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WO1992006103A1 - Synthese d'oligonucleotides - Google Patents

Synthese d'oligonucleotides Download PDF

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Publication number
WO1992006103A1
WO1992006103A1 PCT/GB1991/001687 GB9101687W WO9206103A1 WO 1992006103 A1 WO1992006103 A1 WO 1992006103A1 GB 9101687 W GB9101687 W GB 9101687W WO 9206103 A1 WO9206103 A1 WO 9206103A1
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WIPO (PCT)
Prior art keywords
group
formula
oligonucleotide
oligonucleotides
linker moiety
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PCT/GB1991/001687
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English (en)
Inventor
David Holland
Andrew John Garman
Michael Derek Edge
Michael Joseph Mclean
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Imperial Chemical Industries Plc
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Publication date
Application filed by Imperial Chemical Industries Plc filed Critical Imperial Chemical Industries Plc
Priority to AU86509/91A priority Critical patent/AU665174B2/en
Priority to JP3517413A priority patent/JPH06501692A/ja
Publication of WO1992006103A1 publication Critical patent/WO1992006103A1/fr

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    • 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
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F9/00—Compounds containing elements of Groups 5 or 15 of the Periodic Table
    • C07F9/02—Phosphorus compounds
    • C07F9/06—Phosphorus compounds without P—C bonds
    • C07F9/22—Amides of acids of phosphorus
    • C07F9/24—Esteramides
    • C07F9/2404—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic
    • C07F9/2408—Esteramides the ester moiety containing a substituent or a structure which is considered as characteristic of hydroxyalkyl compounds

Definitions

  • This invention relates to a method for the synthesis of oligonucleotides, to novel compounds which may be used during operation of the method, and to a solid support suitable for use in an automated oligonucleotide synthesiser.
  • PCR polymerase chain reaction
  • Oligonucleotide sequences or derivatives thereof are routinely synthesised for use as linkers, adaptors, building blocks for synthetic genes, synthetic regulatory sequences, probes, primers and other purposes and a number of methods have been developed for producing such sequences. These methods generally rely on the initial attachment of a first suitably protected nucleoside to a solid support by a cleavable linkage followed by sequential reactions of precursors of individual nucleotides to the growing oligonucleotide strand with each addition of a precursor involving a number of chemical reactions. At present the method most generally employed for the production of a lone
  • oligonucleotide is the method based on phosphoramidite chemistry. This is fully described by Caruthers et al in Tetrahedron Letters 1981, 22, (20) pp 1859-62 and European Patent No. 61746 and additionally by Koster et al in US Patent 4725677 (EP 152459) and by M.J. Gait
  • oligonucleotides improvements are desirable which will increase the throughput of such commercial synthesisers, i.e. increase the number of oligonucleotides synthesised per day.
  • nucleotides on a support is provided in the protocol for the Applied Biosystems DNA Synthesiser Model 380B, particularly Section 2 thereof, which is incorporated herein by reference thereto.
  • a method for the synthesis of a plurality of oligonucleotides in which an oligonucleotide is formed by sequential reactions of precursors of individual nucleotides on a support comprising the steps of (a) forming a first oligonucleotide; (b) attaching to said first oligonucleotide a cleavable linker moiety; (c) forming a second oligonucleotide on the cleavable linker moiety; and (d) cleaving the linker moiety to give the desired oligonucleotides.
  • the first oligonucleotide is preferably formed on a support, which is preferably a solid support such as is used in automated oligonucleotide synthesis.
  • the identity of the support is not critical and may be any of the supports used in the automated synthesis of oligonucleotides, for example, modified inorganic polymers such as those disclosed in the US Patent Specification 4,458,066, silica gels, Porasil C, kieselguhr PDMA, polystyrene, polyacrylamide, Silica CPG (LCAA) or controlled pore glass as used in, for example, the Applied Biosystems DNA synthesiser Model 380B.
  • the support can have a precursor of a first nucleotide cleavably attached to it, e.g. a solid support connected to an optionally protected nucleoside by means of a
  • the first oligonucleotide may be formed by conventional technology used for synthesising oligonucleotides, for example by using phosphoramidite chemistry on an automated oligonucleotide synthesiser as described above.
  • the first oligonucleotide is preferably connected to the support by a hydrolysable group (e.g. a base labile group) as is known in the art.
  • the cleavable linker moiety may be attached to the first oligonucleotide by means of a reagent, for example a modified
  • nucleoside or alternatively a reagent which does not contain a nucleoside element, which is capable of connecting to said first oligonucleotide and upon which a second oligonucleotide may be formed, and which can be broken to separate the first and second
  • oligonucleotides under conditions which do not significantly affect the oligonucleotides.
  • the first aspect of the invention may be illustrated by the formation of 2 oligonucleotides using different combinations of the phosphoramidites of 2'-deoxyadenosine (dA), 2'-deoxyguanosine (dG), 2'-deoxycytidine (dC), and 2'-deoxythymidine (dT) separated by the cleavable linker moiety L' built up sequentially in a 3 ' to 5' direction from the 3' hydroxy of ribose on a solid support according to the above method described by M.J. Gait. After synthesis the sequence attached to the solid support is:
  • a preferred first aspect of the present invention provides a method for the synthesis of a plurality of oligonucleotides comprising the steps of:
  • the cleavable linker moiety connects the first and second oligonucleotides by a 3' and a 5' oxygen, more
  • oligonucleotide preferably via a phosphate, phosphite, phosphate ester, phosphite ester or H-phosphonate ester, one on each oligonucleotide.
  • the identity of the first cleavable link is not believed to be critical, it is preferably base labile, and may be for example any of the cleavable links used in automated oligonucleotide synthesisers, such as a link which contains a base labile ester group.
  • the method of invention does not contain a step in which hybridisation of the first or second oligonucleotide with a further oligonucleotide is attempted, for example by contact with a solution containing an oligonucleotide which may be complementary to the first or second oligonucleotide because this is unnecessary.
  • the first aspects of the invention include repetition of steps (b) and (c) any desired number of times, for example 1 to 100 times, or preferably 1 to 5 times, to produce further oligonucleotides which are each connected through a cleavable linker moiety.
  • steps (b) and (c) any desired number of times, for example 1 to 100 times, or preferably 1 to 5 times, to produce further oligonucleotides which are each connected through a cleavable linker moiety.
  • oligonucleotides are formed on the cleavable linker moiety attached to the previously formed oligonucleotide and may be the same as or different to the previously formed oligonucleotides.
  • the cleavable linker moieties may be cleaved, e.g. by base hydrolysis, to give a mixture of individual oligonucleotides which may, if required, be purified and separated.
