[go: up one dir, main page]

WO1993013118A1 - Synthesis of phosphorothioate analogues of oligo- and polynucleotides - Google Patents

Synthesis of phosphorothioate analogues of oligo- and polynucleotides Download PDF

Info

Publication number
WO1993013118A1
WO1993013118A1 PCT/GB1992/002395 GB9202395W WO9313118A1 WO 1993013118 A1 WO1993013118 A1 WO 1993013118A1 GB 9202395 W GB9202395 W GB 9202395W WO 9313118 A1 WO9313118 A1 WO 9313118A1
Authority
WO
WIPO (PCT)
Prior art keywords
sulphur
oligo
defined above
transfer agent
dibenzoyl
Prior art date
Application number
PCT/GB1992/002395
Other languages
French (fr)
Inventor
Colin Bernard Reese
Original Assignee
King's College London
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 King's College London filed Critical King's College London
Publication of WO1993013118A1 publication Critical patent/WO1993013118A1/en

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H1/00—Processes for the preparation of sugar derivatives
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • This invention relates to the synthesis of phosphorothioate analogues of oligo- and poly- nucleotides; more particularly, it relates to the solid phase synthesis of phosphorothioate analogues of, preferably, oligo- and poly- deoxyribonucleotides from phosphora idite building blocks using certain diacyl tri- and tetra-sulphides as sulphur-transfer agents.
  • Antisense oligodeoxyribonucleotides (see, for example, "Oligodeoxynucleotides - Antisense inhibitors of gene expression", Cohen, J.S., Ed., Macmillan, London, 1989), which are complementary to particular RNA targets, may inhibit translation and thereby provide a highly specific method for the inhibition of gene expression.
  • Phosphorothioate analogues of oligonucleotides in which one of the non-bridging oxygen atoms of the internucleotide linkages is replaced by a sulphur atom are much more resistant to nuclease-promoted degradation than corresponding unmodified oligomers, (see, for example, Eckstein, F., Ann. Rev. Biochem. 54 . , 367-402, 1985) , and are therefore potentially valuable antisense therapeutic agents.
  • oligonucleotide phosphorothioates have been prepared by the phosphotriester approach in solution, (see, for example, Burgers, P.M.J. , and Eckstein, F. , Biochemistry, 18., 592-596, 1979; and Kemal, 0., et al, J. Chem. Soc, Chem. Co mun., 591-593, 1983) , they are at present much more conveniently prepared by solid phase synthesis using suitably-protected nucleoside H-phosphonate, (see, for example, Froehler, B.C., Tet.
  • the H-phosphonate approach does not permit the synthesis of oligonucleotides containing both natural phosphodiester and modified (e.g. phosphorothioate diester) internucleotide linkages, and is considered by some workers to be less efficient than the phosphoramidite approach, (see, for example, Iyer, R.P., et a_l, J. Org. Chem., 55., 4693-4699, 1990; and Vu, H. , and Hirschbein, B.L., Tet. Lett., .32 . , 3005-3008, 1991).
  • n 3 or ;
  • X represents RC(O)- or RC(S)- wherein R represents alkyl, cycloalkyl or aryl, (R'O)C(O)- wherein R represents alkyl, (R 2 N)C(0)- or (R 2 N)C(S)- wherein R is as defined above and R 2 NH may be a cyclic 10 secondary amine,
