 Ar), 4.93 (d, 1H, J =13.8 Hz, C H 2 Ar), 4.59 (d, 1H, J= 5.1 Hz), 4.45 (bs, 1H), 4.10–4.03 (m, 1H,H-5'a), 3.89–3.80 (m, 1H, H-5'b), 3.11 (ddd, 1H, J = 5.1, 9.7, 13.8 Hz, H-2'a), 2.61 (dd, 1H, J = 5.2, 13.8 Hz, H-2'b); HRMS (ESI) m / z for C 24 H 22 Calculated value of N6O6 [M]: 490.1601; Measured value [M + H]: + 491.1652.

[0825] Synthesis of dATP-NB-7: A solution of starting material (40 mg, 0.067 mmol) and proton sponge (17.3 mg, 0.0806 mmol) in trimethyl phosphate (300 μL) was heated to 50 °C for 10 min and then cooled to 0 °C. Phosphorus oxychloride (9.42 μL, 0.10 mmol) was added to the above solution. After 2 h, a thoroughly vortexed solution of tributylammonium pyrophosphate (184 mg, 0.336 mmol) and tributylamine (184 μL) in DMF (500 μL) was added to the reaction mixture. The mixture was stirred for 1 h, and then 0.1 M TEAB buffer (pH 8.5, 15 mL) was added. The resulting mixture was stirred for 2 h, and then NH3 solution (10 mL) was added. The resulting mixture was stirred for 16 h and then concentrated under reduced pressure. The resulting residue was diluted with H2O and washed with CH2Cl2. The aqueous layer was concentrated under reduced pressure. The resulting residue was purified by passing it through a C18 column (50 min, 0% B to 90% B, A: 0.1 M TEAB in water / B: ACN). The collected fraction was concentrated and the resulting residue was purified by passing it through a SAX column (50 min, 0% B to 80% B, A: 15% ACN in water; B: 15% ACN in 1 M TEAB). After purification, the collected fraction was concentrated under reduced pressure and the resulting residue was redissolved in water and lyophilized. A colorless syrupy lyophilized product (12.7 mg) was obtained. 1 H NMR (400 MHz, D2O) δ 8.45 (bs, 1H), 8.17 (bs, 1H), 7.95 (d, 1H, J= 7.9 Hz, ArH), 7.69–7.60 (m, 2H, ArH), 7.51–7.42 (m, 1H, ArH), 6.36 (app t, 1H, J = 7.1 Hz, H-1'), 4.96–4.84 (m, 2H,C H 2 Ar), 4.59–4.53 (m, 1H), 4.45–4.38 (m, 1H), 4.19–4.02 (m, 2H, H-5'a, H-5'b), 2.75–2.61 (m, 2H, H-2'a, H-2'b); 31 P NMR (121.4 MHz, D2O) δ –10.8 (bs, 1P), –11.5 (bs, 1P), –23.3 (bs, 1P); HRMS (ESI) m / z for C 17 H 21 N6O 14 Calculated value of P3 [M]: 626.0329; Measured value [M – H]: – 625.0897.

[0826] 6.19.2. Synthesis of L-3'-O-nitrobenzyl-dGTP

[0827]

[0828]

[0829]

[0830] Experimental procedure:

[0831]

[0832] Synthesis of dGTP-NB-1: At room temperature, imidazole (1.68 g, 24.7 mmol) and TBSCl (1.8 g, 12.0 mmol) were added to a solution of β-L-2'-deoxyguanosine (1.0 g, 3.74 mmol) in DMF (10 mL). The mixture was stirred overnight at room temperature and quenched by adding CH3OH (20 mL). The resulting mixture was extracted with EtOAc and water, and the organic layer was dried over Na2SO4, filtered, and concentrated under vacuum. At room temperature, (Boc)2O (4.08 g, 15.5 mmol) and DMAP (1.64 g, 15.5 mmol) were added to a solution of crude residue in DMF (10 mL). The mixture was stirred overnight at room temperature. The mixture was concentrated under reduced pressure, and the residue was diluted with CH2Cl2 and washed with saturated NaHCO3 (aqueous solution). The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to obtain dGTP-NB-1 (1.3 g, 44%) as white foam; 1 HNMR (400 MHz, CDCl3) δ 8.24 (s, 1H,H-8), 6.43 (app t, 1H, J = 6.2 Hz, H-1'), 4.56 (app dt, 1H, J = 5.3, 4.0 Hz,H-3'), 4.01–3.96 (m, 1H, H-4'), 3.87 (dd, 1H, J = 4.0, 11.5 Hz, H-5'a), 3.76(dd, 1H, J = 3.1, 11.5 Hz, H-5'b), 2.53–2.35 (m, 2H, H-2'a, H-2'b), 1.71 (s,9H, Boc t Bu), 1.40 (s, 18H, Boc 2 x t Bu), 0.92 (s, 9H, TBS) t Bu), 0.90 (s, 9H, TBS) t Bu), 0.11–0.06 (m, 12H, TBS of 4 x CH3).

