MODIFIED NUCLEOTIDES, METHODS FOR MAKING AND USING SAME

• US 20080091005A1
• Filed: 07/20/2007
• Published: 04/17/2008
• Est. Priority Date: 07/20/2006
• Status: Abandoned Application

• Current Assignee: Life Technologies Corporation (Thermo Fisher Scientific Incorporated)
• Original Assignee: Applied Biosystems, Inc. (Thermo Fisher Scientific Incorporated), VisiGen Biotechnologies, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Belosludtsev, Yuri, Hardin, Susan, Gao, Xiaolian, Reddy, Mitsu, Yu, Peilin, Deluge, Norha, Wang, Hongyi, Chen, Jer-Kang, Menchen, Steven, Williams, Amy, Lam, Joe, Lincecum, Tommie
• Application Number:
  US11/781,160
• Publication Number:
  US 20080091005A1
• Time in Patent Office: Days
• US Class Current: 536/26.6
• CPC Class Codes: C07H 21/04 with deoxyribosyl as saccharide radical

Abstract

Modified nucleotides are disclosed for use in single molecule sequencing, methods for making the modified nucleotides and method for using the modified nucleotides. Linkers for making the modified nucleotide are also disclosed.

Claims

1. A modified nucleotide of the general formula (I):

DG-E′

-G-E-Nu 

 

(I)

where;

DG is a detectable group,

E and E′

are the same and different group,

G is a linking group, and

Nu is a natural or synthetic nucleotide,

where G comprises a linear or branched alkenyl group or an alkenyl group including a central ring structure.

2. The nucleotide of claim 1, wherein the E and E′

group include a central main group element.

3. The nucleotide of claim 1, wherein the central main group elements are selected from the group consisting of boron (B), carbon (C), nitrogen (N), oxygen (O), silicon (Si), phosphorus (P), sulfur (S), gallium (Ga) and germanium (Ge).

4. The nucleotide of claim 1, wherein G includes a central ring structure.

5. The nucleotide of claim 1, wherein G includes a linear alkenyl group.

6. The nucleotide of claim 1, wherein the nucleotide has the general formula (II):

DG-E′

-R²-A—

R¹-E-Nu 

 

(II)

where;

DG is a detectable group,

E and E′

are the same and different and are a carbon group (C(H)₂, C(HR³) or C(R³)₂), an oxygen atom (O), a sulfur atom (S), an amino group (N(R³)), an phosphano group (P(R³)), a phosphito group (P(OR³)O), a phosphate group (P(O₂)O), a polyphosphate group (P(O₂)O). (n is an integer having a value between 3 and

     12), a silyl group (Si(R³)₂), a siloxyl group (Si(OR³)₂), a carboxy group (C(O)O), a keto group (C(O)), an amido group (C(O)N(R³)), an urea group (N(R³)C(O)N(R³)), a carbonate group (OC(O)O), or an urethane group (OC(O)N(R³), R³ is a hydrogen atom, a carbyl group or is absent depending on the structure,

R¹ and R² are the same or different and are carbenzyl groups,

A is a ring structure, and

Nu is a natural or synthetic nucleotide.

7. The nucleotide of claim 1, wherein the nucleotide has the general formulas (III or IIIa):

DG-E′

-R²-A-R¹-E-P(O₂)OP(O₂)OP(O₂)-Sugar-Base 

 

(III)
DG-E′

-R²-A-R¹-E-P(O₂)OP(OZ¹)OP(OZ²)-Sugar-Base 

 

(IIIa)

where;

DG is a detectable group,

E and E′

are the same and different and are a carbon group (C(H)₂, C(HR³) or C(R³)₂), an oxygen atom (O), a sulfur atom (S), an amino group (N(R³)), an phosphano group (P(R³)), a phosphito group (P(OR³)O), a phosphate group (P(O₂)O), a polyphosphate group (P(O₂)O)_n (n is an integer having a value between 3 and

     12), a silyl group (Si(R³)₂), a siloxyl group (Si(OR³)₂), a carboxy group (C(O)O), a keto group (C(O)), an amido group (C(O)N(R³)), an urea group (N(R³)C(O)N(R³)), a carbonate group (OC(O)O), or an urethane group (OC(O)N(R³), R³ is a hydrogen atom, a carbyl group or is absent depending on the structure,

R¹ and R² are the same or different and are is carbenzyl groups,

A is a ring structure, Sugar is a sugar moiety,

Base is a natural or synthetic nucleotide base and

Z¹ or Z² are the same or different and are groups that either modify incorporation timing or enhancing detection of the detectable group.