  • an easily prepared crystalline solid may 5 be used as an efficient sulphur-transfer agent which appears both to have good solubility properties and to transfer sulphur very rapidly.
  • the presently-preferred reagent may be readily prepared in 81% yield in a one-step reaction from thiobenzoic acid and sulphur monochloride, 0 which are both relatively cheap, commercially-available starting materials; it may be isolated as a stable, pale yellow crystalline solid that is extremely soluble in chloroform, dichloro ethane and tetrahydrofuran, but not in acetonitrile. 5
  • the presently-preferred dibenzoyl tetrasulphide was prepared by an advantageous modification of the original literature procedure.
  • a solution of triethylamine (16.2 ml, 0.12 mol) in dry tetrahydrofuran (100 ml) was added dropwise over a period of 20 minutes to a stirred solution of redistilled thiobenzoic acid (16.5 g, 0.12 mol) and sulphur monochloride (6.0 ml, 0.075 mol) in dry tetrahydrofuran (150 ml) at 0°C (ice-water bath) .
  • the products were stirred for a further period of 1 hour at room temperature and were then filtered.
  • one preferred embodiment of the present invention concerns the use of, in particular, dibenzoyl tetrasulphide as a sulphur-transfer agent, it is not so- limited.
  • dibenzoyl tetrasulphide as a sulphur-transfer agent
  • the main requirements for a sulphur-transfer agent in the context of oligonucleotide phosphorothioate synthesis are as follows:
  • X-S 4 -X and X-S 3 -X may be considered for use as effective sulphur-transfer agents.
  • R represents an aryl group, which may be substituted, for example, with para- or meta- methyl.
  • the preparation of such reagents is within the competence of those skilled in the art.
  • dibenzoyl tetrasulphide As dibenzoyl tetrasulphide was found to be the most reactive of the three reagents here examined in most detail, it was decided to investigate its use in solid phase oligonucleotide synthesis.
  • the efficacy of dibenzoyl tetrasulphide as a sulphur-transfer agent was compared with that of the recently-recommended tetraethylthiura disulphide in the solid phase synthesis of the nonadecadeoxyribonucleoside octadecaphosphorothioate , d[A(S)T(S)T(S)C(S)C(S)G(S)G(S)A(S)C(S)T(S)C(S)G(S)T(S)C(S)- C(S)A(S) C(S) C(S)C(S)A] .
  • Solid phase synthesis was carried out on a 0.2 ⁇ molar scale in an Applied Biosystems 381A DNA Synthesizer. 5'-0-(4,4'-dimethoxytrityl) -2'-deoxy- ribonucleoside 3 '-(2-cyanoethyl) -N,N-diisopropylphosphor- a idites with standard base protecting groups (i.e. benzoyl on adenine and cytosine, and isobutyryl on guanine) were used as building blocks, and lH-tetrazole was used as activating agent.
  • standard base protecting groups i.e. benzoyl on adenine and cytosine, and isobutyryl on guanine
  • Sulphur-transfer was effected in each of the 18 synthetic cycles either with (a) 0.4M dibenzoyl tetrasulphide in commercially-supplied, base-free 0.025% butylated-hydr ⁇ xytoluene(anti-peroxidation stabilizer) - containing tetrahydrofuran for 60 seconds or (b) 0.5M tetraethylthiuram disulphide in acetonitrile for 900 seconds as recommended by Vu and Hirschbein (loc c_it) .