[0833]

[0834] Synthesis of dGTP-NB-2: At room temperature, 1 M TBAF (2 mL, 2.0 mmol) was added to a solution of dGTP-NB-1 (530 mg, 0.67 mmol) in THF (5 mL). The mixture was stirred at room temperature for 30 min and concentrated under reduced pressure. The resulting residue was purified by column chromatography (100% EtOAc) to give dGTP-NB-2-diol (340 mg, 90%) as a white solid. 1 HNMR (400 MHz, CDCl3) δ 7.94 (s, 1H, H-8), 6.31 (dd, 1H, J =5.6, 9.1 Hz, H-1'), 4.96 (dd, 1H, J = 2.9, 10.8 Hz), 4.78 (bs, 1H), 4.16 (bs,1H), 3.92–3.84 (m, 1H, H-5'a), 3.78–3.70 (m, 1H, H-5'b), 3.10–3.01 (m, 1H, H-2'a), 2.32 (ddd, 1H, J = 1.1, 5.4, 13.2 Hz, H-2'b), 1.92 (d, 1H, J = 3.1 Hz, OH), 1.71 (s, 9H, Boc of t Bu), 1.43 (s, 18H, Boc 2 x t Bu). At 0 °C, imidazole (61.2 mg, 0.9 mmol) and TBSCl (108 mg, 0.72 mmol) were added to a solution of dGTP-NB-2-diol (340 mg, 0.6 mmol) in DMF (5 mL). The mixture was stirred at 0 °C and gradually heated to room temperature and stirred for another 5 hours. The mixture was quenched with CH3OH and then concentrated under reduced pressure. The residue was diluted with EtOAc and washed with H2O. The organic layer was dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by silica gel column chromatography to give dGTP-NB-2 (337 mg, 82%) as a white foam. 1 HNMR (400 MHz, CDCl3) δ 8.20 (s, 1H, H-8), 6.46 (app t,1H, J= 6.4 Hz, H-1'), 4.68–4.61 (m, 1H, H-3'), 4.07–4.02 (m, 1H, H-4'), 3.90–3.84 (m, 2H, H-5'a, H-5'b), 2.66–2.58 (m, 1H, H-2'a), 2.57 (ddd, 1H, J =4.2, 6.4, 13.5 Hz, H-2'b), 1.70 (s, 9H, Boc) t Bu), 1.40 (s, 18H, Boc 2 x t Bu), 0.91 (s, 9H, TBS) t Bu), 0.11 (s, 3H, TBS CH3), 0.10 (s, 3H, TBS CH3).

[0835]

[0836] Synthesis of dGTP-NB-3: A solution of TBAOH (0.21 mL, 0.989 mmol, 55% aqueous solution) and NaI (4 mg) in NaOH (1 M, 1 mL) was added to a solution of dGTP-NB-2 (337 g, 0.494 mmol) in CH2Cl2 (5 mL). A solution of 2-nitrobenzyl bromide (534 mg, 2.47 mmol) in CH2Cl2 (3 mL) was added dropwise to the mixture, and the reaction mixture was stirred in the dark at room temperature for two hours. The organic layers were separated, and the aqueous layer was washed twice with CH2Cl2. The combined organic layers were dried over Na2SO4, filtered, and concentrated under reduced pressure. The residue was purified by column chromatography (4:1 to 1:1, hexane–EtOAc) to give dGTP-NB-3 (320 mg, 75%) as a white foam. R f 0.55 (1:1, hexane – EtOAc); 1 HNMR (400 MHz, CDCl3) δ 8.24 (s, 1H, H-8), 8.10 (dd, 1H, J = 1.2, 8.1 Hz,ArH), 7.81 (dd, 1H, J = 0.8, 8.1 Hz, ArH), 7.69 (app dt, 1H, J = 1.2, 8.1Hz, ArH), 7.51–7.45 (m, 1H, ArH), 6.48 (dd, 1H,J = 6.6, 7.9 Hz, H-1'), 4.95(s, 2H, C H 2 Ar), 4.41–4.37 (m, 1H, H-3'), 4.30–4.26 (m, 1H, H-4'), 3.90 (dd,1H, J = 4.3, 11.3 Hz, H-5'a), 3.83 (dd, 1H, J = 3.1, 11.3 Hz, H-5'b), 2.69(ddd, 1H, J = 2.3, 5.7, 13.4 Hz, H-2'a), 2.59 (ddd, 1H, J = 5.7, 8.0, 13.4Hz, H-2'b), 1.71 (s, 9H, Boc) t Bu), 1.40 (s, 18H, Boc 2 x t Bu), 0.90 (s, 9H, TBS) t Bu), 0.11 (s, 3H, TBS CH3), 0.11 (s, 3H, TBS CH3).

[0837]

[0838] Synthesis of dGTP-NB-4: At room temperature, silica gel 60 (10 g, 100–200 mesh) was added to a solution of dGTP-NB-3 (320 mg, 0.39 mmol) in CH2Cl2 (30 mL). The mixture was evaporated to dryness under reduced pressure. The residue was heated to 70–80 °C under vacuum with an oil pump for 5 hours, and then purified by silica gel column chromatography to give dGTP-NB-4 (150 mg, 74%) as a white solid. 1 HNMR (400 MHz, CDCl3) δ 8.02–7.97 (m, 1H, ArH), 7.75–7.68 (m, 2H, ArH, H-8), 7.62–7.54 (m, 1H, ArH), 7.42–7.35 (m, 1H, ArH), 6.17 (app t, 1H, J = 6.2 Hz, H-1'), 5.92 (bs, 2H, N H 2 ), 4.92–4.80 (m, 2H,C H2 Ar), 4.31–4.26 (m, 1H, H-3'), 4.19–4.13 (m, 1H, H-4'), 3.78–3.68 (m, 2H,H-5'a, H-5'b), 2.58–2.43 (m, 2H, H-2'a, H-2'b), 0.90 (s, 9H, TBS t Bu), 0.11(s, 6H, TBS of 2 x CH3).