8. The nucleotide of claim 1, wherein the nucleotide has the general formulas (IV or IVa):

DG-E′

-R²-A-R¹-E-P(O)(OP(O₂)OH)OP(O₂)-Sugar-Base 

 

(IV)
DG-E′

-R²-A-R¹-E-P(O)(OP(OZ¹)OH)OP(OZ²)-Sugar-Base 

 

(IVa)

where;

DG is a detectable group,

E and E′

are the same and different and are a carbon group (C(H)₂, C(HR³) or C(R³)₂), an oxygen atom (O), a sulfur atom (S), an amino group (N(R³)), an phosphano group (P(R³)), a phosphito group (P(OR³)O), a phosphate group (P(O₂)O), a polyphosphate group (P(O₂)O). (n is an integer having a value between 3 and

     12), a silyl group (Si(R³)₂), a siloxyl group (Si(OR³)₂), a carboxy group (C(O)O), a keto group (C(O)), an amido group (C(O)N(R³)), an urea group (N(R³)C(O)N(R³)), a carbonate group (OC(O)O), or an urethane group (OC(O)N(R³), R³ is a hydrogen atom, a carbyl group or is absent depending on the structure,

R¹ and R² are the same or different and are is carbenzyl groups,

A is a ring structure,

Sugar is a sugar moiety,

Base is a natural or synthetic nucleotide base and

Z¹ or Z² are the same or different and are groups that either modify incorporation timing or enhancing detection of the detectable group.

9. The nucleotide of claim 1, wherein the nucleotide has the general formula (V):

DG-E′

-R²-A-R¹-E-P(O)(OP(O₂)OP(O₂)OH)-Sugar-Base 

 

(V)

where;

DG is a detectable group,

E and E′

are the same and different and are a carbon group (C(H)₂, C(HR³) or C(R³)₂), an oxygen atom (O), a sulfur atom (S), an amino group (N(R³)), an phosphano group (P(R³)), a phosphito group (P(OR³)O), a phosphate group (P(O₂)O), a polyphosphate group (P(O₂)O)_n (n is an integer having a value between 3 and

     12), a silyl group (Si(R³)₂), a siloxyl group (Si(OR³)₂), a carboxy group (C(O)O), a keto group (C(O)), an amido group (C(O)N(R³)), an urea group (N(R³)C(O)N(R³)), a carbonate group (OC(O)O), or an urethane group (OC(O)N(R³), R³ is a hydrogen atom, a carbyl group or is absent depending on the structure,

R¹ and R² are the same or different and are is carbenzyl groups,

A is a ring structure,

Sugar is a sugar moiety, and

Base is a natural or synthetic nucleotide base.

10. The nucleotide of claim 1, wherein the nucleotide has the general formula (VI):

DG-E′

-R²-A-R¹-E-Sugar(P(O₂)OP(O₂)OP(O₂)OH)Base 

 

(VI)

where;

DG is a detectable group,

E and E′

are the same and different and are a carbon group (C(H)₂, C(HR³) or C(R³)₂), an oxygen atom (O), a sulfur atom (S), an amino group (N(R³)), an phosphano group (P(R³)), a phosphito group (P(OR³)O), a phosphate group (P(O₂)O), a polyphosphate group (P(O₂)O)_n (n is an integer having a value between 3 and

     12), a silyl group (Si(R³)₂), a siloxyl group (Si(OR³)₂), a carboxy group (C(O)O), a keto group (C(O)), an amido group (C(O)N(R³)), an urea group (N(R³)C(O)N(R³)), a carbonate group (OC(O)O), or an urethane group (OC(O)N(R³), R³ is a hydrogen atom, a carbyl group or is absent depending on the structure,

R¹ and R² are the same or different and are is carbenzyl groups,

A is a ring structure,

Sugar is a sugar moiety, and

Base is a natural or synthetic nucleotide base.