Landscapes

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

Abstract

A process for the solid phase production of a phosphorothioate analogue of an oligo- or polynucleotide from phosphoramidite building blocks in the presence of a sulphur-transfer agent characterised in that the sulphur-transfer agent corresponds to the following general formula: X - Sn - X wherein n represents 3 or 4; and X represents RC(O)- or RC(S)- wherein R represents alkyl, cycloalkyl or aryl, (R'O)C(O)- wherein R' represents alkyl, (R2N)C(O)- or (R2N)C(S)- wherein R is as defined above and R2NH may be a cyclic secondary amine, RSO2- wherein R is as defined above, (RO)2P(O)- or (RO)2P(S)- wherein R is as defined above; is disclosed.

Description

SYNTHESIS OF PHOSPHOROTHIOATE ANALOGUES OF OLIGO- AND POLY- NUCLEOTIDES
This invention relates to the synthesis of phosphorothioate analogues of oligo- and poly- nucleotides; more particularly, it relates to the solid phase synthesis of phosphorothioate analogues of, preferably, oligo- and poly- deoxyribonucleotides from phosphora idite building blocks using certain diacyl tri- and tetra-sulphides as sulphur-transfer agents.
(The present invention is equally applicable to the production of "oligo-" and "poly-" nucleotides. As there is no strict demarcation, the former term will generally be used.)
Antisense oligodeoxyribonucleotides, (see, for example, "Oligodeoxynucleotides - Antisense inhibitors of gene expression", Cohen, J.S., Ed., Macmillan, London, 1989), which are complementary to particular RNA targets, may inhibit translation and thereby provide a highly specific method for the inhibition of gene expression. Phosphorothioate analogues of oligonucleotides in which one of the non-bridging oxygen atoms of the internucleotide linkages is replaced by a sulphur atom are much more resistant to nuclease-promoted degradation than corresponding unmodified oligomers, (see, for example, Eckstein, F., Ann. Rev. Biochem. 54., 367-402, 1985) , and are therefore potentially valuable antisense therapeutic agents.
Although oligonucleotide phosphorothioates have been prepared by the phosphotriester approach in solution, (see, for example, Burgers, P.M.J. , and Eckstein, F. , Biochemistry, 18., 592-596, 1979; and Kemal, 0., et al, J. Chem. Soc, Chem. Co mun., 591-593, 1983) , they are at present much more conveniently prepared by solid phase synthesis using suitably-protected nucleoside H-phosphonate, (see, for example, Froehler, B.C., Tet. Lett., 2_7_, 5575- 5578, 1986) , or phosphoramidite, (see, for example, Stec, W.J., et al, JACS, 106, 6077-6079, 1984; and Stein, C.A., et al. Nucleic Acids Res., JL6., 3209-3221, 1988) , building blocks. While the H-phosphonate approach has the advantage of requiring only one sulphur-transfer step after the whole sequence has been assembled, the phosphoramidite approach requires a sulphur-transfer step in the course of each synthetic cycle. However, the H-phosphonate approach does not permit the synthesis of oligonucleotides containing both natural phosphodiester and modified (e.g. phosphorothioate diester) internucleotide linkages, and is considered by some workers to be less efficient than the phosphoramidite approach, (see, for example, Iyer, R.P., et a_l, J. Org. Chem., 55., 4693-4699, 1990; and Vu, H. , and Hirschbein, B.L., Tet. Lett., .32., 3005-3008, 1991).
Both approaches involve the use of a sulphur- transfer agent and, in the case of the phosphoramidite approach, it is of crucial importance that the reagent should be freely soluble in a suitable organic solvent, such as dichloromethane, acetonitrile or tetrahydrofuran, and that it should effect sulphur-transfer as rapidly as possible so as not unduly to extend the duration of each synthetic cycle. Elemental sulphur, which was first used for this purpose, (see, for example, Stec, loc cit; and Stein, loc cit) , does not have particularly satisfactory solubility properties and also does not effect the conversion of phosphite into phosphorothioate triesters very rapidly. For these reasons, there has recently been a search for more suitable sulphur-transfer agents, leading to recommendations that di-(phenylacetyl) disulphide:
Figure imgf000004_0001
O 3H - 1,2 - benzodithiole - 3 - one 1,1 - dioxide:
Figure imgf000005_0001
and tetraethylthiuram disulphide:
Figure imgf000005_0002
should be used in the phosphoramidite approach to the solid phase synthesis of o1 igodeoxyribonuc1eotide phosphorothioates, (see, for example, Kamer, P.C.J., et al, Tet. Lett., 2JD, 6757-6760, 1989; Iyer, R.P., et a_l, JACS, 112 , 1253-1254, 1990, and Iyer, et aJL, supra; and Vu and Hirschbein, loc cit; respectively) . Although all three of these reagents appear to have good solubility properties, only 3H-l,2-benzodithiole-3-one 1,1-dioxide appears to transfer sulphur very rapidly. However, it is not particularly easy to prepare and this could be disadvantageous if oligonucleotide phosphorothioates prove to be useful in antisense chemotherapy and are consequently required in relatively large quantities.
