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WO2000001711A1 - Support solide reutilisable pour la synthese d'oligonucleotides - Google Patents

Support solide reutilisable pour la synthese d'oligonucleotides Download PDF

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Publication number
WO2000001711A1
WO2000001711A1 PCT/CA1999/000600 CA9900600W WO0001711A1 WO 2000001711 A1 WO2000001711 A1 WO 2000001711A1 CA 9900600 W CA9900600 W CA 9900600W WO 0001711 A1 WO0001711 A1 WO 0001711A1
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Prior art keywords
group
linker arm
unsubstituted
substituted
moiety
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PCT/CA1999/000600
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English (en)
Inventor
Richard T. Pon
Shuyuan Yu
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University Technologies International Inc
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University Technologies International Inc
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Priority claimed from CA 2242649 external-priority patent/CA2242649A1/fr
Application filed by University Technologies International Inc filed Critical University Technologies International Inc
Priority to JP2000558112A priority Critical patent/JP2002519433A/ja
Priority to EP99927625A priority patent/EP1091972A1/fr
Priority to AU44940/99A priority patent/AU4494099A/en
Priority to US09/720,907 priority patent/US7135564B1/en
Publication of WO2000001711A1 publication Critical patent/WO2000001711A1/fr
Anticipated expiration legal-status Critical
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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

Definitions

  • the present invention relates to a reusable solid support for oligonucleotide synthesis. In another of its aspects, the present invention relates to a process for production of such a reusable solid support. In yet another of its aspects, the present invention relates to a process for use of such a reusable solid support.
  • succinyl linker arm has the following general formula:
  • the succmyl group links the growing oligonucleotide from its terminal 3' hydroxyl group by an ester bond to a p ⁇ mary amme on the support, which may be, for example, conventional controlled pore glass (CPG) or silica, by an amide bond.
  • CPG controlled pore glass
  • the desired oligonucleotide is freed or cleaved from the succinyl linker arm hydrolyzmg the ester carbonyl group.
  • the hydrolysis agent is usually concentrated ammonium hydroxide Typically, this reaction can take from 1 -4 hours to complete. With improvements to current solid-phase oligonucleotide synthesizers, this cleavage step can represent 50% or more of the total time require to synthesize the desired oligonucleotide.
  • linker arm Another type of linker arm is disclosed in United States patent 5,112,962 [Letsmger et al. (Letsmger)], the contents of which are hereby incorporated by reference Letsmger teaches a linker arm for solid support synthesis of oligonucleotides and oligonucleotide de ⁇ vatives have the following formula
  • oxalyl linker arm which purportedly release the synthesized oligonucleotide or oligonucleotide de ⁇ vate in a pe ⁇ od of 1-30 minutes in a manner that leaves the oligonucleotide fully protected
  • the oxalyl linker arm purportedly can be rapidly cleaved by 5% ammonium hydroxide m methanol, ammonium hydroxide, wet tertiary amine, t ⁇ ethylamine/alcohol, t ⁇ ethylamme/methanol, t ⁇ ethylamine/ethanol, aqueous t ⁇ methylamme and other bases
  • the oxalyl linker arm of Letsmger suffers fiom its purported advantage
  • the present inventors have discovered that the oxalvl linker arm of Letsmger is susceptible to significant spontaneous hydrolysis (e g spontaneous hydrolysis of ⁇ 10-40° o per month) which
  • linker arm is not reusable after production and cleavage of the desired oligonucleotide
  • conventional linker arms may be regarded as non- recyclable
  • Figure 1 illustrates the conventional use of a succinyl linker arm for the production of an oligonucleotide
  • the support is irreversibly linked to the linker compound (I e . the succinyl moiety) and cannot be reused
  • linker arm for solid support oligonucleotide synthesis, which linker arm is recyclable. More specifically, the art is in need of a linker arm capable of repeated oligonucleotide synthesis/cleavage.