11. The nucleotide of claim 1, wherein the nucleotide has the general formula (VII):

DG-E′

-R²-A-R¹-E-Base-Sugar-P(O₂)OP(O₂)OP(O₂)OH 

 

(VII)

where;

DG is a detectable group,

E and E′

are the same and different and are a carbon group (C(H)₂, C(HR³) or C(R³)₂), an oxygen atom (O), a sulfur atom (S), an amino group (N(R³)), an phosphano group (P(R³)), a phosphito group (P(OR³)O), a phosphate group (P(O₂)O), a polyphosphate group (P(O₂)O)_n (n is an integer having a value between 3 and

     12), a silyl group (Si(R³)₂), a siloxyl group (Si(OR³)₂), a carboxy group (C(O)O), a keto group (C(O)), an amido group (C(O)N(R³)), an urea group (N(R³)C(O)N(R³)), a carbonate group (OC(O)O), or an urethane group (OC(O)N(R³), R³ is a hydrogen atom, a carbyl group or is absent depending on the structure,

R¹ and R² are the same or different and are is carbenzyl groups,

A is a ring structure,

Sugar is a sugar moiety, and

Base is a natural or synthetic nucleotide base.

12. The nucleotide of claim 1, wherein the ring structure A is saturated, unsaturated or aromatic or can include a mixture of saturated, unsaturated, or aromatic rings.

13. The nucleotide of claim 1, wherein the each ring in the ring structure includes from to about 12 main group elements.

14. The nucleotide of claim 1, wherein each carbyl group and each carbenzyl group include from 1 to 40 carbon, where one or more of the carbon atoms can be replaced with a hetero atoms selected from the group consisting of B, C, Si, Ge, N, P. As, O, S, or Se and having sufficient hydrogen atoms to satisfy the valency of the group, where one or more hydrogen atoms can be replaced with F, Cl, Br, I, OR, SR, COR, COOR, CONH₂, CONHR, CONRR′

, or any other monovalent group inert or substantially inert under the substitution/displacement reaction conditions.

15. The nucleotide of claim 1, wherein the nucleotide has the general formula (VIII):

DG-E′

-R-E-Nu 

 

(VIII)

where;

DG is a detectable group,

E and E′

are the same and different and are a carbon group (C(H)₂, C(HR³) or C(R³)₂), an oxygen atom (O), a sulfur atom (S), an amino group (N(R³)), an phosphano group (P(R³)), a phosphito group (P(OR³)O), a phosphate group (P(O₂)O), a polyphosphate group (P(O₂)O)_n (n is an integer having a value between 3 and

     12), a silyl group (Si(R³)₂), a siloxyl group (Si(OR³)₂), a carboxy group (C(O)O), a keto group (C(O)), an amido group (C(O)N(R³)), an urea group (N(R³)C(O)N(R³)), a carbonate group (OC(O)O), or an urethane group (OC(O)N(R³), R³ is a hydrogen atom, a carbyl group or is absent depending on the structure (e.g., E′

or E is a nitrogen atom doubly bonded to DG or to R² or a carbon atom triply bonded to DG or R²),

R is a carbenzyl group, and

Nu is a natural or synthetic nucleotide.

16. The nucleotide of claim 1, wherein the nucleotide has the general formulas (IX or IXa):

DG-E′

-R-E-P(O₂)OP(O₂)OP(O₂)-Sugar-Base 

 

(IX)
DG-E′

-R-E-P(O₂)OP(OZ¹)OP(OZ²)-Sugar-Base 

 

(IXa)

where;

DG is a detectable group,

E and E′

are the same and different and are a carbon group (C(H)₂, C(HR³) or C(R³)₂), an oxygen atom (O), a sulfur atom (S), an amino group (N(R³)), an phosphano group (P(R³)), a phosphito group (P(OR³)O), a phosphate group (P(O₂)O), a polyphosphate group (P(O₂)O)_n (n is an integer having a value between 3 and

     12), a silyl group (Si(R³)₂), a siloxyl group (Si(OR³)₂), a carboxy group (C(O)O), a keto group (C(O)), an amido group (C(O)N(R³)), an urea group (N(R³)C(O)N(R³)), a carbonate group (OC(O)O), or an urethane group (OC(O)N(R³), R³ is a hydrogen atom, a carbyl group or is absent depending on the structure (e.g., E′

or E is a nitrogen atom doubly bonded to DG or to R² or a carbon atom triply bonded to DG or R²),

R is a carbenzyl group,

Sugar is a sugar moiety,

Base is a natural or synthetic nucleotide base, and

Z¹ or Z² are the same or different and are groups that either modify incorporation timing or enhancing detection of the detectable group as described herein.

