WO1993023416A1 - Chemical synthesis of 2',3'-dideoxynucleoside 5'-triphosphates - Google Patents

Chemical synthesis of 2',3'-dideoxynucleoside 5'-triphosphates Download PDF

Info

Publication number
WO1993023416A1
WO1993023416A1 PCT/US1993/004291 US9304291W WO9323416A1 WO 1993023416 A1 WO1993023416 A1 WO 1993023416A1 US 9304291 W US9304291 W US 9304291W WO 9323416 A1 WO9323416 A1 WO 9323416A1
Authority
WO
WIPO (PCT)
Prior art keywords
pyrophosphate
dideoxynucleoside
triphosphates
tri
dissolved
Prior art date
Application number
PCT/US1993/004291
Other languages
French (fr)
Inventor
Jasenka Matulic-Adamic
Leonid Beigelman
Original Assignee
United States Biochemical Corporation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by United States Biochemical Corporation filed Critical United States Biochemical Corporation
Publication of WO1993023416A1 publication Critical patent/WO1993023416A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/06—Pyrimidine radicals
    • C07H19/10—Pyrimidine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H19/00—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof
    • C07H19/02—Compounds containing a hetero ring sharing one ring hetero atom with a saccharide radical; Nucleosides; Mononucleotides; Anhydro-derivatives thereof sharing nitrogen
    • C07H19/04—Heterocyclic radicals containing only nitrogen atoms as ring hetero atom
    • C07H19/16—Purine radicals
    • C07H19/20—Purine radicals with the saccharide radical esterified by phosphoric or polyphosphoric acids

Definitions

  • This invention relates to the chemical synthesis of 2' ,3'-dideoxynucleoside 5'-triphosphates.
  • This invention features preparation of ddNTP ' s in high yield in a "one pot" reaction in which 2',3'- dideoxynucleosides (ddN' s ) are reacted with a phosphorylating agent, e.g. , phosphorusoxychloride, in the presence of a proton removing reagent, e.g. , a proton sponge or molecular sieves, followed by treatment of the resulting onophosphorylated chemical, e.g. , a phosphorodichloridate, with a pyrophosphate, e.g. , tri-n- butylammonium pyrophosphate or tri-n-octylammonium pyrophosphate in acetonitrile.
  • a phosphorylating agent e.g. , phosphorusoxychloride
  • a proton removing reagent e.g. , a proton sponge or molecular sieves
  • the described method is simple and mild enough to synthesize a series of ddNTP ' s .
  • the proton sponge or molecular sieves used in these reactions has a stabilizing influence on the ⁇ -glycosidic bond hydrolysis of these highly acid-sensitive nucleosides thus providing high triphosphate yields. Moreover, it improves the formation of the intermediate nucleoside dichlorophosphates. Chemical manipulations involved are kept to a minimum and only one column chromatography is required to obtain the pure end product. In addition, the method can be scaled up for multigram synthesis.
  • the invention features a method for chemical synthesis of 2' ,3'-dideoxynucleoside 5'-triphosphates from 2',3'-dideoxynucleosides without purification of intermediate products by reacting the nucleosides with a phosphorusoxychloride in the presence of a proton removing agent, e.g. , a proton sponge, molecular sieves, or a base such as pyridine, 2,6-lutidine (2,6-dimethylpyridine) and 2,4,6-collidine (2,4,6- trimethylpyridine) , and subsequent treatment with a pyrophosphate.
  • a proton removing agent e.g. , a proton sponge, molecular sieves, or a base
  • a base such as pyridine, 2,6-lutidine (2,6-dimethylpyridine) and 2,4,6-collidine (2,4,6- trimethylpyridine
  • the proton sponge is 1,8-bis(dimethylamino)-naphthalene and the pyrophosphate is tri-n-butylammonium pyrophosphate or tri-n- octylammonium pyrophosphate dissolved in acetonitrile; or the molecular sieves are referred to as molecular sieves 3A or 4A; in particular the proton sponge and nucleosides are dissolved in triethylphosphate or triethylphosphate- tri ethylphosphate, e.g. , at 120-125°C.
  • the method of this invention features use of a reagent which removes protons (an acid scavenger) from solution to prevent glycosidic bond cleavage by HCl liberated by reaction with P0C1 3 .
  • the synthetic method includes phosphorylation of a dideoxynucleoside with a phosphorylating agent in the presence of such a reagent. In this way a high yield of the desired ddNTP is achieved.
  • Tributylamine (4 eq) and 0.25 M tri-n-butylammonium pyrophosphate (4 eq) in acetonitrile were added at 0°C.
  • 1 M triethylammonium bicarbonate (TEAB) buffer (10 ml/mmole) was added (water or aqueous ammonium hydroxide can also be used) , and the solution was set at room temperature (about 24°C) for 24 hours. It was then concentrated .in vacuo at 30°C and coevaporated twice with methanol. The residue was dissolved in 0.05 M TEAB (pH 7.5) (100 ml/mmole). Purification:

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Health & Medical Sciences (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Biochemistry (AREA)
  • Biotechnology (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Molecular Biology (AREA)
  • Saccharide Compounds (AREA)

Abstract

Method for synthesis of 2',3'-dideoxynucleoside 5'-triphosphates by contacting a 2',3'-dideoxynucleoside with a phosphorylating agent in the presence of a proton removing reagent.

Description

DESCRIPTION
CHEMICAL SYNTHESIS OF 2 ' , 3 ' -DIDEOXYNUCLEOSIDE 5'-TRIPHOSPHATES
Background of the Invention This invention relates to the chemical synthesis of 2' ,3'-dideoxynucleoside 5'-triphosphates.
Ludwig, 16 Acta Biochim. et Biophvs. Acad. Sci. Hung. 131, 1981, describes a method for synthesis of adenosine 5'-triphosphate and 2'-deoxyadenosine 5'-tri- phosphate by treatment of 5'-phosphodichloridate with tri- n-butylammonium pyrophosphate in dimethylformamide. Kovacz and Otvos, 29 Tetrahedron Lett. 4525, 1988, describe synthesis of 2'-deoxyuridine-5'-triphosphates using a proton sponge and a pyrophosphate dissolved in dimethylformamide.
Summary of the Invention
This invention features preparation of ddNTP ' s in high yield in a "one pot" reaction in which 2',3'- dideoxynucleosides (ddN' s ) are reacted with a phosphorylating agent, e.g. , phosphorusoxychloride, in the presence of a proton removing reagent, e.g. , a proton sponge or molecular sieves, followed by treatment of the resulting onophosphorylated chemical, e.g. , a phosphorodichloridate, with a pyrophosphate, e.g. , tri-n- butylammonium pyrophosphate or tri-n-octylammonium pyrophosphate in acetonitrile.
The described method is simple and mild enough to synthesize a series of ddNTP ' s . The proton sponge or molecular sieves used in these reactions has a stabilizing influence on the Ν-glycosidic bond hydrolysis of these highly acid-sensitive nucleosides thus providing high triphosphate yields. Moreover, it improves the formation of the intermediate nucleoside dichlorophosphates. Chemical manipulations involved are kept to a minimum and only one column chromatography is required to obtain the pure end product. In addition, the method can be scaled up for multigram synthesis.
Thus, in a first aspect the invention features a method for chemical synthesis of 2' ,3'-dideoxynucleoside 5'-triphosphates from 2',3'-dideoxynucleosides without purification of intermediate products by reacting the nucleosides with a phosphorusoxychloride in the presence of a proton removing agent, e.g. , a proton sponge, molecular sieves, or a base such as pyridine, 2,6-lutidine (2,6-dimethylpyridine) and 2,4,6-collidine (2,4,6- trimethylpyridine) , and subsequent treatment with a pyrophosphate.
In preferred embodiments, the proton sponge is 1,8-bis(dimethylamino)-naphthalene and the pyrophosphate is tri-n-butylammonium pyrophosphate or tri-n- octylammonium pyrophosphate dissolved in acetonitrile; or the molecular sieves are referred to as molecular sieves 3A or 4A; in particular the proton sponge and nucleosides are dissolved in triethylphosphate or triethylphosphate- tri ethylphosphate, e.g. , at 120-125°C.
Other features and advantages of the invention will be apparent from the following description of the preferred embodiments thereof, and from the claims. Description of the Preferred Embodiments The drawing will first briefly be described.
Drawin :
The figure is a diagrammatic representation of the chemical synthesis of ddNTP's by the method of this invention. Synthesis:
Referring to the figure, in general, the method of this invention features use of a reagent which removes protons (an acid scavenger) from solution to prevent glycosidic bond cleavage by HCl liberated by reaction with P0C13. Thus, the synthetic method includes phosphorylation of a dideoxynucleoside with a phosphorylating agent in the presence of such a reagent. In this way a high yield of the desired ddNTP is achieved.
The following is an example of a method for synthesis and purification of ddNTP ' s . This example is not limiting to the invention, and those of ordinary skill in the art will recognize that other equivalent methods can be used to produce the desired chemicals. Example:
2' ,3'-dideoxynucleosides (5 mmol) and 1,8- bis(dimethylamino)-naphthalene (proton sponge; molecular sieves 3A or 4A can also be used) were dissolved in triethylphosphate or triethylphosphate-trimethylphosphate 1:1, if necessary by heating at 120-125°C for 4-6 minutes. The solution was quickly cooled to 0°C and phosphorylchloride (1.1 equivalents (eq) ) added. The reaction mixture was stirred at 0-4°C for 1-5 hours. Tributylamine (4 eq) and 0.25 M tri-n-butylammonium pyrophosphate (4 eq) in acetonitrile were added at 0°C. After 1 minute of vigorous stirring 1 M triethylammonium bicarbonate (TEAB) buffer (10 ml/mmole) was added (water or aqueous ammonium hydroxide can also be used) , and the solution was set at room temperature (about 24°C) for 24 hours. It was then concentrated .in vacuo at 30°C and coevaporated twice with methanol. The residue was dissolved in 0.05 M TEAB (pH 7.5) (100 ml/mmole). Purification:
The above solution was applied on the column of DEAE Sephadex A-25 (HC03 ~form) and chromatography was performed with a linear gradient of 2 L each 0.05 M and 1 M TEAB (pH 7.5), flow rate 2 ml/min, fraction size 25 ml. Fractions containing triphosphates were combined and concentrated in vacuo at 30°C. Reverse phase HPLC on C18 column (elution with 100 mM TEAB (pH 6.7) containing a linear gradient of acetonitrile from 0% to 20% in 25 minutes) revealed that this product was chromatographically homogeneous. The syrupy residue was dissolved in water and lyophilized. This procedure was repeated once more, and the remaining solid dissolved in methanol and treated with a solution of sodium perchlorate in acetone. The mixture was centrifuged, supernatant liquor decanted, and the precipitate washed 3 times with acetone. The remaining solid was dissolved in water and lyophilized to give 2',3'-dideoxynucleoside 5'- triphosphates (sodium" salts) as white solids in 60-85% yield.
Other embodiments are within the following claims.