  • oligonucleootide preferably includes an oligodeoxyribonucleotide, an oligoribonucleotide and analogues thereof (for example those which bear protecting groups), including those with methyl-phosphonate and phosphorothioate or phosphorodithioate diester backbones, and
  • oligonucleotides with oligodeoxyribonucleotides especially the
  • 2'-oligodeoxyribonucleotides being more usually synthesised by the method of the invention.
  • the preferred oligonucleotides are
  • oligodeoxyribonucleotides are essentially single stranded, and are preferably from at least two, more preferably at least 5, especially from 10 to 200 bases long.
  • the method of the invention gives the advantage of more effective use of the apparatus and subsequently reducing the cost of production and purification of oligonucleotides.
  • the DNA synthesiser can produce two or more oligonucleotides (which may be the same or different) on any one of its columns without being re-programmed between each oligonucleotide.
  • oligonucleotides which may be the same or different
  • the DNA synthesiser can go on to produce another without any intervention by an operative. This can significantly increase the productivity of such apparatus.
  • the method of the invention is particularly useful for the synthesis of primers for the Polymerase Chain Reaction (PCR) technique.
  • PCR Polymerase Chain Reaction
  • oligonucleotides synthesised are for this purpose.
  • Such primers are typically required in pairs and the method of the invention is convenient since it allows production of oligonucleotides in pairs. This is particularly an advantage when using single column synthesisers and/or for heavily used facilities for out-of hours working.
  • the precursors of the individual nucleotides are nucleoside phosphoramidites which are protected at the 5' oxygen atom and are optionally base protected.
  • Methods of protecting nucleoside bases are known in the art, for example by a protecting group which is removable by treatment with mild acid or alkali.
  • Adenine and cytosine may be protected by an optionally substituted N-benzoyl group and Guanine by an N-isobutyryl group.
  • Adenocsine and Guanine may also be protected by a dimethylformamide or phenoxyacetyl group, and cytosine by an isobutyryl group.
  • the protecting groups are desirably removed after separation of the protected oligonucleotide from the support. Cleavage of the linker moiety may be effected before, during or after the removal of the protecting groups depending upon the chemistry employed. It is preferred that the protecting groups are removable by treatment with aqueous base, particularly concentrated ammonia solution.
  • the linker is cleavable under basic or alkaline conditions so that protecting group removal and cleavage of linker moieties can be effected in one step. Typical basic conditions employed, are to mix the protected
  • oligonucleotide with concentrated ammonia for example at around 55°C for up to 24 hours, especially from about 5 to 24 hours. It is preferred that a linker moiety is chosen such that cleavage is completed under these conditions.
  • Other bases preferably volatile bases may be employed to effect cleavage. These may conveniently be organic amines in water, for example piperidine or methylamine, preferably at a concentration from
  • oligoribonucleotides precursors are for example the same as for oligodeoxyribonucleotides except that on the 2' position of the ribose there is a protected hydroxyl group, for example a tertiary butyl dimethyl silyloxy group or 1-[(2-chloro-4-methyl) phenyl]-4-methoxy piperidin-4-yloxy group which is abbreviated to CTMP, as described by T.S. Rao et al in Tet. Lett., 28, 4897 (1987).
  • oligonucleotides i.e. as primers for PCR
  • oligonucleotides having a 5' phosphate group see e.g. Higuchi & Ockman (1989), Nucl. Acid Res. 17(14), p5865). Therefore a synthetic method that gives rise to an oligonucleotide having a 5' phosphate group is of vailue.
  • a further advantageous use for oligonucleotides having a 5' phosphate group is in the chemical synthesis of genes where 5' phosphorylated oligonucleotides are desired.
  • cleavable linker moiety used in the method of the invention preferably comprise either (I) a moiety whose cleavage gives rise to OH groups at both the 5' and 3' ends of the desired
  • oligonucleotides or (II) a moiety whose cleavage gives rise to a free 3' OH group on one oligonucleotide and a 5' phosphate group on another oligonucleotide.
  • step (d) preferably yields desired oligonucleotides each having at the 3' and 5' position a group selected from hydroxy and phosphate.
  • the reagents currently used to synthesise oligonucleotides include the protected nucleoside phosphoramidites. It would therefore be convenient if the cleavable linker moiety used in the present methods is attached by means of a modified nucleoside.
  • the invention also provides a modified nucleoside reagent of general structure (II) which is capable of connecting to said first oligonucleotide and upon which a second oligonucleotide may be formed:
  • Nuc is a nucleoside in which the base optionally is protected
  • Z is a protecting group attached to the 5' oxygen of Nuc; -O-PA is a phosphoramidite group, a phosphate ester group, a
  • L' is a cleavable linker moiety, which is preferably a
  • L' may be cleaved or split into two or more parts by treatment with base, for example with aqueous alkali, ammonium hydroxide or piperidine.
  • the modified nucleoside of formula Z-Nuc-L'-O-PA is preferably of Formula (III):
  • optionally protected base such as optionally protected uracil, thymine, cytosine, adenine or guanine, or analogues thereof, and D is H or a protected hydroxyl group.
  • phosphate ester groups and H-phosphonate groups these may be mentioned groups which, in the free acid form, are respectively of formula:
  • Z 3 is a protecting group, preferably a base labile protecting group, for example 2-chlorophenyl or 2,4-dichlorophenyl.
  • -O-PA is a phosphoramidite of general structure:
  • R 4 and R 5 are each independently optionally substituted alkyl, especially C 1-4 -alkyl; optionally substituted aralkyl, especially optionally substituted benzyl; cycloalkyl and cycloalkylalkyl containing up to ten carbon atoms, such as cyclopentyl or cyclohexyl; or R 4 and R 5 taken together with the nitrogen atom to which they are attached form an optionally substituted pyrollidine or piperidine or R 4 and R 5 when
  • R 4 and R 5 are preferably iso-propyl.
  • R 6 represents a hydrogen atom or a protecting group, for example a phosphate protecting group.
  • phosphate protecting groups there may be mentioned optionally substituted alkyl groups, for example methyl, 2-cyanoethyl, 2-chlorophenyl,
  • R 6 is methyl or, more preferably, 2-cyanoethyl.
  • Nuc in the structure II above represents the conventional nucleoside and deoxynucleosides (deoxy)cytidine, (deoxy)adenosine, (deoxy)guanosine, (ribo)thymidine or (deoxy)uridine as well as analogues thereof.
  • the base portion of the nucleoside optionally is protected by a protecting group.
  • the amine substituent in adenine, cytosine and guanine may be protected by any of the protecting groups used in the art (for example, as described in E Ohtsuka et al, Nucleic Acids Research, (1982), 10, 6553-6570).
  • nucleotide chemists include particularly isobutyryl and
  • nucleosides in which the base is protected include, for example, N 4 -benzoylcytosine,
  • N 6 -benzoyladenine and N 2 -isobutyrylguanine are protected by protection, for example thymine and uracil.