17. The nucleotide of claim 1, wherein the nucleotide has the general formulas (X or Xa):

DG-E′

-R-E-P(O)(OP(O₂)OH)OP(O₂)-Sugar-Base 

 

(X)
DG-E′

-R-E-P(O)(OP(OZ¹)OH)OP(OZ²)-Sugar-Base 

 

(Xa)

where;

DG is a detectable group,

E and E′

are the same and different and are a carbon group (C(H)₂, C(HR³) or C(R³)₂), an oxygen atom (O), a sulfur atom (S), an amino group (N(R³)), an phosphano group (P(R³)), a phosphito group (P(OR³)O), a phosphate group (P(O₂)O), a polyphosphate group (P(O₂)O)_n (n is an integer having a value between 3 and

     12), a silyl group (Si(R³)₂), a siloxyl group (Si(OR³)₂), a carboxy group (C(O)O), a keto group (C(O)), an amido group (C(O)N(R³)), an urea group (N(R³)C(O)N(R³)), a carbonate group (OC(O)O), or an urethane group (OC(O)N(R³), R³ is a hydrogen atom, a carbyl group or is absent depending on the structure (e.g., E′

or E is a nitrogen atom doubly bonded to DG or to R² or a carbon atom triply bonded to DG or R²),

R is a carbenzyl group,

Sugar is a sugar moiety,

Base is a natural or synthetic nucleotide base, and

Z¹ or Z² are the same or different and are groups that either modify incorporation timing or enhancing detection of the detectable group as described herein.

18. The nucleotide of claim 1, wherein the nucleotide has the general formula (XI):

DG-E′

-R-E-P(O)(OP(O₂)OP(O₂)OH)-Sugar-Base 

 

(XI)

where;

DG is a detectable group,

E and E′

are the same and different and are a carbon group (C(H)₂, C(HR³) or C(R³)₂), an oxygen atom (O), a sulfur atom (S), an amino group (N(R³)), an phosphano group (P(R³)), a phosphito group (P(OR³)O), a phosphate group (P(O₂)O), a polyphosphate group (P(O₂)O)_n (n is an integer having a value between 3 and

     12), a silyl group (Si(R³)₂), a siloxyl group (Si(OR³)₂), a carboxy group (C(O)O), a keto group (C(O)), an amido group (C(O)N(R³)), an urea group (N(R³)C(O)N(R³)), a carbonate group (OC(O)O), or an urethane group (OC(O)N(R³), R³ is a hydrogen atom, a carbyl group or is absent depending on the structure (e.g., E′

or E is a nitrogen atom doubly bonded to DG or to R² or a carbon atom triply bonded to DG or R²),

R is a carbenzyl group,

Sugar is a sugar moiety, and

Base is a natural or synthetic nucleotide base.

19. The nucleotide of claim 1, wherein the nucleotide has the general formula (XII):

DG-E′

-R-E-Sugar(P(O₂)OP(O₂)OP(O₂)OH)Base 

 

(XII)

where;

DG is a detectable group,

E and E′

are the same and different and are a carbon group (C(H)₂, C(HR³) or C(R³)₂), an oxygen atom (O), a sulfur atom (S), an amino group (N(R³)), an phosphano group (P(R³)), a phosphito group (P(OR³)O), a phosphate group (P(O₂)O), a polyphosphate group (P(O₂)O)_n (n is an integer having a value between 3 and

     12), a silyl group (Si(R³)₂), a siloxyl group (Si(OR³)₂), a carboxy group (C(O)O), a keto group (C(O)), an amido group (C(O)N(R³)), an urea group (N(R³)C(O)N(R³)), a carbonate group (OC(O)O), or an urethane group (OC(O)N(R³), R³ is a hydrogen atom, a carbyl group or is absent depending on the structure (e.g., E′

or E is a nitrogen atom doubly bonded to DG or to R² or a carbon atom triply bonded to DG or R²),

R is a carbenzyl group,

Sugar is a sugar moiety, and

Base is a natural or synthetic nucleotide base.

20. The nucleotide of claim 1, wherein the nucleotide has the general formula (XIII):

DG-E′

-R-E-Base-Sugar-P(O₂)OP(O₂)OP(O₂)OH 

 

(XIII)

where;

DG is a detectable group,

E and E′

are the same and different and are a carbon group (C(H)₂, C(HR³) or C(R³)₂), an oxygen atom (O), a sulfur atom (S), an amino group (N(R³)), an phosphano group (P(R³)), a phosphito group (P(OR³)O), a phosphate group (P(O₂)O), a polyphosphate group (P(O₂)O)_n (n is an integer having a value between 3 and

     12), a silyl group (Si(R³)₂), a siloxyl group (Si(OR³)₂), a carboxy group (C(O)O), a keto group (C(O)), an amido group (C(O)N(R³)), an urea group (N(R³)C(O)N(R³)), a carbonate group (OC(O)O), or an urethane group (OC(O)N(R³), R³ is a hydrogen atom, a carbyl group or is absent depending on the structure (e.g., E′

or E is a nitrogen atom doubly bonded to DG or to R² or a carbon atom triply bonded to DG or R²),

R is a carbenzyl group,

Sugar is a sugar moiety, and

Base is a natural or synthetic nucleotide base.