Claims

1. Method for synthesis of 2 ',3'- dideoxynucleoside 5'-triphosphates, comprising the step of contacting a 2',3 '-dideoxynucleoside with a phosphorylating agent in the presence of a proton removing reagent.
2. The method of claim 1 wherein said phosphorylating agent is phosphorusoxychloride.
3. The method of claim 1 wherein said proton removing reagent is a proton sponge or molecular sieves.
4. The method of claim 3 wherein said proton sponge is bis-(dimethylamino) -naphthalene.
5. The method of any of claims 1-4, further comprising the step of adding a pyrophosphate.
6. The method of claim 5 wherein said pyrophosphate is tri-n-butylammonium pyrophosphate.
7. The method of claim 5 wherein said pyrophosphate is tri-n-octylammonium pyrophosphate.
8. The method of claim 5 wherein said pyrophosphate is dissolved in acetonitrile.
9. The method of claim 1 wherein said 2' ,3'- dideoxynucleoside 5'-triphosphate and said phosphorylating agent are dissolved in triethylphosphate or triethylphosphate-trimethylphosphate prior to or during said contacting.
PCT/US1993/004291 1992-05-13 1993-05-06 Chemical synthesis of 2',3'-dideoxynucleoside 5'-triphosphates WO1993023416A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
US88248092A 1992-05-13 1992-05-13
US07/882,480 1992-05-13

Publications (1)

Publication Number Publication Date
WO1993023416A1 true WO1993023416A1 (en) 1993-11-25

Family

ID=25380670

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/US1993/004291 WO1993023416A1 (en) 1992-05-13 1993-05-06 Chemical synthesis of 2',3'-dideoxynucleoside 5'-triphosphates

Country Status (2)

Country Link
AU (1) AU4236793A (en)
WO (1) WO1993023416A1 (en)

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000056750A1 (en) * 1999-03-19 2000-09-28 Parker Hughes Institute One pot synthesis of 5'-hydroxy phosphorylated nucleoside derivatives, and compounds formed thereby
CN114369128A (en) * 2020-10-14 2022-04-19 北京红惠新医药科技有限公司 Process for preparing nicotinamide mononucleotide

Non-Patent Citations (20)