  • Z in the above formulae represents a protecting group for the 5'-hydroxyl group of the nucleoside, especially an acid labile protecting group.
  • Suitable protecting groups will be apparent to those skilled in the art and include those discussed in 'Protective
  • protecting groups include, tetrahydropyranyl e.g.
  • tetrahydropyran-2-yl 4-methoxytetrahydropyranyl e.g. 4-methoxytetrahydropyran-2-yl, methoxytrityl (preferably for oligoribonucleotide synthesis only), dimethoxytrityl, pixyl, isobutyloxycarbonyl, t-butyl dimethylsilyl and like protecting groups.
  • Z is
  • -O-PA is a H-phosphonate or a phosphoramidite
  • these are oxidised to respectively a phosphate diester or phosphate tri-ester groups during operation of the method, for example using aqueous iodine or peroxide.
  • the oxidation is preferably performed after step (c) and before step (d), whilst in the case of phosphoramidite it is preferably performed during step (a) and step (c).
  • L' preferably comprises or consists of three sections (i), (ii) and (iii) which have the structures discussed below.
  • Section (i) suitably comprises a group which upon cleavage, for example by hydrolysis, leads to the generation of a 3' hydroxyl group at the 3' terminus of the oligonucleotide to which it was previously attached.
  • groups there may be mentioned carbonyl, CONH and imidate groups.
  • section (i) is a -C- group.
  • Section (ii) can be any spacer group, preferably a spacer group compatible with automated oligonucleotide synthesis, for
  • section (ii) is a divalent organic spacer group, for example of 2 to 15 atoms in length, preferably 2 to 6 atoms in length.
  • the preferred divalent organic spacer group comprises or consists of one or more substituted or unsubstituted methylene groups optionally interrupted by other groups such as 1,2-, 1,3-, or
  • Section (iii) may comprise a group capable of giving rise to beta-elimination of a phosphate ester group.
  • This group can be of two types, Type A or Type B:
  • Type A is of the structure:
  • Q2 is a electron withdrawing group such as -SO 2 - and R 1 , R 2 and R 3 are each independently H or a non-electron withdrawing group such as alkyl, especially C 1-4 -alkyl, or a substituent that is not itself a leaving group in a beta-elimination reaction and does not otherwise interfere when a phosphate ester group, introduced by means of -O-PA, is eliminated.
  • R 1 may be an electron-withdrawing group.
  • Type B is of the general formula:
  • R 1 , R 2 and R 3 are each independently H or
  • section (iii) is a group of formula
  • each R 7 independently is H or C 1-4 -alkyl
  • R 8 & R 9 is a single bond and the other is H or C 1-4 -alkyl
  • R 10 & R 11 are each independently H or C 1-4 -alkyl or R 10 together with
  • R 11 and the carbon atoms to which they are attached form an optionally substituted 4,5,6 or 7 membered alicyclic or heterocyclic ring;
  • Z 2 is a protecting group, preferably a base labile protecting group.
  • group (i) may be sufficiently electronegative to serve as element Q2 in group (iii).
  • group (ii) may be sufficiently electronegative to serve as element Q2 in group (iii).
  • the 3' oxygen of Nuc is attached directly to group (iii) and group (ii) is obviated.
  • the sections are preferably linked together in the order (i), (ii), (iii) with (i) and (iii) being connected to nucleoside (Nuc) and -O-PA respectively, as shown in structure (III).
  • W is a divalent organic spacer group as defined in Section (ii), especially -CH 2 CH 2 -CO.OCH 2 CH 2 -.
  • L' is of formula (IV):
  • the modified nucleoside is preferably of the formula (V):
  • Compounds of Formula (II) or (III) wherein -O-PA is a phosphoramidite group may be prepared by reacting a compound of formula Z-Nuc-L'-OH with a compound of formula X -PA in CH 2 Cl 2 using diisopropylethylamine as base, wherein PA is a phosphoramidite as defined above for -O-PA except that -O- is absent, and X is a
  • the compound of Formula (II) or (III) may be prepared by reaction of a compound of formula Z-Nuc-L'-OH with the triazolide of the corresponding free phosphate ester using a method analogous to that described in the above book by M.J. Gait.
  • the compound of Formula (II) or (III) may be prepared by reaction of a compound of formula Z-Nuc-L'-OH with PCI in the presence of 1,2,4-triazole using a method analogous to that described by B.C.
  • the compound of the formula Z-Nuc-L'-OH may be prepared in two steps by reaction of a compound of formula Z-Nuc-OH with the anhydride of a suitable bifunctional carboxylic acid, for example succinic acid, followed by coupling of the acid derivative so
  • the carboxylic acid may be activated toward reaction with a hydroxyl group by methods known in the art, for example by in situ formation of the symmetrical anhydride by the condensation of two molecules of the carboxylic acid derivative via the intercession of a coupling agent such as for example 1,3-dicylclohexyl carbodiimide.
  • the reaction of the hydroxy compound with the activated carboxylic acid derivative may be performed in an aprotic solvent in the presence of one molar equivalent of base.
  • the compound of formula Z-Nuc-L'-OH so produced is preferably purified from the reaction mixture by some suitable means, for example chromatography.
  • the compound of formula Z-Nuc-OH may be prepared by reaction of a compound of formula HO-Nuc-OH with a compound of formula Z-X 1 (wherein X 1 and Z are as defined above) preferably in an aprotic solvent in the presence of a molar equivalent of base.
  • Z-X 1 is a compound that reacts preferentially at only one of the two (or three if HO-Nuc-OH is a ribonucleoside) available hydroxyl groups.
  • Z-X 1 reacts selectively with the primary hydroxyl at the 5'- position of HO-Nuc-OH.
  • a convenient modified nucleoside is of formula (VI):
  • the cleavable linker moiety L' can be either introduced by means of a reagent (for example of structure II) which comprises a nucleoside that will become the 3' nucleotide of the second oligonucleotide or by means of a reagent that does not contain a nucleoside element.
  • a reagent for example of structure II
  • the modified nucleosides of general structure (II) are of great value for introducing a cleavable linker moiety as described in the method of the invention. However, five such nucleosides are required depending on whether the 3' nucleoside of a second desired nucleotide is A, G, T, C or U.
  • a single reagent which does not contain a nucleoside element, which is capable of connecting the first and second oligonucleotides together and is compatible with phosphoramidite chemistry or other chemistry used in oligonucleotide synthesis, for example in DNA synthesisers, and is capable of being completely removed from the oligonucleotides, for example by treatment with ammonium hydroxide.