[0839]

[0840] Synthesis of dGTP-NB-6: 1 M TBAF (0.58 mL, 0.58 mmol) was added to a solution of dGTP-NB-4 (150 mg, 0.29 mmol) in THF (2 mL) at room temperature. The mixture was stirred at room temperature for 30 min. The mixture was evaporated and the residue was dissolved in CH3OH (5 mL). DMF–DMA (0.5 mL) was added to the mixture at room temperature, and then the mixture was heated to 50 °C. The mixture was stirred at 50 °C for 4 h, and then evaporated under reduced pressure. The residue was purified by silica gel column chromatography to give dGTP-NB-6 (60 mg, 45%) as a white solid. 1 HNMR (400 MHz, CDCl3) δ8.64 (s, 1H), 8.07 (s, 1H), 8.05–7.97 (m, 1H, ArH), 7.82–7.75 (m, 1H, ArH), 7.75–7.69 (m, 1H, ArH), 7.59–7.52 (m, 1H, ArH), 6.34 (dd, 1H, J = 5.8, 8.3Hz, H-1'), 5.00 (dd, 1H, J = 12.4 Hz, C H 2 Ar), 4.90 (dd, 1H, J = 12.4 Hz, C H 2 Ar), 4.46–4.41 (m, 1H, H-3'), 4.24–4.19 (m, 1H, H-4'), 3.84–3.70 (m, 2H,H-5'a, H-5'b), 3.19 (s, 3H, (C H 3 )2N), 3.17 (s, 3H, (C H 3 ) 2N), 2.82–2.72 (m, 1H,H-2'a) 2.66–2.62 (m, 1H, H-2'b).

[0841]

[0842] Synthesis of dGTP-NB-7: A solution of starting material (60 mg, 0.131 mmol) and proton sponge (42.2 mg, 0.197 mmol) in trimethyl phosphate (300 μL) was heated to 50 °C for 10 min and then cooled to 0 °C. Phosphorus oxychloride (9.42 μL, 0.10 mmol) was added to the above solution. After 2 h, a thoroughly vortexed solution of tributylammonium pyrophosphate (368 mg, 0.672 mmol) and tributylamine (368 μL) in DMF (1 mL) was added to the reaction mixture. The mixture was stirred for 1 h, and then 0.1 M TEAB buffer (pH 8.5, 15 mL) was added to the mixture. The resulting mixture was stirred for 2 h, and then NH3 solution (10 mL) was added to the mixture. The resulting mixture was stirred for 16 h and then concentrated under reduced pressure. The resulting residue was diluted with H2O and washed with CH2Cl2. The aqueous layer was concentrated under reduced pressure. The resulting residue was purified by passing it through a C18 column (50 min, 0% B to 90% B, A: 0.1 M TEAB in water / B: ACN). The resulting residue was then purified by passing it through a SAX column (50 min, 0% B to 80% B, A: 15% ACN in water / 15% ACN in 1 M TEAB). After purification, a colorless syrupy product (18 mg) was obtained. 1 H NMR (400 MHz, D2O) δ 8.10 (bs, 1H, H-8), 8.00–7.91 (m, 1H, ArH), 7.67–7.61 (m, 2H, ArH), 7.50–7.42 (m, 1H, ArH), 6.17 (dd, 1H, J = 5.9, 8.7 Hz, H-1'), 4.94–4.83 (m, 2H, C H 2Ar), 4.56–4.51 (m, 1H), 4.40–4.33(m, 1H), 4.15–4.00 (m, 2H, H-5'a, H-5'b), 2.74–2.65 (m, 1H, H-2'a) 2.62–2.54(m, 1H, H-2'b); 31 P NMR (121.4 MHz, D2O) δ –10.9 (bs, 1P), –11.5 (d, 1P, J =19.4 Hz), –23.3 (bs, 1P).

[0843] 6.19.3. Synthesis of L-3'-O-nitrobenzyl-dTTP

[0844]

[0845]

[0846]

[0847] Experimental procedure:

[0848]

[0849] Synthesis of dTTP-NB-2: β -L-deoxythymidine (2.0 g, 8.256 mmol, 1.0 equivalent) was dissolved in anhydrous DMF (30 mL). Imidazole (843 mg, 12.4 mmol, 1.5 equivalent) and tert-butyldimethylsilyl chloride (1.368 g, 9.08 mmol, 1.1 equivalent) were added to the mixture under nitrogen at 0 °C. The reaction mixture was stirred at room temperature for 4 h. Cold water was then added and the mixture was extracted with EtOAc (2 × 20 mL). The combined organic layers were dried over anhydrous Na₂SO₄, concentrated, and the resulting residue was subjected to column chromatography using 5%–7% MeOH in CH₂Cl₂ as the eluent to give dTTP-NB-2 (2.21 g, 75%) as a white solid. NMR (500 MHz, CD₃OD) δ 7.60 (q, J = 1.0 Hz, 1H), 6.24(dd, J = 7.9, 5.9 Hz, 1H), 4.34 (dt, J = 5.5, 2.6 Hz, 1 H), 3.94 (q,J = 2.8Hz, 1H), 3.90 – 3.86 (m, 1H), 3.83 – 3.79 (m, J = 11.4, 3.2 Hz, 1H), 2.26 –2.21 (m, J = 13.4, 5.9, 2.6 Hz, 1H), 2.11 (ddd, J = 13.6, 8.0, 6.0 Hz, 1H),1.85 (d, J = 1.2 Hz, 3H), 0.92 (s, 9H), 0.12 – 0.09 (m, 6H). ES-MS (ESI) m / z for C 16 H 29 N2O5Si + The calculated value [M + H] + 357.1840; Measured value 357.1720

[0850]