The present invention relates to a process for the solid phase production of phosphorothioate analogues of oligo- or poly- nucleotides from phosphoramidite building blocks in the presence of a sulphur-transfer agent characterised in that the sulphur-transfer agent corresponds to the following general formula:
X - S„ - X
wherein n represents 3 or ;
and
5 X represents RC(O)- or RC(S)- wherein R represents alkyl, cycloalkyl or aryl, (R'O)C(O)- wherein R represents alkyl, (R2N)C(0)- or (R2N)C(S)- wherein R is as defined above and R2NH may be a cyclic 10 secondary amine,
RS02- wherein R is as defined above, (R0)2P(0)- or (RO)2P(S)- wherein R is as defined above.
15 According to a preferred embodiment of the present invention, it has now been found that dibenzoyl tetrasulphide:
Figure imgf000006_0001
an easily prepared crystalline solid (see, for example, Bloch, I., and Berg ann, M. , Ber. , 5_3_, 961-977, 1920), may 5 be used as an efficient sulphur-transfer agent which appears both to have good solubility properties and to transfer sulphur very rapidly. Furthermore, the presently-preferred reagent may be readily prepared in 81% yield in a one-step reaction from thiobenzoic acid and sulphur monochloride, 0 which are both relatively cheap, commercially-available starting materials; it may be isolated as a stable, pale yellow crystalline solid that is extremely soluble in chloroform, dichloro ethane and tetrahydrofuran, but not in acetonitrile. 5
2 Ph — C -- SjCI; Et3N. t≤trarivαroturan / S s. ^~Ph
V-.
SH 7 Ph S S'
'.0 In fact, the presently-preferred dibenzoyl tetrasulphide was prepared by an advantageous modification of the original literature procedure. A solution of triethylamine (16.2 ml, 0.12 mol) in dry tetrahydrofuran (100 ml) was added dropwise over a period of 20 minutes to a stirred solution of redistilled thiobenzoic acid (16.5 g, 0.12 mol) and sulphur monochloride (6.0 ml, 0.075 mol) in dry tetrahydrofuran (150 ml) at 0°C (ice-water bath) . The products were stirred for a further period of 1 hour at room temperature and were then filtered. The filtrate was concentrated under reduced pressure and the residue was extracted with warm ethyl acetate (200 ml) . After filtration, the extract was cooled and concentrated under reduced pressure. The residue was crystallized from ethyl acetate to give dibenzoyl tetrasulphide. [Observed: C, 49.5, H, 2.95; S, 37.5. Calculated for C14H10O2S4: C, 49.7; H, 3.0; S, 37.9%], m.p. 84-85°C; yield, 16.4 g (81%) ; <SC[CDC13 ,90.6 MHz] 128.0, 128.9, 134.4, 134.9, 186.8.
Although one preferred embodiment of the present invention concerns the use of, in particular, dibenzoyl tetrasulphide as a sulphur-transfer agent, it is not so- limited. As will be generally appreciated by those skilled in the art, the main requirements for a sulphur-transfer agent in the context of oligonucleotide phosphorothioate synthesis are as follows:
(1) it should be a readily-prepared solid and preferably crystalline;
(2) it should be indefinitely stable in the solid state and relatively stable in solution at or below room temperature; if it does decompose slowly in solution, the decomposition products should also be soluble in the solvent used;
(3) it should be very soluble (say, at least 0.5M) in one or more of the solvents, including acetonitrile, tetrahydrofuran and dichloromethane, that are commonly used in solid phase oligonucleotide synthesis;
(4) in the case of solid phase oligodeoxyribonucleotide synthesis by the phosphite triester approach, (i.e. by using standard nucleoside phosphoramidite 2-cyanoethyl ester building blocks) , it should be possible to transfer sulphur to the phosphite triester groups within ca. 60 seconds in each synthetic cycle to such an extent that the internucleotide linkages of unblocked oligonucleotide products containing up to 20 nucleoside residues are at least 99% phosphorothioates, as determined by 31P-NMR spectroscopy.