  • Z 1 is a protecting group
  • R 1 , R 2 , R 3 , Q1, Q2 and -O-PA are as hereinbefore defined;
  • each R 7 independently is H or C 1-4 -alkyl
  • R 8 and R 9 are a single bond by means of which the group of formula (Vila) is attached to E, and the other is H or
  • Z 2 is a protecting group, preferably a base labile protecting group
  • R 10 & R 11 are each independently H or C 1-4 -alkyl or R 10 together with
  • R 11 and the carbon atoms to which they are attached form an optionally substituted 4, 5, 6 or 7 membered alicyclic or heterocyclic ring;
  • E is a single covalent bond or a spacer group; and provided that when A 1 and A 2 are both of Formula (Vlld) E is a spacer group.
  • the protecting group represented by Z is preferably an acid labile protecting group, more preferably an acid labile protecting group listed above for Z, especially dimethoxytrityl.
  • Z 2 is a base labile protecti group it is preferably selected from the base labile protecting gr ps disclosed in the abovementioned book by T.W. Green, especially a silyl group, for example t-butyl dimethylsilyl, or more preferably an acyl group such as a
  • R 1 , R 2 , R 3 , R 7 , R 8 , R 9 , R 10 and R 11 is H or
  • C 1-4 -alkyl it is preferably methyl, more preferably H.
  • Q 2 is
  • E is a spacer group it is preferably a spacer group as hereinbefore defined in section (ii), more preferably an optionally substituted alkyl, alicycic or aryl group, especially phenylene or an alkyl group containing up to 6 carbon atoms.
  • the preferred alicyclic ring is a 5 or 6 membered ring, for example a cyclohexyl or cyclopentyl ring.
  • the preferred heterocyclic ring is a 5 or 6 membered ring, for example furanyl or pyranyl ring.
  • E is a single covalent bond, -(CH 2 ) m -, -CO.NH(CH 2 ) m NH-CO.-,
  • G is -(CH 2 ) m -, aryl, especially phenyl, or an alicyclic group such as cyclohexyl,
  • each m independently has a value of from 1 to 6, preferably 2 to 6, especially 2.
  • a 1 and A 2 are both selected from Formula (Vllb) or (VIIc) it is preferred that E is of formula -(CH 2 ) m - or
  • E is of formula G as hereinbefore defined, especially -(CH 2 ) m - or phenyl.
  • E is of formula -(CH 2 ) m -, -G-CO.-O-(CH 2 ) m - or -G-O-CO.-(CH 2 ) m - wherein m and G are as hereinbefore defined.
  • a 1 is of Formula (Vila) and A 2 is of Formula (Vlld) it is preferred that E is of formula -(CH 2 ) m -, -O.CO.-G- or -G-CO.-O-G- wherein m and G are as hereinbefore defined.
  • E is of formula -(CH 2 ) m -, -(CH 2 ) m -O-CO.-G- or -(CH 2 ) m -CO.-O-G- wherein m and G are as hereinbefore defined.
  • E is of formula -(CH 2 ) m - or
  • a 1 is of Formula (Vlld) and A 2 is of Formula (Vila) it is preferred that E is of formula -(CH 2 ) m -, -G-O-CO.-G- or -G-CO.-O-G- wherein m and G are as hereinbefore defined.
  • E is of formula -(CH 2 ) m - or -(CH 2 ) m -OCO.-G- or -(CH 2 ) m -CO.-O- (CH 2 ) m - wherein m and G are as hereinbefore defined.
  • a 1 and A 2 are each independently selected from Formula (Vila), (Vllb) and (Vlld) wherein the carbon atom marked with an asterisk is attached to the oxygen atom shown in Formula (VII).
  • the compounds of formula (VII) are suitable reagents for attaching a cleavable linker moiety of formula -A 1 -E-A 2 - between a first and second oligonucleotide as described by the method of the invention.
  • oligonucleotide of formula d(AGCTA) results having a 5'-OH group
  • a 2 is of Formula (Vllb) or (VIIc) d(AGCTA) results having a
  • the method of the invention provides the great benefit of enabling one to select whether the first, second, and subsequent oligonucleotides prepared according to the method of the invention have a hydroxy group at the 3' position and a hydroxy or phosphate group at the 5' position.
  • Compounds of Formula (VII) wherein -O-PA is a phosphoramidite may be prepared by reacting a compound of formula Z 1 -O-A 1 -E-A 2 -OH with a compound of formula X -PA in CH Cl using di(N-isopropyl)ethylamine as base.
  • PA is preferably a phosphoramidite as defined above for -O-PA except that -O- is absent, and Z 1 , A 1 , E and A 2 are as hereinbefore defined, and X 1 is a leaving group, for example Cl or Br.
  • the compound of Formula (VII) may be prepared by reaction of a compound of formula Z 1 -O-A 1 -E-A 2 -OH with the triazolide of the corresponding free phosphate ester using a method analogous to that described in the above book by M.J. Gait.
  • the compound of ormula (VII) may be prepared by reaction in a compound of formula Z 1 -O-A 1 -E-A 2 -OH with PCI, in the presence of 1,2,4-triazole using a method analogous to that described by B.C.Froehler et al, Nucleic Acid Research, (1986), 14, 5399-5407.
  • the compound of formula Z 1 -O-A 1 -E-A 2 -OH may be prepared by deprotection of a compound of formula Z 1 -O-A 1 -E-A 2 -O-TBDMS, wherein
  • TBDMS is a t-butyldimethyl silyl group (which is removable using tetrabutyl ammonium fluoride in THF) or other protecting group which is removable under neutral conditions.
  • the compound of formula Z 1 -O-A 1 -E-A 2 -O-TBDMS may be prepared by reaction of the compound of formula Z 1 -O-A 1 -E-A 2 -O-TBDMS (wherein A is as defined for A 1 except that Z 2 , when present, is H and A 4 is as defined for A 2 except that
  • Z 2 when present, is H) with a compound of formula Z 2 -X 1 wherein X 1 is a leaving group, for example Cl or Br (e.g. a C 1-4 -alkanoyl halide such as acetyl chloride or propanoyl bromide, or an optionally
  • Z 1 -O-A 1 -E-A 2 -O-TBDMS may be prepared by reaction of Z 1 -O-A 3 -E-A 4 -OH with TBDMS-Cl.
  • Z 1 -O-A 3 -E-A 4 -OH may be prepared by reaction of
  • the compound of formula Z 1 -O-A 1 -E-A 2 -OH may be prepared by reaction of a compound of formula Z 1 -O-A 1 -E-CO 2 H with a compound of formula
  • the compound of formula Z 1 -O-A 1 -E-CO 2 H may be prepared by the reaction of the compound of formula Z 1 -O-A 1 -OH with an activated form of the compound of formula HO 2 C-E-CO 2 H, preferably an aprotic solvent in the presence of a molar equivalent of base.
  • the dicarboxlic acid may be activated to attack by the hydroxyl group by being present as the acid anhydride, the acid chloride or some other suitable derivative, or the reaction may be mediated by the presence of a coupling agent as described above.