[0851] Synthesis of dTTP-NB-3: dTTP-NB-2 (1.6 g, 4.49 mmol, 1.0 equivalent) was dissolved in dry pyridine (16 mL) at 0 °C under a nitrogen atmosphere. TMSCl (1.7 mL, 13.4 mmol, 3 equivalent) was added. The reaction mixture was allowed to slowly reach room temperature and stirred for 3 h. After the reaction was complete, the mixture was evaporated under reduced pressure and immediately sealed under nitrogen. 15 mL of DMA was added to the reaction mixture, followed by triethylamine (5.2 mL, 35.9 mmol, 8 equivalent) at 0 °C, and then PhCOCl (1.05 mL, 8.98 mmol, 2 equivalent). The reaction mixture was stirred for 4 h. After the reaction was complete (monitored by TLC), the reaction mixture was cooled to 0 °C, brine was added, and the mixture was stirred vigorously for another 3 h. The reaction mixture was extracted three times with EtOAc. The combined organic layers were dried on anhydrous Na2SO4, concentrated, and the resulting residue was subjected to column chromatography using 30% EtOAc / Hex as the eluent to give dTTP-NB-3 (1.2 g, 58%) as a white solid after concentration. NMR (500 MHz, ) δ 7.94 – 7.90 (m, 2H), 7.76 (q, J = 1.0 Hz, 1H), 7.72 –7.67 (m, 1H), 7.55 – 7.51 (m, 2H), 6.22 (dd, J = 7.7, 6.0 Hz, 1H), 4.39 –4.34 (m, 1H), 3.97 (q, J = 2.9 Hz, 1H), 3.94 – 3.89 (m, 1H), 3.87 – 3.81 (m,1H), 2.33 – 2.27 (m, 1H), 2.24 – 2.17 (m, 1H), 1.92 (d, J = 1.2 Hz, 3H), 0.94 (s, 9H), 0.14–0.13 (m, 6H). ES-MS (ESI) m / z for C 23 H 33 N2O6Si + The calculated value [M + H] + 461.2103; measured value 461.2145.

[0852]

[0853] Synthesis of dTTP-NB-4: At 0 °C, under a nitrogen atmosphere and in the dark, 2-nitrobenzyl bromide (718 mg, 3.32 mmol, 1.3 equivalents) dissolved in dry DCM (20 mL) was added to a stirred solution of dTTP-NB-3 (1.18 g, 2.56 mmol, 1.0 equivalents) in DCM (24 mL). While strictly maintaining the mentioned order of addition, 1 M NaOH aqueous solution (10 mL) and Bu4NOH (60%; 10 mL) were added. The reaction mixture was allowed to stir for 3 h. The reaction mixture was diluted with water and then extracted with CH2Cl2 (3 × 50 mL). The combined organic layers were washed with brine and dried over anhydrous Na2SO4. After filtration and concentration, the crude product was further purified by rapid column chromatography (15% ethyl acetate in hexane) to give 800 mg of a yellow, viscous material of a mixture of dTTP-NB-4 and dTTP-NB-5 (in a 2:1 ratio), as indicated by LC-MS. No further purification was required; the mixture was used directly as the starting material in the next step. ES-MS (ESI) m / z against C 30 H 38 N3O8Si + The calculated value [M + H] + 596.2423; measured value 596.2468.

[0854]

[0855] Synthesis of dTTP-NB-7: A 30% ammonium hydroxide solution (1.44 ml; 12.32 mmol) was added to a mixture of dTTP-NB-4 and dTTP-NB-5 (1.18 g) in ethanol (15 ml). The reaction mixture was stirred at room temperature in the dark for 1 h and then subjected to evaporation. The residue was extracted with CH2Cl2 (3 × 50 mL3). The organic layers were combined and washed with brine and dried over anhydrous Na2SO4. After concentration, the residue was further purified by rapid column chromatography (40% to 60% ethyl acetate in hexane) to give dTTP-NB-7 (330 mg, 90% yield) as a yellow solid. ¹H NMR (500 MHz, CDCl3) δ 8.64 (broad s, 1H), 8.07 (dd, J = 8.2, 1.2 Hz, 1H), 7.78 (dd, J =7.8, 0.9 Hz, 1H), 7.66 (td, J= 7.7, 1.3 Hz, 1H), 7.51 – 7.49 (m, 1H), 7.48 –7.43 (m, 1H), 6.34 (dd, J = 8.8, 5.4 Hz, 1H), 4.94 – 4.86 (m, 2H), 4.25 –4.19 (m, 2H), 3.93-3.88 (m, 1H), 3.83 – 3.79 (m, 1H), 2.51 (ddd, J = 13.5,5.4, 1.4 Hz, 1H), 2.05 – 1.99 (m, 1H), 1.91 (d, J = 1.2 Hz, 3H), 0.91 (s,1H), 0.11 – 0.10 (m, J = 2.4 Hz, 6H). ES-MS (ESI) m / z for C 23 H 34 N3O7Si + The calculated value [M+H] + 492.2161; Measured value 492.2323.

[0856]

[0857] Synthesis of dTTP-NB-8: At 0 °C, a solution of TBAF in THF (1.0 M; 2.78 mL; 2.78 mmol) was added to a solution of dTTP-NB-7 (330 mg; 0.67 mmol) in 8 mL of anhydrous THF. The reaction mixture was allowed to warm to room temperature and stirred for 2 h in the absence of air and light. The mixture was poured into cold water (50 mL), and the resulting mixture was extracted with 10% MeOH / DCM (3 × 50 mL). The organic layers were combined, washed with brine, and dried over anhydrous Na2SO4. After concentration, the residue was purified by rapid column chromatography (1%–7% CH3OH in CH2Cl2) to give dTTP-NB-8 (170 mg, 67% yield) as a white solid. 1 H NMR (500 MHz,, CDCl3) 8.64 (br s, 1H), 8.05(dd, J = 8.2, 1.1 Hz, 1H), 7.76 – 7.70 (m, 1H), 7.65 (td, J = 7.6, 1.2 Hz,1H), 7.49 – 7.43 (m, 1H), 7.40 (d,J = 1.2 Hz, 1H), 6.15 (dd, J = 7.9, 6.2Hz), 4.90 (q, J = 14.1 Hz, 2H), 4.36 (dt, J = 5.8, 2.8 Hz, 1H), 4.18 (q, J =2.8 Hz, 1H), 3.98-3.92 (m, 1H), 3.85-3.78 (m, 1H), 2.75 – 2.55 (m, 1H), 2.48– 2.35 (m, 1H), 1.91 (d, J = 1.2 Hz, 3H). ES-MS (ESI) m / z for C 17 H 20 N3O7Si + The calculated value [M + H] + 378.1296; measured value 378.1306.