* Cited by examiner, † Cited by third party
Title
A.M. MICHELSON, "The Chemistry of Nucleosides and Nucleotides", Published 1963, by ACADEMIC PRESS (NEW YORK, NY), pages 132, 133, 197, 225. *
ACTA BIOCHIM. BIOPHYS. ACAD SCI. HUNG., Vol. 16 (3-4), issued 1981, J. LUDWIG, "A New Route to Nucleoside 5'-Triphosphates", pp. 131-133. *
BIOORG. KHIM., Vol. 13(12), issued 1987, BOGACHEV, "Improved Synthesis of Deoxynucleoside 5'-0-Monothiophosphates and 5'-0-(1-Thio)Triphosphates", pp. 1683-1686. *
BIOORG. KHIM., Vol. 9(1), issued 1983, SAMUKOV et al., "Synthesis of 2',3'-Dideoxynucleosides", pp. 52-59. *
J. AM. CHEM. SOC., Vol. 88(7), issued 05 April 1966, MCCARTHY et al., "Purine Nucleosides. XIV. Unsaturated Furanosyl Adenine Nucleosides Prepared via Base-Catalyzed Elimination Reactions of 2'-Deoxyadenosine Derivatives", pp. 1549-1553. *
J. HETEROCYCLIC CHEM., Vol. 26, issued 1989, KASKAR et al., "A New Synthesis of 2',3'-Dideoxycytidine", pp. 1531-1533. *
J. MED. CHEM., Vol. 31, issued 1988, HERDEWIJN et al., "Synthesis and Anti-HIV Activity of Different Sugar-Modified Pyrimidine and Purine Nucleosides", pp. 2040-2048. *
J. ORG. CHEM., Vol. 39(1), issued 1974, JAIN et al., "Reactions of 2-Acyloxybutyryl Halides with Nucleosides. IV. A Facile Synthesis of 2',3' - Unsaturated Nucleosides Using Chromous Acetate", pp. 30-38. *
J. ORG. CHEM., Vol. 51, issued 1986, GAO et al., "Transient Protection 2. One-Flask Synthesis of 6-0[(4-Nitrophenyl)EthylÜ-2'-Deoxyguanosine Nucleosides", pp. 755-758. *
J. ORG. CHEM., Vol. 54(3), issued 1989, LUDWIG et al., "Rapid and Efficient Synthesis of Nucleosides 5'-0-(1-Thiotriphosphates), 5'-Triphosphates and 2',3'-Cyclophosphorothioates Using 2-Chloro-4H-1.3.2.-Benzodioxaphosphorin-4-One", pp. 631-635. *
J. ORG. CHEM., Vol. 54(9), issued 1989, CHU et al., "General Synthesis of 2', 3'-Dideoxynucleosides and 2', 3'-Didehydro-2', 3'-Dideoxynucleosides", pp. 2217-2225. *
NATURE, Vol. 344, issued 05 April 1990, PERRAULT et al., "Mixed Deoxyribo- and Ribooligonucleotides with Catalytic Activity", pp. 565-567. *
NUCLEOSIDES & NUCLEOTIDES, Vol. 8(5&6), issued 1989, AUER et al., "Synthesis and Biological Applications of 2',3'-Dideoxynucleoside-5'-0(o-Thio)Triphosphates", pp. 849-853. *
NUCLEOSIDES & NUCLEOTIDES, Vol. 9(7), issued 1990, STARRETT et al., "The Use of Acetyl Bromide for the Multigram Synthesis of the Anti-HIV Agent 2',3'-Didehydro-2',3'-Dideoxycytidine(d4C)", pp. 885-897. *
NUCLEOSIDES & NUCLEOTIDES, Vol. 9(8), issued 1990, BHAT et al., "An Improved Synthesis of 2',3'-Dideoxycytidine", 1061-1065. *
PROC. NAT. ACAD. SCI. USA, Vol. 74(12), issued December 1977, SANGER et al., "DNA Sequencing with Chain-Terminating Inhibitors", pp. 5463-5467. *
SYNTHETIC COMMUNICATIONS, Vol. 15(5), issued 1985, PRISBE et al., "A Novel and Efficient Preparation of 2',3'-Dideoxynucleosides", pp. 401-409. *
SYNTHETIC COMMUNICATIONS, Vol. 20(7), issued 1990, HUANG et al., " A Practical Synthesis of 2'-Deoxyuridine from Uridine", pp. 1039-1046. *
TETT. LETT., Vol. 29(36), issued 1988, (G. Br.), KOVACS et al., "Simple Synthesis of 5-Vinyl- and 5-Ethynyl-2'-Deoxyuridines-5'-Triphosphates", pp. 4525-4528. *
ZORBACH et al., "Synthetic Procedures in Nucleic Acid Chemistry", Vol. 1, Published 1968, by J. WILEY & SONS, (NEW YORK), "3'-0-Acetyl-2'-Deoxyadenosine", pp. 172-175. *