  • the compound of formula Z 1 -O-A 1 -OH may be prepared by the reaction of the compound of formula HO-A 1 -OH with Z 1 -Cl (or some other suitably activated form of Z 1 ) in an anhydrous aprotic solvent in the presence of a molar equivalent of base.
  • Formula (VII), and precursors thereof, Z 1 , A 1 , E. A 2 , O, PA and Z 2 are as hereinbefore defined except where stated otherwise and DCCI is
  • a compound comprising two or more oligonucleotides linked, preferably by 3' and 5' oxygen atoms, by a group or groups containing a cleavable linker moiety of formula -A 1 -E-A 2 - or -L'- wherein A 1 , E, and L' and A 2 are as hereinbefore defined. It is preferred that the cleavable linker moiety and formula -A 1 -E-A 2 - or -L'- is connected to each oligonucleotide via a H-phosphonate, phosphate, phosphite, phosphate ester or phosphite ester linkage. It is preferred that one of the oligonucleotides is connected to a support.
  • phosphite linkages are of formula -P(-OR 6 )- wherein R 6 is as
  • nucleoside precursors as the sole means for introducing nucleotide elements into the oligonucleotide.
  • a compound of Formula (II) or (VII) may also be used to convert a support which does not have a first cleavable link attached to it to a support which does have a first cleavable link attached.
  • a further aspect of the present invention comprises a method for the preparation of solid support bearing a cleavable link by condensation of a solid support which does not bear a cleavable with a compound of Formula (II) or (VII) as hereinbefore defined.
  • the solid support is preferably one of the conventional supports which has hydroxyl or amino groups, preferably hydroxyl groups, for example one of the aforementioned solid supports used in automated oligonucleotide synthesis.
  • Reaction with a reagent of Formula (II) or (VII) may be performed by analogous method to these which are known.
  • the cleavable link may be introduced by means of an automated nucleic acid synthesiser, in the same manner as for the nucleotide precursors.
  • use of a reagent of Formula (II) or (VII) use of a reagent of Formula
  • (II) or (VII) which does not contain a beta-elinination moiety are preferred, since these do not give rise after cleavage to undesirable phosphorylation of the solid support.
  • a convenient feature of the reagents which do not contain the beta-elimination group is that the hydroxyl group of hydroxyl containing supports is re-generated allowing the possibility of re-use of the support for the synthesis of further oligonucleotides.
  • a general advantage of this further aspect of the invention is the avoidance of purchasing or synthesising the support with the first nucleoside attached. This is especially advantageous for the synthesis of oligonucleotides of non-conventional structure where the support containing the first attached nucleoside may not be readily obtainable.
  • a still further aspect of the present invention provides a solid support, suitable for use in an automated oligonucleotide synthesiser, of Formula (VIII) or (IX):
  • SUP is a solid support, preferably a solid support having hydroxy or
  • P 1 is of the formula: -
  • R 6 and Z 3 are as hereinbefore defined.
  • SUP is preferably one of the aforementioned solid supports used in automated oligonucleotide synthesis.
  • Reagent M 1 is of formula (VI) wherein B is thymidinyl.
  • This compound was prepared by the method described by Gait et al in Nucleic Acids Research (1980), 8(5), 1090.
  • oligodeoxyribonucleotides of sequence TCTAACAGCTGATCTL'CAGCTGATCC was prepared on an Applied Biosystems 380B DNA Synthesiser from 5'-dimethoxytrityl-N-4-benzoyl-2'-deoxycytidine bound to controlled pore glass via 3' -OH and a
  • Synthesiser to introduce reagent M which will introduce TL' in the sequence above.
  • the procedure consisted briefly of: (1) removal of the dimethoxytrityl group with 3% trichloroacetic acid in dichloromethane; (2) coupling of 5'-0-(4,4'-dimethoxytrityl)-2'- deoxythymidin-3'-yl 2-(2-[(2-cyanoethoxy)N,N-(diisopropylamino) phosphanyloxyjethylsulfonyl) ethyl succinate (0.1 M solution in 1,2 dichloroethane:anhydrous acetonitrile, 10:9) activated by tetrazole for 1 minute; (3) iodine oxidation of the intermediate phosphite linkage to a phosphate linkage; (4) a capping step with acetic anhydride.
  • the detritylated oligodeoxyribonucleotide sequence was cleaved from the solid support and also cleaved at the cleavable link moiety (L') and completely deprotected by treatment with ammonium hydroxide solution (sp.gr. 0.88) for 16h. at 55 C.
  • ammonium hydroxide solution was evaporated and the residue was dissolved in sterile water (1 ml). This product was analysed by hplc using a
  • Partisil SAX 10 micron column Jones Chromatography
  • eluant A 60% formamide
  • eluant B 0.3 M potassium dihydrogen orthophosphate in 60% formamide with a gradient of 0-85% eluant B in 30 minutes.
  • the chromatography revealed the presence of three oligonucleotide sequences in approximately equal amounts which eluted from the column after 10.5, 13.9 and 14.8 minutes. These products were identified by comparing their elution times with those of independently synthesised
  • oligonucleotide sequences and were shown to be oligonucleotides of sequence: dCAGCTGATCC (elution time 10.5 minutes); - 5'-phosphorylated dCAGCTGATCC (elution t: e 13.9 minutes) and dTCTAACAGCTGATCT (elution time 14.8 minutes).
  • the sequence eluting after 10.5 minutes was a result of a partial coupling reaction of reagent M to the
  • the method of the invention was used in the synthesis of two oligodeoxyribonucleotide P.C.R. primers from one deoxynucleoside bound to a solid phase.
  • cleavage from the solid support After completion of the synthesis, cleavage from the solid support, cleavage of the cleavable linker moiety and removal of the base protecting groups were all achieved by incubation in ammonia solution, according to normal oligo synthesis protocols.
  • the ammoniacal solution containing the pair of oligomers was then lyophilized, the residue was redissolved in 1 ml of water and the DNA concentration was determined spectrophotometrically.
  • the mixture of oligodeoxynucleotides thus produced is referred to hereinafter as "primer mix 1".
  • Two other PCR primers were prepared individually by standard means to serve as a comparison for the efficiencies of the PCR's
  • primer mix 1 performed using primer mix 1.
  • sequences of the primers are:
  • Oligo 1 5'-CTATTCAAAATCGGAGCTCTAAGAT 3' Oligo 2: 5'-TAGGGATTTGATTTTACGAGAGA 3'
  • each tube contained final
  • Each tube also contained a final concentration of 50 micromolar each of dATP, dGTP, dCTP, dTTP.
  • In each tube was contained 30 ng of Chlamydia trachomatis (Serovar L2) genomic DNA and 2.5 units of Taq DNA polymerase.