As indicated above, in addition to the presently- preferred dibenzoyl tetrasulphide and the almost equally effective but somewhat less soluble dibenzoyl trisulphide, (see, for example, Bloch and Bergmann, loc cit) , other diacyl tetra- and tri-sulphides: X-S4-X and X-S3-X, respectively, may be considered for use as effective sulphur-transfer agents. The latter compounds may be derived from (i) other carboxylic acids (i.e. X = RC(0)-, wherein R represents an alkyl, cycloalkyl or aryl group) ; (ii) thiocarboxylic acids (i.e. X = RC(S)-, wherein R represents an alkyl, cycloalkyl or aryl group) , (iii) carbonate esters (i.e. X = (RO)C(O)-, wherein R represents alkyl); (iv) carbamic acids and thiocarbamic acids (i.e. X = (R2N)C(0)- or (R2N)C(S)-, when R represents an alkyl, cycloalkyl or aryl group, and R2NH may be a cyclic secondary amine); (v) sulphonic acids (i.e. X = RS02-/ wherein R represents an alkyl, cycloalkyl or aryl group) ; and (vi) phosphate or phosphorothioate diesters (i.e. X = (R0)2P(0)- or X = (RO)2P(S)-, wherein R represents an alkyl, cycloalkyl or aryl group) . In the case of tri- and tetra- sulphides derived from simple carboxylic acids (i.e. X = R(C0)-), it is preferred that R represents an aryl group, which may be substituted, for example, with para- or meta- methyl. The preparation of such reagents is within the competence of those skilled in the art.
The following further illustrates the present invention:
The conversion of tri ethyl phosphite, (MeO)3P, (<S (CDC13) 142.0) into trimethyl phosphorothioate, (MeO)3P=S, (5 (CDCI3) 73.7) in CDCI3 solution at 35°C was investigated by 31P-NMR spectroscopy. A 0.1M solution of trimethyl phosphite was allowed to react in turn with 2.0 mol. equiv. of (i) dibenzoyl tetrasulphide, BzSSSSBz, (ii) dibenzoyl trisulphide BzSSSBz, and (iii) dibenzoyl disulphide BzSSBz, (see, for example, Organic Syntheses, Coll. Vol. Ill, 116- 118, 1955) . While reactions (i) and (ii) went to completion in less than 1 minute to give the phosphorothioate as the sole product, reaction (iii) required more than 24 minutes
(tι ~ 4.5 minutes) to go to completion. (It cannot be assumed that dibenzoyl tetrasulphide is quantitatively converted by trimethyl phosphite into dibenzoyl trisulphide as dibenzoyl trisulphide also reacts rapidly with trimethyl phosphite. It has not been ascertained which sulphur atom, i.e. one adjacent or one not adjacent to a carbonyl group, is transferred from the tetrasulphide to the phosphite.)
As dibenzoyl tetrasulphide was found to be the most reactive of the three reagents here examined in most detail, it was decided to investigate its use in solid phase oligonucleotide synthesis. The efficacy of dibenzoyl tetrasulphide as a sulphur-transfer agent was compared with that of the recently-recommended tetraethylthiura disulphide in the solid phase synthesis of the nonadecadeoxyribonucleoside octadecaphosphorothioate , d[A(S)T(S)T(S)C(S)C(S)G(S)G(S)A(S)C(S)T(S)C(S)G(S)T(S)C(S)- C(S)A(S) C(S) C(S)A] . Solid phase synthesis was carried out on a 0.2 μmolar scale in an Applied Biosystems 381A DNA Synthesizer. 5'-0-(4,4'-dimethoxytrityl) -2'-deoxy- ribonucleoside 3 '-(2-cyanoethyl) -N,N-diisopropylphosphor- a idites with standard base protecting groups (i.e. benzoyl on adenine and cytosine, and isobutyryl on guanine) were used as building blocks, and lH-tetrazole was used as activating agent. Sulphur-transfer was effected in each of the 18 synthetic cycles either with (a) 0.4M dibenzoyl tetrasulphide in commercially-supplied, base-free 0.025% butylated-hydrσxytoluene(anti-peroxidation stabilizer) - containing tetrahydrofuran for 60 seconds or (b) 0.5M tetraethylthiuram disulphide in acetonitrile for 900 seconds as recommended by Vu and Hirschbein (loc c_it) . In both experiments (a) and (b) , the crude d[DMTrA(S)T(S)T(S)C(S) - C(S)G(S)G(S)A(S)C(S)T(S)C(S)G(S)T(S)C(S)C(S)A(S)C(S)C(S)A] was examined by 31P-NMR spectroscopy after ammonolytic release from the solid support and deacylation. In accompanying Figure la (relating to experiment (a) ) the ratio of the integrals of the signals at δ ca. 56.2 (assigned to phosphorothioate phosphorus) and δ ca . -0.3 (assigned to phosphate phosphorus) is estimated to be ca. 99:1, and in accompanying Figure lb (relating to experiment (b) ) , this ratio is estimated to be ca. 98:2. When treatment with 0.5M tetraethylthiuram disulphide was decreased to 450 seconds per synthetic cycle, the latter ratio fell to ca. 92.5:7.5.
It appears from the above results that the presently-preferred dibenzoyl tetrasulphide transfers sulphur at least twenty times as rapidly as tetraethylthiuram disulphide. On a 0.2 μmolar scale, this allowed the duration of the synthetic cycle to be decreased from ca. 23 to ca. 9 minutes. It was apparent from LC analysis (accompanying Figure 2) that the quality of the synthetic nonadecadeoxyribonucleoside octadecaphosphoro- thioate obtained was similar in both cases. The isolated yields obtained were also similar. It is possible that with the optimisation of conditions, solvents and sulphur- transfer agents, for example, cycle times may be further reduced and required concentrations reduced.
Preliminary results obtained in the synthesis of phosphorothioate analogues of oligoribonucleotides suggest that sulphur transfer occurs much more slowly and that dibenzoyl tetrasulphide is also a suitable reagent to use for this purpose. Hence, the present invention is particularly directed to the production of oligodeoxyribo- nucleotides.