  • R 8 is selected from the group consisting of a substituted or unsubstituted C,-C 20 alkyl group, a substituted or unsubstituted C 5 - C 30 aryl group and a substituted or unsubstituted C 5 -C 40 alkylaryl group
  • X 3 and X 4 are the same or different and are selected from the group consisting of -0-, -S-, -S(O) 2 - and -N(R 12 )-
  • R 12 is selected from the group consisting of a substituted or unsubstituted C,-C 20 alkyl group, a substituted or unsubstituted C 5 -C 30 aryl group and a substituted or unsubstituted C 5 -C 40 alkylaryl group
  • Y is selected from the group consisting of:
  • each step of the de ⁇ vatization described m the previous paragraph has the potential of incompletely de ⁇ vatizing each HX 4 - moiety on the support thereby increasing the likelihood of a heterogeneous surface Practically, it becomes necessary to block or cap unde ⁇ vatized HX 4 - moieties so that the linker moiety does interact with them Thus, the disadvantage is additional labour and cost required to effect de ⁇ vatization of the solid support
  • a linker arm based on the de ⁇ vatized support desc ⁇ bed by Pon et al is not as resistant to partial cleavage du ⁇ ng regeneration as a de ⁇ vatized suppo ⁇ having a more fully saturated moiety
  • It is an object of the present invention provide a novel linker arm for solid support oligonucleotide synthesis which obviates or mitigates at least one of the above-mentioned disadvantages of the prior art.
  • the present invention provides a reusable linker arm for solid support oligonucleotide synthesis, the linker arm comprising the following formula:
  • Z is a linker moiety and T is an organic radical.
  • the present invention provides a reusable linker arm for solid support oligonucleotide synthesis, the linker arm comprising the following formula:
  • the present invention provides a process for production of a reusable linker arm for oligonucleotide synthesis having the following formula:
  • the present invention provides a process for production of a reusable linker arm for oligonucleotide synthesis having the following formula:
  • the present invention provides a process for producing an oligonucleotide having a desired sequence comprising the steps of: (i) reacting a linker arm having the formula:
  • Z is a linker moiety and T is an organic radical, with at least one oligonucleoside base until an oligonucleotide having the desired sequence is produce;
  • oligonucleotide is intended to have a broad meaning and encompasses conventional oligonucleotides, backbone-modified oligonucleotides (e.g. phosphorothioate, phosphorodithioate and methyl-phophonate analogs useful as oligotherapeutic agents) and oligonucleotide derivatives such as oligonucleotide-peptide conjugates.
  • backbone-modified oligonucleotides e.g. phosphorothioate, phosphorodithioate and methyl-phophonate analogs useful as oligotherapeutic agents
  • oligonucleotide derivatives such as oligonucleotide-peptide conjugates.
  • Figure 1 illustrates a specific process pathway for conventional oligonucleotide synthesis
  • Figures 2 and 3 illustrate specific preferred embodiments of the present invention. BEST MODE FOR CARRYING OUT THE INVENTION
  • Figure 1 illustrates a conventional process for solid support oligonucleotide synthesis
  • a linking compound such as succmic acid (while succmic acid is illustrated, succmic anhyd ⁇ de may also be used)
  • succmic anhyd ⁇ de may also be used
  • the reaction results in the formation of an amide linkage between the linking compound and the support to produce succmyl-support conjugate
  • the succmyl-support conjugate is reacted with a desired initial nucleoside to produce a linker arm
  • DMT is dimethyoxyt ⁇ tyl
  • B is the nucleobase
  • R' is H (for deoxy ⁇ bonucleosides) or OR (for ⁇ bonucleosides) wherein R is H or a conventional blocking/ protecting group
  • the reaction results in the formation of an ester linkage between the linking compound and the desired initial nucleoside at the 3' position of the latter
  • the linker arm is then used m conventional oligonucleotide synthesis (e g in a conventional automated synthesizer) to produce an oligonucleotide of desired sequence attached to the linker arm
  • the oligonucleotide is then cleaved from the linker by hydrolysis This serves to cleave the ester bond thereby freeing the oligonucleotide and an amine- termmated, non-reusable linker arm
  • a support having a hydroxy-termmated functionality may be combined with a conventional linking compound to produce linker arm which may used to synthesize an oligonucleotide of desired sequence
  • linker arm may be regenerated or recycled after cleavage of the oligonucleotide of desired sequence
  • the reusable linker arm of the present invention has the following formula- Z— 0— T [SUPPORT]
  • Z is a linker moiety and T is an organic radical.