[0858]

[0859] Synthesis of dTTP-NB-9: A solution of starting material (60 mg, 1.0 equivalent, 0.159 mmol) and proton sponge (41 mg, 1.2 equivalent) was dried overnight in a vacuum oven under P2O5. Subsequently, the reaction mixture was sealed under nitrogen, and trimethyl phosphate (0.53 µL) was added and the solution was completely dissolved, then cooled to 0 °C. POCl3 (22.3 µL, 1.5 equivalent) was added to the above solution and stirred at the same temperature for 1 h. After 1 h, a well-vortexed solution of tributylammonium pyrophosphate (329 mg, 0.9 mmol) and tributylamine (0.29 µL) in DMF (1.2 mL) was added to the reaction mixture and stirred at room temperature for 20 min. Then, 0.1 M TEAB buffer (pH 8.0, 5 mL) was added to the mixture and stirred for 2 h. After that, NH3 solution (5 mL) was added and the resulting mixture was stirred for another 3 h, then concentrated under reduced pressure. The resulting residue was purified by passing it through a C18 column (50 min, 0% B to 90% B, A: 0.1 M TEAB in water / B: ACN). The collected fraction was concentrated and the resulting residue was purified by passing it through a SAX column (50 min, 0% B to 80% B, A: 15% ACN in water; B: 15% ACN in 1 M TEAB). After purification, the collected fraction was concentrated under reduced pressure and the resulting residue was redissolved in water and lyophilized. A white foamy lyophilized product (33 mg) was obtained. 1 H NMR (400 MHz, D2O) δ7.97 (d, J = 8.6 Hz, 1H), 7.70 – 7.62 (m, 3H), 7.51 – 7.46 (m, 1H), 6.23 (dd,J = 9.2, 5.6 Hz, 1H), 4.88 (q, J = 12.8 Hz, 2H), 4.47 (d, J = 5.5 Hz, 1H), 4.32 – 4.27 (m, 1H), 4.16 – 4.05 (m, 2H), 2.41 (dd, J = 13.8, 6.0 Hz, 1H), 2.29 – 2.18 (m, 1H), 1.83 (d, J = 0.8 Hz, 3H); 31 P NMR (121.4 MHz, D2O) δ –7.33 (d, 1P), –10.89 (d, 1P), –21.77 (t, 1P); HRMS (ESI) m / z for C 17 H 21 N3O 16Calculated value of P3 [M]: 616.0140; Measured value [M – H]: – 616.0145.

[0860] 6.19.4. Synthesis of L-3'-O-nitrobenzyl-dCTP

[0861]

[0862]

[0863]

[0864] Experimental procedure:

[0865]

[0866] Synthesis of dCTP-NB-1: TBDMSCl (212 mg; 1.28 mmol) was added to a stirred solution of β-L-2'-deoxycytidine 1 (0.26 g; 1.40 mmol) in dry pyridine (9 mL) and the mixture was stirred ...

Claims

1. A nucleotide or nucleoside comprising: a. A pentose sugar selected from (3R,4S)-3,4,5-trihydroxypentanal and (4R)-4,5-dihydroxypentanal; wherein the H of the 5' hydroxyl group is substituted with one or more phosphate groups; and wherein the H of the 3' hydroxyl group, if present, is optionally substituted with a cleavable protecting group; b. Nitrogenous bases, and c. Optional cleavable marker comprising a cleavable linker and a marker; It contains at least one of a cleavable protecting group and a cleavable label.

2. The nucleotide of claim 1, wherein, The cleavable marker is attached to the nitrogenous base, the 3'O, or the 5' phosphate group.

3. The nucleotide of claim 1, having a structure according to formula I: Equation I The bases are nitrogen-containing bases, and R' is a cleavable protecting group.

4. The nucleotide of claim 1, having a structure according to formula II. Formula II Where the base is a nitrogen-containing base; R' is a cleavable protecting group or H; R2-mark is a cleavable mark containing a cleavable linker R2 and the mark.

5. The nucleotide of claim 1, having a structure according to formula III: Formula III The bases are nitrogen-containing bases; the R2-label is a cleavable label containing a cleavable linker R2 and a label.

6. The nucleotide as described in claim 1, 2, 4, or 5, wherein, The marker is selected from the following group: 。 7. The nucleotide according to any one of claims 1-4, wherein, The cleavable protecting group is selected from allyl, dimethyl disulfide, nitrobenzyl, and azide protecting groups.

8. The nucleotide of claim 7, wherein, The cleavable protecting group is selected from: 。 9. The nucleotide as described in claim 1, 2, 4, or 5, wherein, The cleavable linker is photocleavable, cleavable by contact with water-soluble phosphine, or cleavable by a water-soluble catalyst containing a transition metal.

10. The nucleotide of claim 9, wherein, The cleavable linker comprises an allyl or azide group.

11. The nucleotide of claim [139], wherein, The cleavable linker comprises: ,or 。 12. The nucleotide of claim 3, wherein the nucleotide is selected from: and Wherein R' is selected from: 。 13. The nucleotide of claim 4, wherein the nucleotide is selected from: and ； Wherein R' is selected from: H, ;and R2 includes: ,or 。 14. The nucleotide of claim 5, wherein the nucleotide is selected from: and ； Where R2 includes: ,or 。 15. The nucleotide of claim 4, wherein the nucleotide is selected from: and ; Wherein R' is selected from: H、 。 16. The nucleotide of claim 5, wherein the nucleotide is selected from: and ； Wherein R' is selected from: H、 。 17. A mirror-image nucleic acid polymerase comprising a sequence having at least 90% sequence identity with SEQ ID NO: 1, wherein the polymerase comprises D-type amino acids.