Claims

Claims ; -
1. A process for the solid phase production of a phosphorothioate analogue of an oligo- or poly- nucleotide from phosphoramidite building blocks in the presence of a sulphur-transfer agent characterised in that the sulphur- transfer agent corresponds to the following general formula:
X - Sn„ - X
wherein n represents 3 or 4; and
X represents RC(0)- or RC(S)- wherein R represents alkyl, cycloalkyl or aryl,
(R 0)C(0)- wherein R' represents alkyl, (R2N)C(0)- or (R2N)C(S)- wherein R is as defined above and R2NH may be a cyclic secondary amine, RS02- wherein R is as defined above,
(R0)2P(0)- or (R0)2P(S)- wherein R is as defined above.
2. A process as claimed in claim l wherein R represents aryl, which may be substituted.
3. A process as claimed in claim 1 or claim 2 wherein the sulphur-transfer agent is dibenzoyl tetra- or tri¬ sulphide.
4. A process as claimed in any of claims 1 to 3 wherein the oligo- or poly- nucleotide is an oligo- or poly- deoxyribonucleotide.
PCT/GB1992/002395 1991-12-23 1992-12-23 Synthesis of phosphorothioate analogues of oligo- and polynucleotides WO1993013118A1 (en)

Applications Claiming Priority (2)

Application Number Priority Date Filing Date Title
GB919127243A GB9127243D0 (en) 1991-12-23 1991-12-23 Synthesis of phosphorothioate analogues of oligonucleotides
GB9127243.5 1991-12-23

Publications (1)

Publication Number Publication Date
WO1993013118A1 true WO1993013118A1 (en) 1993-07-08

Family

ID=10706722

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/GB1992/002395 WO1993013118A1 (en) 1991-12-23 1992-12-23 Synthesis of phosphorothioate analogues of oligo- and polynucleotides

Country Status (2)

Country Link
GB (1) GB9127243D0 (en)
WO (1) WO1993013118A1 (en)

Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0723973A1 (en) * 1995-01-25 1996-07-31 King's College London Nucleoside phosphorothioate derivatives, synthesis and use thereof
US6172217B1 (en) 1996-12-27 2001-01-09 Isis Pharmaceuticals Inc. Method of synthesizing phosphorothioate oligonucleotides
EP1023310A4 (en) * 1997-10-15 2001-05-16 Isis Pharmaceuticals Inc Improved synthesis of sulfurized oligonucleotides
US7378516B2 (en) 1997-10-15 2008-05-27 Isis Pharmaceuticals, Inc. Synthesis of sulfurized oligonucleotides
JP2008266331A (en) * 2004-04-05 2008-11-06 Alnylam Pharmaceuticals Inc Methods and reagents used for oligonucleotide synthesis and purification

Non-Patent Citations (5)