  • T contains at least one carbon.
  • T is a C,-C 300 organic moiety, more preferably a C,-C 200 organic moiety, most preferably a C C ⁇ organic moiety.
  • T may be a saturated or unsaturated organic moiety.
  • T may contain one or more heteroatoms.
  • T may comprise at least one heteroatom selected from N and O.
  • the organic moiety in T comprises at least one moiety having the formula:
  • the organic moiety in T comprises at least one moiety having the formula:
  • the organic moiety in T comprises at least one moiety having the formula: ⁇ i l ⁇
  • the organic moiety m T comp ⁇ ses at least one moiety having the formula
  • the organic moiety in T comp ⁇ ses at least one moiety having the formula
  • T may be unsubstituted or substituted
  • the organic moiety of T may be substituted by at least one moiety selected from the group comp ⁇ smg a C ] -C 40 alkyl group, a C 5 -C 40 aryl group, a C,-C 40 alkoxy group, a C,-C 40 ester group, a C,-C 40 hydroxy group, a C 2 -C 40 acrylate group and a C,-C 40 alkylaryl group
  • T has the formula
  • T has the formula:
  • a is 0 or 1
  • Q is an organic moiety
  • R 8 is hydrogen or a protecting group
  • b is an integer having a value of 0-40.
  • a may be 0 and R 8 may be hydrogen.
  • a may be 1 and R 8 may be a protecting group.
  • Non-limiting examples of protecting groups may be selected from the group comprising acetyl, chloroacetyl, methoxyacetyl, t-butyl phenoxyacetyl, phenoxyacetyl, trityl, methoxytrityl, dimethoxytrityl (DMT), dialkylphosphite, pivalyl-isobutyloxycarbonyl, f-butyldimethylsilyl, phenoxyacetal, 9- phenylxanthen-9-yl (pixyl), tetrahydropyranyl, methoxytetrahydropyranyl, methoxymethyl, benzyloxymethyl, methoxyethoxymethyl, methylthiomethyl, dialkylphosphate, levulinyl, dimethylphenylsilyl, trimethylsilyl, isopropyl- dimethylsilyl, diisopropylmethylsilyl, diethylisopropyls
  • Q may be a moiety having the formula: - io-
  • q, r, s, t and u are the same or different and each is an integer having a value of 0-40 and R a is selected from the group comprising hydrogen, hydroxyl, a C,-C 40 alkyl group, a C 5 -C 40 aryl group, a C 1 -C 40 alkoxy group, a C,-C 40 ester group, a C,-C 40 hydroxy group, a C 2 -C 40 acrylate group and a C 5 -C 40 alkylaryl group.
  • s is 0, q, r and u are the same or different and each is an integer having a value of 1-10, t is an integer of 1-5 and R is hydroxyl.
  • T has the formula:
  • R 8 is selected from the group comprising hydrogen, hydroxyl, a C,-C 40 alkyl group, a C 5 -C 40 aryl group, a C,-
  • C 40 alkoxy group a C [ -C 40 ester group,a C,-C 40 hydroxy group, a C 2 -C 40 acrylate group and a C 5 -C 40 alkylaryl group
  • b is an integer having a value of 0-40.
  • Q is a C,-C 100 organic moiety.
  • Q may be a saturated organic moiety or an unsaturated organic moiety. It is preferreed that Q is a C,-C 100 organic moiety comprising at least one heteroatom selected from N and O.
  • the organic moiety Q comprises at least one moiety having the formula: O
  • the organic moiety Q comprises at least one moiety having the formula:
  • organic moiety Q comp ⁇ ses at least one moiety having the formula:
  • organic moiety Q comp ⁇ ses at least one moiety having the formula:
  • organic moiety comp ⁇ ses at least one moiety having the formula:
  • the organic moiety Q may unsubstituted or substituted.