  • PCR primers in each tube were as follows: tube 1, 100 pmoles oligo 1, 100 pmoles oligo 2; tube 2, 75 pmoles oligo 1, 100 pmoles oligo 2; tube 3, 50 pmoles oligo 1, 100 pmoles oligo 2; tube 4, 25 pmoles oligo 1, 100 pmoles oligo 2; tube 5, 10 pmoles oligo 1, 100 pmoles oligo 2; tube 6, 100 pmoles primer mix 1; tube 7, 200 pmoles primer mix 1.
  • Lanes 1-7 contain samples from tubes 1-7 described above, respectively. Lane M, O x 174 size markers. EXAMPLE 4
  • reagent M2 The structure of reagent M2 is as follows:
  • DMT is:
  • Step 1) Preparation of 1-O-(4,4'-dimethoxytrityl) threitol.
  • Step 2) Preparation of 1-O-(4.4'-dimethoxytrityl)-4-O-(tert.)- butyldimethylsilyl threitol.
  • Step 3 Preparation of 1-O- (4, 4' -dimethoxytrityl) -2 , 3 , -di-O-benzoyl -1-O (tert . ) butyldimethylsilyl threitol.
  • Step 4) Preparation of 1-O-(4,4'-dimethoxytrityl)-2,3- di-O-benzoyl threitol.
  • step 3 The product from step 3) (2.7g, 3.6mmol) was dissolved in a mixture of tetrahydrofuran (Aldrich), pyridine and water (100ml, 8:1:1
  • An oligonucleotide was formed on a solid support via a first
  • cleavabl linker moiety was attached to the first oligonucleotide by means of reagent M2.
  • Reagent M2 was dissolved in anhydrous acetonitrile to a concentration of 0.1M, and a bottle containing this solution was attached to one of the spare reagent ports on the DNA synthesizer .
  • the synthesiser was then programmed to synthesise the following sequence:
  • the synthesiser achieves the steps of a) forming a first oligonucleotide of sequence (5'-3') TAGGGATTTGATTTTACGA
  • X is a first cleavable link contained in the succinylglycylglycyl-aminopropyl spacer.
  • the synthesiser also performs the cleavage of the first cleavable link X by the ammonia treatment as in step d) in the method of the invention.
  • the eluted oligonucleotide in the ammonia solution was incubated at 55°C for 16 hours and evaporated to dryness under reduced pressure.
  • the residue was redissolved in 1ml of water, and 100 ⁇ l of this solution were mixed with 100 ⁇ l of piperidine and incubated at 55°C for between 16 and 72 hours.
  • oligonucleotides 2) and 3) are of identical length and sequence to the products expected from cleavage of the oligonucleotide containing the cleavable link.
  • oligonucleotides so produced was determined by the incorporation of a radioactive phosphorus at these positions as described below.
  • oligonucleotides After treatment of the oligonucleotides with either concentrated ammonium hydroxide or 50% piperidine the solutions were lyophilized and the oligonucleotides were redissolved in water to a concentration of approximately 1 mg/ml. One microlitre of this solution was then added to an Eppendorf tube containing water (6 ⁇ l), 10x reaction buffer ("One Phor All", Pharmacia, 1 ⁇ l), [gamma 32 P] adenosine triphosphate (Amersham, l ⁇ l) and T4 Polynucleotide kinase (Pharmacia, 1 ⁇ l). This mixture was then incubated at 37°C for one hour.
  • Ethanol (30 ⁇ l) was added, the contents were mixed by repeated inversion of the tube, and the sample was incubated at -70 °C for 15 minutes.
  • the tube was spun in an Eppendorf centrifuge (Model 5415) at 14000 rpm for 15 minutes, and the supernatant was discarded.
  • the pellet was dried briefly in vacuo and was redissolved in 10 ⁇ l of a solution containing 80% formamide, 0.1% bromophenol blue, 0.1% xylene cyanol and 10 ⁇ M EDTA. This solution was loaded into one of the wells of a denaturing polyacrylamide gel (8% acrylamide, 50% urea) adjacent to the appropriate radiolabelled size markers, and the gel was run at 40W for approximately two hours. The locations and sizes of labelled DNA fragments were determined by autoradiography. The presence of strong bands co-migrating with bands due to
  • oligonucleotide One microlitre of the solution of oligonucleotide described above was added to an Eppendorf tube containing water (5 ⁇ 1), 5x reaction buffer ("TdT Tailing Buffer", BRL, 2 ⁇ 1), [alpha P] 2'-deoxyadenosine triphosphate (Amersham, 1 ⁇ l) and terminal deoxynucleotidyl transferase (BRL, 1 ⁇ 1). This mixture was then incubated at 37°C for one hour.
  • TdT Tailing Buffer BRL, 2 ⁇ 1
  • BRL 5x reaction buffer
  • [alpha P] 2'-deoxyadenosine triphosphate Amersham, 1 ⁇ l
  • BRL terminal deoxynucleotidyl transferase
  • Radiolabelled DNA was recovered by precipitation from ethanol and then analyzed by denaturing gel electrophoresis exactly as described above for 5'- end-labelled fragments.
  • step (d) The mixture of oligonucleotides produced in step (d) described above was analyzed by reverse-phase HPLC on a Waters ⁇ Bondapak C18 column using a linear gradient from 0-30% buffer B in buffer A over 45 minutes where buffer A was 0.1M triethylammonium acetate (pH 7.5) and buffer B was 80% acetonitrile in 0.1M triethylammonium acetate (pH 7.5).
  • a control oligonucleotide of 19 residues (18 phosphates) had a retention time of 28 minutes, an oligonucleotide of 25 residues (24 phosphates) had a retention time of 31 minutes and an oligonucleotide of 44 residues (43 phosphates) had a retention time of 34 minutes under these
  • reagent M3 1-O-(4,4'-dimethoxytrityl)-1,2-dihydroxyethan -2-yl-1-(1,4-dicarboxy) butanoate-4-(1,2-dihydroxy-2- ⁇ 2-cyanoethyl-N,N- diisopropylphosphoramidite ⁇ ethan-1-yl ester.
  • Reagent M3 The structure of Reagent M3 is as follows:
  • Step 2 Preparation of 1-O-(4.4'-dimethoxytrityl)-1,2-dihydroxyethan- 2-yl-1-(1,4-dicarboxy) butanoate DMT-O-CH 2 CH 2 -O -C-CH 2 CH 2 -C-OH
  • step 1) The product from step 1) (9g, 24.7mmol) was dissolved in dry pyridine (100ml) and succinic anhydride (Aldrich, 2.72g, 27.2mmol) was added. When dissolution was complete, 4-(N,N-dimethylamino) pyridine (50mg) was added and the solution was stirred at room temperature overnight. The solvent was evaporated under reduced pressure and residual pyridine was removed by repeated co-evaporation with toluene. The residue was dissolved in dichloromethane (300ml) and this solution was washed with three equal volumes of ice-cold 10% citric acid and one volume of water.