Cited By (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2000056750A1 (en) * 1999-03-19 2000-09-28 Parker Hughes Institute One pot synthesis of 5'-hydroxy phosphorylated nucleoside derivatives, and compounds formed thereby
US6465641B1 (en) 1999-03-19 2002-10-15 Parker Hughes Institute One pot synthesis of 5′-hydroxy phosphorylated nucleoside derivatives, and compounds formed thereby
CN114369128A (en) * 2020-10-14 2022-04-19 北京红惠新医药科技有限公司 Process for preparing nicotinamide mononucleotide

Also Published As

Publication number Publication date
AU4236793A (en) 1993-12-13

Similar Documents

Publication Publication Date Title
Roseman et al. Nucleoside polyphosphates. XI. 1 An improved general method for the synthesis of nucleotide coenzymes. Syntheses of uridine-5', cytidine-5'and guanosine-5'diphosphate derivatives
US6309836B1 (en) Compounds for protecting hydroxyls and methods for their use
EP0212536B1 (en) 7-deaza-2'-deoxyguanosin-nucleotides, process for their preparation and their use in nucleic-acid sequencing
DE2122529C2 (en)
Yu et al. A hydrolytic procedure for ribonucleosides and its possible application to the sequential degradation of RNA
Matulic-Adamic et al. Synthesis of 5'-deoxy-5'-difluoromethyl phosphonate nucleotide analogs
Ralph et al. Studies on Polynucleotides. XVIII. 1 Experiments on the Polymerization of Mononucleotides. The Synthesis and Characterization of Deoxyguanosine Oligonucleotides 2
Lynch et al. A chemical synthesis of nicotinamide adenine dinucleotide (NAD+)
Bartlett et al. Stereochemical course of polymerization catalyzed by avian myeloblastosis virus reverse transcriptase.
Hobbs et al. A general method for the synthesis of 2'-azido-2'-deoxy-and 2'-amino-2'-deoxyribofuranosyl purines
Kitade et al. 8-Methyladenosine-substituted analogues of 2-5A: synthesis and their biological activities
Drummond et al. Deoxyribonucleoside-3′, 5′ Cyclic Phosphates. Synthesis and Acid-Catalyzed and Enzymic Hydrolysis
Pontis et al. Synthesis of D-fructopyranose 2-phosphate and D-fructofuranose 2-phosphate
Imai et al. Expedient chemical synthesis of sequence-specific 2', 5'-oligonucleotides
WO1993023416A1 (en) Chemical synthesis of 2',3'-dideoxynucleoside 5'-triphosphates
Percival et al. Applications of enzymes in the synthesis and hydrolytic study of 2-deoxy-α-D-glucopyranosyl phosphate
Augustyns et al. Influence of the Incorporation of 1‐(2, 3‐Dideoxy‐β‐D‐Erythro‐Hexopyranosyl)‐Thymine on the Enzymatic Stability and Base‐Pairing Properties of Oligodeoxynucleotides
Ohtsuka et al. Polynucleotides. VIII. New method for the synthesis of protected deoxyribooligonucleotides with 5'-phosphate
US3701772A (en) Production of nucleotide anhydrides
Nadeau et al. Use of ribonucleosides as protecting groups in synthesis of polynucleotides with phosphorylated terminals
WO1993023415A1 (en) CHEMICAL SYNTHESIS OF 2',3'-DIDEOXYNUCLEOSIDE 5'-(α-THIO) TRIPHOSPHATES
EP0407816A2 (en) Base modified nucleosides
Chladek et al. Synthesis and properties of nucleoside 5'-phosphoazidates derived from guanosine and adenosine nucleotides: effect on elongation factors G and Tu dependent reactions
JPH0435480B2 (en)
Hayatsu et al. N-Sulfomethylation of guanine, adenine and cytosine with formaldehyde-bisulfite. A selective modification of guanine in DNA

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AT AU BB BG BR CA CH CZ DE DK ES FI GB HU JP KP KR LK LU MG MN MW NL NO NZ PL PT RO RU SD SE SK UA

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): AT BE CH DE DK ES FR GB GR IE IT LU MC NL PT SE

121 Ep: the epo has been informed by wipo that ep was designated in this application
122 Ep: pct application non-entry in european phase
REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

NENP Non-entry into the national phase

Ref country code: CA