18. The polymerase of claim 17, wherein, The polymerase is composed of D-type amino acids.

19. The polymerase of claim 17 or 18, comprising a sequence having at least 95% sequence identity with SEQ ID NO:

1.

20. The polymerase of claim 19, comprising a sequence having at least 96%, 97%, 98%, or 99% sequence identity with SEQ ID NO:

1.

21. The polymerase according to any one of claims 17-20, comprising one or more modifications at one or more amino acid sites selected from E276, K317, N424 and S651 compared to SEQ ID NO:

1.

22. The polymerase of claim 21, comprising one or more substitutions selected from E276A, K317G, N424A and S651A compared to SEQ ID NO:

1.

23. The polymerase of claim 22, comprising the substitutions of E276A, K317G, N424A, and S651A compared to SEQ ID NO:

1.

24. The polymerase according to any one of claims 17-23, comprising one or more modifications at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733 and I744 compared to SEQ ID NO:

1.

25. The polymerase of claim 24, wherein, It comprises, compared with SEQ ID NO: 1, one or more substitutions from Ile to Ala, Val, Leu or Tyr at one or more amino acid sites selected from I80, I127, I171, I176, I191, I228, I256, I264, I268, I400, I597, I610, I618, I630, I642, I715, I733 and I744.

26. The polymerase of claim 25, comprising one or more substitutions selected from I80V, I127V, I171A, I176V, I191V, I228V, I256V, I264A, I268L, I400V, I597V, I610V, I618A, I630L, I642V, I715Y, I733V, and I744V compared to SEQ ID NO:

1.

27. The polymerase of claim 26, comprising substitutions of I80V, I127V, I171A, I176V, I191V, I228V, I256V, I264A, I268L, I400V, I597V, I610V, I618A, I630L, I642V, I715Y, I733V, and I744V compared to SEQ ID NO:

1.

28. The polymerase according to any one of claims 17-27, comprising one or more modifications at one or more amino acid sites selected from M129, I130, G131, D141, E143, L408, Y409, P410, A485, T514 and I521 compared to SEQ ID NO:

1.

29. The polymerase of claim 28, comprising one or more modifications at one or more amino acid sites selected from D141, E143, Y409 and A485 compared to SEQ ID NO:

1.

30. The polymerase of claim 29, comprising one or more substitutions selected from D141A, E143A, Y409V and A485L compared to SEQ ID NO:

1.

31. The polymerase of claim 30, comprising substitutions of D141A, E143A, Y409V, and A485L compared to SEQ ID NO:

1.

32. The polymerase of claim 28, comprising one or more modifications at one or more amino acid sites selected from D141, E143, L408, Y409, P410, A485, T514 and I521 compared to SEQ ID NO:

1.

33. The polymerase of claim 32, comprising one or more substitutions selected from D141A, E143A, L408A, Y409A, P410I, A485V, T514S and I521L compared to SEQ ID NO:

1.

34. The polymerase of claim 33, comprising substitutions of D141A, E143A, L408A, Y409A, P410I, A485V, T514S and I521L compared to SEQ ID NO:

1.

35. The polymerase of claim 28, comprising one or more substitutions selected from M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S, I521L compared to SEQ ID NO: 1, or a modification of adding D between I130 and G131.

36. The polymerase of claim 35, comprising the substitution of M129L, D141A, E143A, L408A, Y409A, P410I, A485V, T514S and I521L compared to SEQ ID NO: 1 and the addition of D between I130 and G131.

37. The polymerase according to any one of claims 17-36, comprising a sequence selected from SEQ ID NOs: 2-7.

38. The polymerase of claim 37, comprising sequences selected from SEQ ID NOs: 2-7.

39. A method for synthesizing a mirror-image nucleic acid polymerase comprising the natural chemical linking (NCL) of two or more precursor fragments of a D-peptide selected from any one of SEQ ID NOs: 37-56.

40. The method of claim 39, further comprising: One or more cysteine ​​residues are desulfurized to one or more alanine residues, optionally wherein said one or more cysteine ​​residues are selected from Cys 500 Cys 539 Cys 595 Cys 651 and Cys 714 .

41. The method of claim 39 or 40, further comprising: In Cys 506 and Cys 509 The Acm protection at the location is removed.

42. A mirror-image nucleic acid polymerase, which is generated by the method according to any one of claims 39-41.

43. A mirror-image nucleic acid polymerase, which is generated by the method described in Example 13.

44. A method for replicating L-polynucleotides, comprising the following steps: The L-polynucleotide is incubated with a mixture comprising (i) the L-polynucleotide, (ii) the L-primer, (iii) the L-dNTP, (iv) the polymerase as described in any one of claims 17-38, 42 and 43, and (v) a buffer solution to induce the replication of the L-polynucleotide.

45. The method of claim 44, wherein, The L-polynucleotide is DNA or RNA.

46. ​​The method according to any one of claims 44-45, wherein, The mixture contains L-dATP, L-dGTP, L-dCTP and L-dTTP.

47. The method according to any one of claims 44-46, wherein, The buffer solution contains 50 mM Tris-HCl, 20 mM MgCl2, 1 mM DTT and 50 mM KCl at pH 7.

5.