  • succinic anhydride Aldrich, 2.72g, 27.2mmol
  • Step 3 Preparation of 1-O-(4,4'-dimethoxytrityl)-1,2-dihydroxyethan -2-yl -1-(1,4-dicarboxy)butanoate-4-(1,2-dihydroxy)ethan-1-yl ester.
  • step 2 The product from step 2) (8g, 17.24mmol) was dissolved in dry pyridine (200ml) containing 1,2-dihydroxyethane (6.2g, 100mmol). To this solution was added 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride (Aldrich, 3.75g, 19.5mmol). The solution was stirred at room temperature overnight when TLC in dichloromethane: methanol (19:1) showed there to be no starting material present. The solvent was removed under reduced pressure and residual pyridine was removed by repeated co-evaporation with toluene. The residue was redissolved in ethyl acetate and washed with three equal volumes of saturated sodium chloride solution and one volume of water.
  • 1-(3-dimethylaminopropyl)-3-ethyl carbodiimide hydrochloride Aldrich, 3.75g, 19.5mmol
  • step 3 The product from step 3) (2g, 3.9mmol) was dissolved in dry
  • dichloromethane 50ml and the solution was stirred under a stream of dry argon.
  • dry diisopropylethylamine 2.7ml, 16mmol
  • 2-cyanoethyl-N,N-diisopropylaminochloropho ⁇ phine (1.05ml, 4.72mmol).
  • the solution was stirred at room temperature under a stream of dry argon for 30 minutes when TLC in dichloromethane: methanol (19:1) showed there to be no starting material present.
  • the reaction was quenched by addition of dry methanol (5ml) and the solution was diluted with ethyl acetate (200ml).
  • oligodeoxyribonucleotides bound to a solid support except that reagent M3 was used, dissolved in anhydrous acetonitrile to a concentration of 0.15M, in place of reagent M2.
  • step (d) The two oligonucleotides found after step (d) were analysed as described in Example 5 and found to be identical by eleetrophore ⁇ is and HPLC to those described in Example 5, demonstrating scission of the cleavable link.
  • Reagent M4 The structure of Reagent M4 is as follows:
  • Step 1) Preparation of 1-0-(4,4'-dimethoxytrityl)-1,2-dihydroxyethan-2yl 2-(2-hydroxyethylsulfonyl)ethyl succinate.
  • 1,3-N,N-dicyclohexylcarbodiimide (383mg, 0.5meq) and the mixture was stirred at room temperature for 45 minutes.
  • Dicyclohexylurea was filtered off and washed with dichloromethane (4ml). The filtrate and washings were combined and evaporated under reduced pressure to a yellow oil which was redissolved in dry pyridine (15ml).
  • sulphonyldiethanol (1.54g, 2.7meq; prepared by toluene azeotropic dehydration of 65% aqueous material supplied by Aldrich) in dry pyridine (5ml).
  • the solution was stirred at room temperature for 23 hours and evaporated under reduced pressure. Residual pyridine was removed by repeated co-evaporation with toluene.
  • the residual oil was redissolved in dichloromethane: methanol (19:1) and applied to a silica column.
  • step 1) The product from step 1) (0.54g, 0.9mmol) was dissolved in dry
  • dichloromethane 50ml and the solution was stirred under a stream of dry argon.
  • dry diisopropylethylamine (0.61ml, 3.6mmol)
  • 2-cyanoethyl-N,N-diisopropylaminochlorophosphine (0.24ml, 1.08mmol).
  • the solution was stirred at room temperature under a stream of dry argon for 30 minutes when TLC in dichloromethane: methanol (19:1) showed there to be no starting material present.
  • the reaction was quenched by addition of dry methanol (5ml) and the solution was diluted with ethyl acetate (200ml).
  • Example 5 The method of Example 5 was repeated to synthesise two oligonucleotides bound to a solid support, except that reagent M4 was dissolved in anhydrous acetonitrile to a concentration of 0.15M and was used in place of reagent M2.
  • step (d) The two oligonucleotides found after step (d) were analysed as described in example 5 and found to be identical to those described in Example 5, demonstrating scission of the cleavable link.
  • reagent M5 The structure of reagent M5 is as follows:
  • Step 1) Preparation of 1,4-bis-(1-O-[4,4'-dimethoxytrityl]-1,2- dihydroxyethan-2-yl)-benzene-1,4-dicarboxylate.
  • Step 2 Preparation of 1-0-(4,4'-Dimethoxytrityl) -1,2-dihydroxyethan -2-yl -1-(1,4-dicarboxy)benzoate-4-(1.3-dihydroxy)ethan-1-yl ester.
  • step 1 The product from step 1 (2.7g, 3.5mmol) was dissolved in dry
  • step 2 The product from step 2 (0.3g, 0.54mmol) was dissolved in dry
  • Example 5 The method of Example 5 was- repeated to synthesise two
  • oligodeoxyribonucleotides bound to a solid support except that in place of reagent M2 there was used a 0.13M solution of reagent M5 in anhydrous acetonitrile.

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Abstract

On propose une méthode de synthèse d'une pluralité d'oligonucléotides dans laquelle on forme un oligonucléotide par des réactions séquentielles de précurseurs de nucléotides individuels, comportant les phases suivantes: a) formation d'un premier oligonucléotide; B) fixation sur ledit premier oligonucléotide d'une fraction de liaison clivable; c) formation d'un deuxième oligonucléotide sur la fraction de liaison clivable; et d) clivage éventuel du groupe de liaison pour obtenir les oligonucléotides voulus. L'invention concerne également des réactifs nucléosides et non nucléosides aptes à incorporer des fractions de liaison clivables pendant la synthèse automatisée d'oligonucléotides, dont le clivage produit des oligonucléotides ayant un groupe hydroxy ou phosphate en position 3' ou 5', ainsi que des supports solides aptes à être utilisés dans les synthétiseurs d'oligonucléotides automatisés.