21. The nucleotide of claim 1, wherein the each carbyl group and each carbenzyl group include from 1 to 40 carbon, where one or more of the carbon atoms can be replaced with a hetero atoms selected from the group consisting of B, C, Si, Ge, N, P. As, O, S, or Se and having sufficient hydrogen atoms to satisfy the valency of the group, where one or more hydrogen atoms can be replaced with F, Cl, Br, I, OR, SR, COR, COOR, CONH₂, CONHR, CONRR′

, or any other monovalent group inert or substantially inert under the substitution/displacement reaction conditions.

22. A method for preparing gamma phosphate modified nucleotide triphosphates comprising the steps of:

cyclizing a nucleotide triphosphate in N,N-dicyclohexylcarbodiimide (DCC) to form a cyclized nucleotide triphosphate,

contacting the cyclized nucleotide triphosphate with an α

,ω

-diamino linker, where the linker includes a linking group comprising a linear or branched carbenzyl group or a carbenzyl group including a central ring structure, to form a linker gamma phosphate modified nucleotide triphosphate, and

contacting the linker gamma phosphate modified nucleotide triphosphate with a carboxylc acid, a carboxylic acid chloride or a carboxylic acid anhydride including a detectable group having a detectable property to form a detectable group, linker gamma phosphate modified nucleotide triphosphate.

23. A method for preparing gamma phosphate modified nucleotide triphosphates comprising the steps of:

contacting a nucleotide triphosphate with an α

,ω

-diamino linker, where the linker includes a linking group comprising a linear or branched carbenzyl group or a carbenzyl group including a central ring structure, in N-ethyl-N′

-(3-dimethylaminopropyl)carbodiimide (EDC) to form a linker gamma phosphate modified nucleotide triphosphate, and contacting the linker gamma phosphate modified nucleotide triphosphate with a carboxyic

acid, a carboxylic acid chloride or a carboxylic acid anhydride including a detectable group having a detectable property to form a detectable group, linker gamma phosphate modified nucleotide triphosphate.

24. A method for preparing gamma phosphate modified nucleotide triphosphates comprising the steps of:

contacting a protected linker of the general formula Q-NH-G-OH with a phosphate donor to form a phosphate terminated linker of the general formula Q-NH-G-OP(O)(OH)₂,

where Q is a protecting group and G a linker comprising a linear or branched carbenzyl group or a carbenzyl group including a central ring structure,

contacting the phosphate terminated linker, Q-NH-G-OP(O)(OH)₂, with imidazole to form an imidazole activated phosphate terminated linker, Q-NH-G-OP(O)(OH)-imidazole, contacting the imidazole activated phosphate terminated linker, Q-NH-G-OP(O)(OH)-imidazole with a nucleotide polyphosphate to form a protected, linker terminal phosphate modified nucleotide polyphosphate, Q-NH-G-O—

[P(O)(OH)]_n—

O-Nuc, where n is an integer having a value between 3 and 12,

deprotecting the protected gamma phosphate functionalized nucleotide polyphosphate, Q NH-G-O—

[P(O)(OH)]_n—

O-Nuc, to form an unprotected linker, gamma phosphate modified nucleotide polyphosphate, H₂N-G-O—

[P(O)(OH)]_n—

O-Nuc, and

contacting the unprotected linker, gamma phosphate modified nucleotide polyphosphate, H₂N-G-O—

[P(O)(OH)]_n—

-Nuc, with a carboxylc acid, a carboxylic acid chloride or a carboxylic acid anhydride including a detectable group (DG) having a detectable property to form a detectable group gamma phosphate modified nucleotide triphosphate, DG-HN-G-O—

[P(O)(OH)]_n—

O-Nuc.