48. The method according to any one of claims 44-47, wherein, The incubation step includes PCR.

49. A method for sequencing L-polynucleotides, comprising the following cycle: a. Incubate a mixture comprising (i) the L-polynucleotide, (ii) the L-primer, (iii) the L-3'-O-R'-dNTP-R2-label, (iv) the polymerase as described in any one of claims 17-38, 42 and 43, and (v) a buffer solution to obtain the replication product; b. Detecting the signal from the L-3'-O-R'-dNTP-R2-tagged marker incorporated into the replication product; and c. Inducing the cleavage of the R' and R2 groups of the L-3'-O-R'-dNTP-R2-labeled group incorporated into the replication product.

50. The method of claim 49, wherein, The cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times.

51. The method according to any one of claims 49-50, wherein, The L-3'-O-R'-dNTP-R2-label includes L-3'-O-R'-dATP-R2-label, L-3'-O-R'-dTTP-R2-label, L-3'-O-R'-dGTP-R2-label, and L-3'-O-R'-dCTP-R2-label, wherein each label is different.

52. The method according to any one of claims 49-51, wherein, The L-3'-O-R'-dNTP-R2- marker has the structure according to Formula II as defined in any one of claims 4, 6-13 and 15.

53. The method of claim 52, wherein, The L-3'-O-R'-dNTP-R2- label is a nucleotide as described in claim 13 or 15.

54. The method according to any one of claims 49-53, wherein, The signal is a fluorescence signal.

55. A method for sequencing L-polynucleotides, comprising the following steps: a. Incubate a mixture comprising (i) the L-polynucleotide, (ii) the L-primer, (iii) the L-dNTP, (iv) the L-ddNTP-R2-label or the L-3'-O-R'-dNTP-R2-label, (v) the polymerase as described in any one of claims 17-38, 42 and 43, and (vi) a buffer solution to obtain the replication product; b. Separate the replication product; and c. Detect the signal from the L-ddNTP-R2- tag or L-3'-O-R'-dNTP-R2- tag incorporated into the replication product.

56. The method of claim 55, wherein, The L-dNTPs include L-dATP, L-dTTP, L-dGTP, and L-dCTP.

57. The method of claim 55 or 56, wherein, The L-ddNTP-R2-label includes L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label and L-ddCTP-R2-label, where each label is different.

58. The method according to any one of claims 55-57, wherein, The L-ddNTP-R2-marker has Formula III as defined in any one of claims 5, 6, 9-11.

59. The method of claim 58, wherein, The L-ddNTP-R2-label is a nucleotide as described in claim 14 or 16.

60. The method of claim 55 or 56, wherein, The L-3'-O-R'-dNTP-R2-label includes L-3'-O-R'-dATP-R2-label, L-3'-O-R'-dTTP-R2-label, L-3'-O-R'-dGTP-R2-label, and L-3'-O-R'-dCTP-R2-label, wherein each label is different.

61. The method according to any one of claims 55-56 and 60, wherein, The L-3'-O-R'-dNTP-R2-marker has Formula II as defined in any one of claims 4, 6-13 and 15.

62. The method of claim 61, wherein, The L-3'-O-R'-dNTP-R2- label is a nucleotide as described in claim 13 or 15.

63. The method according to any one of claims 55-62, wherein, The signal is a fluorescence signal.

64. The method according to any one of claims 55-63, wherein, The incubation step includes PCR.

65. The method according to any one of claims 55-64, wherein, The separation step includes separating the replicated product by size.

66. A method for sequencing L-polynucleotides, comprising the following cycle: a. Incubate a mixture comprising (i) the L-polynucleotide, (ii) the L-primer, (iii) the L-3'-O-R'-dNTP, (iv) the L-ddNTPs-R2-labeled or L-dNTPs-R2-labeled, (v) the polymerase as described in any one of claims 17-38, 42 and 43, and (vi) a buffer solution to obtain the replication product; b. Detecting the signal from the L-ddNTP-R2-tagged or L-3'-O-R'-dNTP-R2-tagged marker incorporated into the replication product; and c. Inducing the cleavage of: i) the R' group of the L-3'-O-R'-dNTP and the R2 group of the L-ddNTPs-R2-labeled group incorporated into the replication product; or ii) the R' group of the L-3'-O-R'-dNTP, the R' and R2 groups of the L-3'-O-R'-dNTP-R2-labeled group incorporated into the replication product.

67. The method of claim 66, wherein, The cycle is repeated at least 3, 5, 10, 50, 100, 150, 200, 250, 300, 400, 500, or 1000 times.

68. The method of any one of claims 66-67, wherein, The L-3'-O-R'-dNTPs include L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, and L-3'-O-R'-dCTP.

69. The method of any one of claims 66-68, wherein, The L-3'-O-R'-dNTP has the structure according to Formula I as defined in any one of claims 3, 7 and 8.

70. The method of claim 69, wherein, The L-3'-O-R'-dNTP is the nucleotide as described in claim 12.

71. The method according to any one of claims 66-70, wherein, The L-ddNTP-R2-label includes L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label and L-ddCTP-R2-label, where each label is different.

72. The method according to any one of claims 66-66, wherein, The L-ddNTP-R2-marker has the structure according to Formula III as defined in any one of claims 5, 6 and 9-11.

73. The method of claim 72, wherein, The L-ddNTP-R2-label is a nucleotide as described in claim 14 or 16.

74. The method according to any one of claims 66-70, wherein, The L-3'-O-R'-dNTP-R2-label includes L-3'-O-R'-dATP-R2-label, L-3'-O-R'-dTTP-R2-label, L-3'-O-R'-dGTP-R2-label, and L-3'-O-R'-dCTP-R2-label, wherein each label is different.

75. The method according to any one of claims 66-71 and 74, wherein, The L-3'-O-R'-dNTP-R2- marker has the structure according to Formula II as defined in any one of claims 4, 6-13 and 15.