PCT/GB1991/001687 1990-10-04 1991-10-01 Synthese d'oligonucleotides WO1992006103A1 (fr)

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WO1993020092A1 (fr) * 1992-04-03 1993-10-14 Zeneca Limited Synthese d'oligonucleotides
WO1993020091A1 (fr) * 1992-04-03 1993-10-14 Zeneca Limited Support pour synthese d'oligonucleotides
EP0552766A3 (en) * 1992-01-22 1994-09-07 Hoechst Ag Oligonucleotide analogues, their preparation and use
EP0552767A3 (en) * 1992-01-22 1994-09-07 Hoechst Ag 3'-derivatised oligonucleotide analogues with non-nucleotidic groups, their preparation and use
WO1997023497A1 (fr) * 1995-12-22 1997-07-03 University Technologies International Inc. Bras de liaison pour la synthese d'oligonucleotides sur un support solide et procede pour le former
EP0816368A1 (fr) * 1996-07-02 1998-01-07 Glen Research Corporation Phosphorylation d'oligonucléotides par voie chimique et réactifs utilisés
WO2000001711A1 (fr) * 1998-07-02 2000-01-13 University Technologies International Inc. Support solide reutilisable pour la synthese d'oligonucleotides
US6033909A (en) * 1992-01-22 2000-03-07 Hoechst Aktiengesellschaft Oligonucleotide analogs, their preparation and use
WO2000034299A1 (fr) * 1998-12-09 2000-06-15 Biochip Technologies Gmbh Clivage d'oligonucleotides/de polynucleotides synthetises chimiquement, au niveau d'un site predetermine
WO2002026756A3 (fr) * 2000-09-25 2002-08-22 Picoliter Inc Groupements d'oligonucleotides a hybridation partielle et preparation de ceux-ci a l'aide d'energie acoustique concentree
WO2002020537A3 (fr) * 2000-09-08 2002-09-06 Univ Technologies Int Phosphoramidites lieurs pour la synthese des oligonucleotides
US7098326B2 (en) 2002-01-23 2006-08-29 Sigma-Aldrich Co. Methods for the integrated synthesis and purification of oligonucleotides
US7427678B2 (en) 1998-01-08 2008-09-23 Sigma-Aldrich Co. Method for immobilizing oligonucleotides employing the cycloaddition bioconjugation method
US7615629B2 (en) 2002-12-31 2009-11-10 Sigma-Aldrich Co. Methods and compositions for the tandem synthesis of two or more oligonucleotides on the same solid support
EP2845587A3 (fr) * 2008-12-22 2015-07-29 Pola Chemical Industries Inc. Inhibiteur de production de mélanine
WO2019175126A1 (fr) 2018-03-14 2019-09-19 F. Hoffmann-La Roche Ag Dérivés d'acide lna-dicarboxylique et procédé de préparation correspondant
US11021503B2 (en) 2017-05-23 2021-06-01 Hoffmann-La Roche Inc. Process for GalNAc oligonucleotide conjugates

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EP0304215A2 (fr) * 1987-08-18 1989-02-22 Chiron Corporation Réactifs de phosphorylation et leur méthode d'utilisation
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Cited By (25)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0552766A3 (en) * 1992-01-22 1994-09-07 Hoechst Ag Oligonucleotide analogues, their preparation and use
EP0552767A3 (en) * 1992-01-22 1994-09-07 Hoechst Ag 3'-derivatised oligonucleotide analogues with non-nucleotidic groups, their preparation and use
EP1142902A3 (fr) * 1992-01-22 2001-10-17 Hoechst Aktiengesellschaft Analogues des oligonucléotides dérivées en position 3', avec des groupes non-nucléotidiques, leur préparation et leur utilisation
US5646261A (en) * 1992-01-22 1997-07-08 Hoechst Aktiengesellschaft 3'-derivatized oligonucleotide analogs with non-nucleotidic groupings, their preparation and use
US6033909A (en) * 1992-01-22 2000-03-07 Hoechst Aktiengesellschaft Oligonucleotide analogs, their preparation and use
WO1993020092A1 (fr) * 1992-04-03 1993-10-14 Zeneca Limited Synthese d'oligonucleotides
WO1993020091A1 (fr) * 1992-04-03 1993-10-14 Zeneca Limited Support pour synthese d'oligonucleotides
US5393877A (en) * 1992-04-03 1995-02-28 Zeneca Limited Linkers for the synthesis of multiple oligonucleotides in seriatim from a single support attachment
US5552535A (en) * 1992-04-03 1996-09-03 Zeneca Limited Multiple oligonucleotide containing oligomers and the cleanable linkers used in their preparation
AU674847B2 (en) * 1992-04-03 1997-01-16 Avecia Limited Synthesis of oligonucleotides
US6015895A (en) * 1995-12-22 2000-01-18 University Technologies International Inc. Linker arm for solid support oligonucleotide synthesis and process for production thereof
WO1997023497A1 (fr) * 1995-12-22 1997-07-03 University Technologies International Inc. Bras de liaison pour la synthese d'oligonucleotides sur un support solide et procede pour le former
US5959090A (en) * 1996-07-02 1999-09-28 Glen Research Corporation Chemical phosphorylation of oligonucleotides and reactants used therefor
EP0816368A1 (fr) * 1996-07-02 1998-01-07 Glen Research Corporation Phosphorylation d'oligonucléotides par voie chimique et réactifs utilisés
US7427678B2 (en) 1998-01-08 2008-09-23 Sigma-Aldrich Co. Method for immobilizing oligonucleotides employing the cycloaddition bioconjugation method
WO2000001711A1 (fr) * 1998-07-02 2000-01-13 University Technologies International Inc. Support solide reutilisable pour la synthese d'oligonucleotides
WO2000034299A1 (fr) * 1998-12-09 2000-06-15 Biochip Technologies Gmbh Clivage d'oligonucleotides/de polynucleotides synthetises chimiquement, au niveau d'un site predetermine
WO2002020537A3 (fr) * 2000-09-08 2002-09-06 Univ Technologies Int Phosphoramidites lieurs pour la synthese des oligonucleotides
US6806051B2 (en) 2000-09-25 2004-10-19 Picoliter Inc. Arrays of partially nonhybridizing oligonucleotides and preparation thereof using focused acoustic energy
WO2002026756A3 (fr) * 2000-09-25 2002-08-22 Picoliter Inc Groupements d'oligonucleotides a hybridation partielle et preparation de ceux-ci a l'aide d'energie acoustique concentree
US7098326B2 (en) 2002-01-23 2006-08-29 Sigma-Aldrich Co. Methods for the integrated synthesis and purification of oligonucleotides
US7615629B2 (en) 2002-12-31 2009-11-10 Sigma-Aldrich Co. Methods and compositions for the tandem synthesis of two or more oligonucleotides on the same solid support
EP2845587A3 (fr) * 2008-12-22 2015-07-29 Pola Chemical Industries Inc. Inhibiteur de production de mélanine
US11021503B2 (en) 2017-05-23 2021-06-01 Hoffmann-La Roche Inc. Process for GalNAc oligonucleotide conjugates
WO2019175126A1 (fr) 2018-03-14 2019-09-19 F. Hoffmann-La Roche Ag Dérivés d'acide lna-dicarboxylique et procédé de préparation correspondant

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AU8650991A (en) 1992-04-28
CA2093356A1 (fr) 1992-04-05
JPH06501692A (ja) 1994-02-24
GB9021625D0 (en) 1990-11-21
AU665174B2 (en) 1995-12-21

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