* Cited by examiner, † Cited by third party
Title
BERICHTE DER DEUTSCHEN CHEMISCHEN GESELLSCHAFT vol. 53, 1920, WEINHEIM DE pages 961 - 977 I.BLOCH ET AL. 'Uber Trisulfide und Tetrasulfide einiger Carbonsauren. VI. Mitteilung uber Wasserstoffpersulfide.' cited in the application *
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. vol. 106, no. 20, 3 October 1984, GASTON, PA US pages 6077 - 9 W.J.STEC ET AL. 'Automated Solid-Phase Synthesis, Separation and Stereochemistry of Phosphorothioate Analogues of Oligodeoxyribonucleotides.' cited in the application *
JOURNAL OF THE AMERICAN CHEMICAL SOCIETY. vol. 112, no. 3, 31 January 1990, GASTON, PA US pages 1253 - 4 R.P.IYER ET AL. '3H-1,2-Benzodithiole-3-one 1,1'-Dioxide as an Improved Sulfurizing Reagent in the Solid-Phase Synthesis of Oligodeoxyribonucleoside Phosphorothioates.' cited in the application *
TETRAHEDRON LETTERS. vol. 30, no. 48, 1989, OXFORD GB pages 6757 - 6760 P.C.J.KAMER ET AL. 'An efficient Approach Toward the Synthesis of Phosphorothioate Diesters via the Schonberg reaction.' cited in the application *
TETRAHEDRON LETTERS. vol. 32, no. 26, June 1991, OXFORD GB pages 3005 - 8 H.VU ET AL. 'Internucleotide Phosphite Sulfurization with Tetraethylthiuram Disulfide. Phosphorothioate Oligonucleotide Synthesis via Phosphoramidite Chemistry.' cited in the application *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0723973A1 (en) * 1995-01-25 1996-07-31 King's College London Nucleoside phosphorothioate derivatives, synthesis and use thereof
US5708161A (en) * 1995-01-25 1998-01-13 King's College London Nucleoside phosphorothioate derivatives, synthesis and use thereof
US6172217B1 (en) 1996-12-27 2001-01-09 Isis Pharmaceuticals Inc. Method of synthesizing phosphorothioate oligonucleotides
US6403781B2 (en) 1996-12-27 2002-06-11 Isis Pharmaceuticals, Inc. Method of synthesizing phosphorothioate oligonucleotides
US6780989B2 (en) 1996-12-27 2004-08-24 Isis Pharmaceuticals, Inc. Diribonucleoside Phosphoramidites
EP1023310A4 (en) * 1997-10-15 2001-05-16 Isis Pharmaceuticals Inc Improved synthesis of sulfurized oligonucleotides
US7378516B2 (en) 1997-10-15 2008-05-27 Isis Pharmaceuticals, Inc. Synthesis of sulfurized oligonucleotides
US7723511B2 (en) 2000-01-11 2010-05-25 Isis Pharmaceuticals, Inc. Synthesis of sulfurized oligonucleotides
JP2008266331A (en) * 2004-04-05 2008-11-06 Alnylam Pharmaceuticals Inc Methods and reagents used for oligonucleotide synthesis and purification
US8058448B2 (en) 2004-04-05 2011-11-15 Alnylam Pharmaceuticals, Inc. Processes and reagents for sulfurization of oligonucleotides
US8063198B2 (en) 2004-04-05 2011-11-22 Alnylam Pharmaceuticals, Inc. Processes and reagents for desilylation of oligonucleotides
US8431693B2 (en) 2004-04-05 2013-04-30 Alnylam Pharmaceuticals, Inc. Process for desilylation of oligonucleotides

Also Published As

Publication number Publication date
GB9127243D0 (en) 1992-02-19

Similar Documents

Publication Publication Date Title
Rao et al. Dibenzoyl tetrasulphide-A rapid sulphur transfer agent in the synthesis of phosphorothioate analogues of oligonucleotides
US5571902A (en) Synthesis of oligonucleotides
US6020475A (en) Process for the synthesis of oligomeric compounds
DE3855055T2 (en) Chemical blocking by phosphithylation during oligonucleotide synthesis
US5151510A (en) Method of synethesizing sulfurized oligonucleotide analogs
US5705621A (en) Oligomeric phosphite, phosphodiester, Phosphorothioate and phosphorodithioate compounds and intermediates for preparing same
US7723511B2 (en) Synthesis of sulfurized oligonucleotides
US6294664B1 (en) Synthesis of oligonucleotides
US7273933B1 (en) Methods for synthesis of oligonucleotides
DE3689715T2 (en) Methods and reagents for the in vitro synthesis of oligonucleotides.
US5071974A (en) Compositions and methods for the synthesis of oligonucleotides having 5&#39;-phosphorylated termini
US5292875A (en) Method of synthesizing sulfurized oligonucleotide analogs
WO1998008858A1 (en) Improved reagents and process for synthesis of oligonucleotides containing phosphorodithioate internucleoside linkages
EP1023310B2 (en) Improved synthesis of sulfurized oligonucleotides
Farschtschi et al. Preparation of a deoxynucleoside thiophosphoramidite intermediate in the synthesis of nucleoside phosphorodithioates
US6001982A (en) Synthesis of oligonucleotides
EP0749436A1 (en) Compositions and methods for use in the synthesis of oligonucleotides
DE69733150T2 (en) PROCESS FOR THE PRODUCTION OF NUCLEOTIDES OR OLIGONUCLEOTIDES PHOSPHORAMITIDES
WO1993013118A1 (en) Synthesis of phosphorothioate analogues of oligo- and polynucleotides
US5606049A (en) Method of preparing 2&#39;-O-methyl cytidine monomers useful in oligomer synthesis
EP0475443B1 (en) Chemical synthesis of oligonucleotides
US6103891A (en) Method for the synthesis of nucleotide or oligonucleotide phosphoramidites
US20020010326A1 (en) Novel sulfurizing reagent 3-phenyl-1,2,4-dithiazoline-5-one

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): JP US

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

DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
122 Ep: pct application non-entry in european phase