25. A method for preparing gamma phosphate modified nucleotide triphosphates comprising the steps of:

contacting a linker of the general formula Q-NH-G-OH with a sulfonate donor to form a sulfonate terminated linker, Q-NH-G-OS(O)₂CH₃,

contacting the sulfonate terminated linker, Q-NH-G-OS(O)₂CH₃, with phosphate donor to form a phosphate terminated linker, Q-NH-G-OP(O)(OH)₂,

contacting the phosphate terminated linker, Q-NH-G-OP(O)(OH)₂, with imidazole to form an activated phosphate terminated linker, Q-NH-G-OP(O)(OH)-Imidazole, contacting the imidazole activated phosphate terminated linker, Q-NH-G-OP(O)(OH)-Imidazole, with a nucleotide polyphosphate to form a protected linker, terminal phosphate modified nucleotide polyphosphate, Q-NH-G-O—

[P(O)(OH)]_n—

-Nuc, where n is an integer having a value between 3 and 12,

deprotecting the protected gamma phosphate functionalized nucleotide polyphosphate, Q-NH-G-O—

[P(O)(OH)]_n—

O-Nuc, to form an unprotected linker, terminal phosphate modified nucleotide polyphosphate, H₂N-G-O—

[P(O)(OH)]_n—

O-Nuc, and

contacting the unprotected linker, terminal phosphate modified nucleotide polyphosphate, H₂N-G-O—

[P(O)(OH)]_n—

O-Nuc, with a carboxylc acid, a carboxylic acid chloride or a carboxylic acid anhydride including a detectable group having a detectable property to form a detectable group, linker, terminal phosphate modified nucleotide triphosphate, DG-HN-G-O—

[P(O)(OH)]_n—

O-Nuc.

26. A method for preparing gamma phosphate modified nucleotide triphosphates comprising the steps of:

contacting a linker of the general formula H₂N-G-OH with trifluoro acetic acid (TFA) to form a TFA terminated linker, TFA-NH-G-OH,

contacting the TFA terminated linker, TFA-NH-G-OH, with a cyclized nucleotide triphosphate in the presence of a base to form a TFA terminated linker, gamma phosphate modified nucleotide triphosphate, TFA-NH-G-[P(O)(OH)]₃—

O-Nuc,

deprotecting the TFA terminated linker, gamma phosphate modified nucleotide triphosphate, TFA-NH-G-[P(O)(OH)]₃—

O-Nuc, to form a linker, gamma phosphate modified nucleotide triphosphate, H₂N-G-[P(O)(OH)]₃—

O-Nuc, and

contacting the a linker, gamma phosphate modified nucleotide triphosphate, H₂N-G-[P(O)(OH)]₃—

O-Nuc, with a carboxylc acid, a carboxylic acid chloride or a carboxylic acid anhydride including a detectable group having a detectable property to form a detectable group, linker, terminal phosphate modified nucleotide triphosphate, DG-HN-G-O—

[P(O)(OH)]₃—

O-Nuc.

27. A method for preparing gamma phosphate modified nucleotide triphosphates comprising the steps of:

contacting a nucleotide diphosphate salt, Nuc-O—

P(O)(O⁻

)—

O—

P(O)(O⁻

)₂M₃, with a tetracarbyl ammonium salt, R₄N⁺X⁻

, to form a nucleotide diphosphate tetracarbyl ammonium salt, Nuc-O—

P(O)(O⁻

)—

O—

P(O)(O⁻

)₂(R₄N⁺)₃,

contacting an N-TFA-protected, α

-amino, ω

-hydroxy linker, TFA-N(H)-G-OH, with sufficient POCl₃ to form an N-TFA-protected, α

-amino, ω

-dichlorophosphite linker, TFA-N(H)-G-OP(O)Cl₂,

contacting the nucleotide diphosphate tetracarbyl ammonium salt, Nuc-O—

P(O)(O⁻

)—

O—

P(O)(O⁻

)₂(R₄N⁺)₃, with the N-TFA-protected, α

-amino, ω

-dichlorophosphite linker, TFA-N(H)-G-OP(O)Cl₂, to form a TFA-protected, α

-amino, ω

-gamma phosphate modified nucleotide triphosphate tetracarbyl ammonium salt, TFA-NH-G-O—

[P(O)(O⁻

)]₃—

O-Nuc((R₄N⁺)₃,

deprotecting the TFA-protected, α

-amino, ω

-gamma phosphate modified nucleotide triphosphate tetracarbyl ammonium salt, TFA-NH-G-O—

[P(O)(O⁻

)]₃—

O-Nuc((R₄N⁺)₃, to form a linker, gamma phosphate modified nucleotide triphosphate, α

-amino, ω

-gamma phosphate modified nucleotide triphosphate tetracarbyl ammonium salt, H₂N-G-O—

[P(O)(O⁻

)]₃—

O-Nuc((R₄N⁺)₃.