76. The method of claim 72, wherein, The L-3'-O-R'-dNTP-R2- label is a nucleotide as described in claim 13 or 15.

77. The method of any one of claims 66-76, wherein the signal is a fluorescence signal.

78. A mirror-terminal deoxynucleotidyl transferase comprising a sequence having at least 90% sequence identity with SEQ ID NO: 36, wherein the transferase comprises D-amino acids.

79. The transferase of claim 78, wherein, The transferase is composed of D-type amino acids.

80. The transferase of claim 78 or 79, comprising a sequence having at least 95% sequence identity with SEQ ID NO:

36.

81. The transferase of claim 80, comprising a sequence having at least 96%, 97%, 98% or 99% sequence identity with SEQ ID NO:

36.

82. The transferase according to any one of claims 78-81, comprising one or more modifications at one or more amino acid sites of SEQ ID NO:

36.

83. The transferase of claim 82, comprising two, three, four, five, six, seven, eight, nine, ten, eleven or twelve amino acid substitutions compared to SEQ ID NO:

36.

84. The transferase according to any one of claims 78-83, comprising a precursor fragment having a sequence selected from SEQ ID NOs: 57-78 or a modified thereof.

85. A method for synthesizing a mirror-terminal deoxynucleotidyl transferase comprising the natural chemical linking (NCL) of two or more precursor fragments of a D-peptide selected from any one of SEQ ID NOs: 57-78.

86. The method of claim 85, further comprising desulfurizing one or more cysteine ​​residues into one or more alanine residues.

87. The method of claim 86, wherein, The one or more cysteine ​​residues are selected from Cys 47 Cys 108 Cys 162 Cys 224 Cys 268 and Cys 317 .

88. The method of claim 85 or 86, further comprising Cys 26 Cys 59 Cys 87 Cys 173 Cys 249 Cys 275 and Cys 309 The Acm protection at the location is removed.

89. A mirror-terminal deoxynucleotidyl transferase, generated by the method described in any one of claims 85-88.

90. A mirror-terminal deoxynucleotidyl transferase, generated by the method described in Example 14 or 19.

91. A method for synthesizing L-polynucleotides, comprising the following steps: The synthesis of the L-polynucleotide is induced by incubating a mixture comprising (i) an L-primer, (ii) an L-dNTP or L-ddNTP, (iii) a transferase as described in any one of claims 78-84, 89 and 90, and (iv) a buffer.

92. The method of claim 91, wherein, The L-polynucleotide is DNA or RNA.

93. The method as claimed in claim 91 or 92, wherein, The mixture contains L-dATP, L-dGTP, L-dCTP, or L-dTTP.

94. The method of claim 93, wherein, The L-dNTPs include (i) L-dATP or L-dTTP and (ii) L-dGTP or L-dCTP.

95. The method of claim 93, wherein, The L-dNTPs include L-dATP, L-dGTP, L-dCTP, and L-dTTP.

96. The method of claim 91 or 92, wherein, The mixture contains radiolabeled L-ddNTPs, optionally L-ddATP, L-ddGTP, L-ddCTP, or L-ddTTP.

97. The method of claim 96, wherein, The mixture contains L-3'-OR-dNTP, optionally wherein the L-3'-OR-dNTP is L-3'-OR-dATP, L-3'-OR-dGTP, L-3'-OR-dCTP or L-3'-OR-dTTP.

98. The method of claim 91 or 92, wherein, The L-ddNTP contains either the L-ddNTP-R2- tag or the L-3'-O-R'-dNTP-R2- tag.

99. The method of any one of claims 91-98, further comprising the step of stopping the reaction by heating or by adding a chelating agent.

100. The method of claim 99, wherein, The chelating agent is EDTA.

101. A kit for replicating or synthesizing L-polynucleotides, comprising (i) a polymerase as described in any one of claims 17-38, 42 and 43 or a transferase as described in any one of claims 78-84, 89 and 90, and (ii) an optional buffer.

102. The kit of claim 101, further comprising L-dNTP.

103. The kit of claim 102, wherein, The L-dNTPs include L-dATP, L-dGTP, L-dCTP, L-dTTP, or L-UTP.

104. The kit according to any one of claims 101-103, further comprising an L-3'-O-R'-dNTP-R2- label.

105. The kit of claim 104, wherein, The L-3'-O-R'-dNTP-R2- label includes L-3'-O-R'-dATP-R2- label, L-3'-O-R'-dTTP-R2- label, L-3'-O-R'-dGTP-R2- label, L-3'-O-R'-dCTP-R2- label, or L-3'-O-R'-dUTP-R2- label.

106. The kit according to any one of claims 101-105, wherein, The L-3'-O-R'-dNTP-R2-marker has Formula II as defined in any one of claims 4-11.

107. The kit of claim 106, wherein, The L-3'-O-R'-dNTP-R2- label is a nucleotide as described in 13 or 15.

108. The kit according to any one of claims 101-107, further comprising an L-ddNTP-R2- label.

109. The kit of claim 108, wherein, The L-ddNTP-R2-label includes L-ddATP-R2-label, L-ddTTP-R2-label, L-ddGTP-R2-label, L-ddCTP-R2-label, or L-ddUTP-R2-label.

110. The kit according to any one of claims 101-109, wherein, The L-ddNTP-R2-marker has Formula III as defined in any one of claims 5, 6 and 9-11.

111. The kit of claim 110, wherein, The L-ddNTP-R2-label is a nucleotide as described in claim 14 or 16.

112. The kit according to any one of claims 101-111, further comprising L-3'-O-R'-dNTP.

113. The kit of claim 112, wherein, The L-3'-O-R'-dNTPs include L-3'-O-R'-dATP, L-3'-O-R'-dTTP, L-3'-O-R'-dGTP, and L-3'-O-R'-dCTP.

114. The kit according to any one of claims 101-113, wherein, The L-3'-O-R'-dNTP has Formula I as defined in claim 3 or 12.

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