  • the organic moiety Q may be substituted by at least one moiety selected from the group comprising a C,-C 40 alkyl group, a C 5 -C 40 aryl group, a C,-C 40 alkoxy group, a C ⁇ -C 40 ester group, a C,- C 40 hydroxy group, a C 2 -C 40 acrylate group and a C 5 -C 40 alkylaryl group.
  • Q has the formula:
  • each of x, y and z is an integer having a value of 1-40.
  • Z is a linker moiety.
  • Z is derived from a linker compound have the general formula HO-Z-OH (Formula I below).
  • the nature of the linker compound is not particularly restricted.
  • linker moiety Z has the formula:
  • this linker moiety may be derived from succinic acid or succinic anhydride.
  • linker moiety Z has the following formula:
  • this linker moiety may be derived from diglycolic acid or diglycolic anhydride.
  • linker moiety Z has the following formula:
  • this linker moiety may be derived from oxalic acid or oxalyl chloride.
  • linker moiety Z has the following formula:
  • R 1 , R 2 and R 3 are the same or different and are selected from the group consisting of hydrogen, halide, a substituted or unsubstituted C,-C 20 alkyl group, a substituted or unsubstituted C 5 -C 30 aryl group and a substituted or unsubstituted C 5 -C 40 alkylaryl group;
  • R 4 and R 5 are the same or different and are selected from the group consisting of hydrogen, a substituted or unsubstituted C,-C 20 alkyl group, a substituted or unsubstituted C 5 -C 30 aryl group and a substituted or unsubstituted C 5 -C 40 alkylaryl group;
  • X 1 is selected from the group consisting of -O-, -C(O)-, -S-, -S(O) 2 - and -N(R)-;
  • R is selected hydrogen, a substituted or unsubstituted C,-
  • X 2 is selected from the group consisting of -O-, -S-, -C(O)-, -S(O) 2 - and -N(R)-
  • R is selected from the group comprising hydrogen, a substituted or unsubstituted C,-C 20 alkyl group, a substituted or unsubstituted C 5 - C 30 aryl group and a substituted or unsubstituted C 5 -C 40 alkylaryl group
  • R 6 and R 7 are the same or different and are selected from the group consisting of hydrogen, a substituted or unsubstituted C ⁇ -C 20 alkyl group, a substituted or unsubstituted C 5 -C 30 aryl group and a substituted or unsubstituted C 5 -C 40 alkylaryl group
  • m is 0, 1 or 2.
  • B 1 preferably is selected from the group consisting of hydrogen, halide, a substituted or unsubstituted C,-C 20 alkyl group, a substituted or unsubstituted C 5 -C 30 aryl group and a substituted or unsubstituted C 5 -C 40 alkylaryl group.
  • at least one, more preferably each, of R, R 4 , R ⁇ R 6 and R 7 is hydrogen and preferably at least, more preferably both, of m and n are 1.
  • each of R 1 , R 2 and R 3 is hydrogen and that X 1 and X 2 are both -O-.
  • the most prefe ⁇ ed form of linker moiety Z is derived from hydroquinone-O,O'-diacetic acid.
  • linker moiety Z has the following formula:
  • R ⁇ R b and R' are the same or different and are selected from the group consisting of hydrogen, a substituted or unsubstituted C,-C 20 alkyl group, a substituted or unsubstituted C 5 -C 30 aryl group and a substituted or unsubstituted C 5 -C 40 alkylaryl group
  • Y is selected from the group consisting of O, S, SO 2 and O-((CH 2 ),-O) q , 1 is an integer less than or equal to 60, q is an integer in the range of 1 - 1000, n and m are the same or different and are 1 or 2, with the proviso that, when Y is O, at least one of n and m is 2.
  • 1 is an integer in the range of 1 - 10
  • q is an integer in the range of 1 - 1000.
  • the SUPPORT in the above formula is a conventional solid support.
  • the nature of the solid support is not particularly restricted and is within the purview of a person skilled in the art.
  • the solid support may be an inorganic substance.
  • suitable inorganic substances may be selected from the group consisting of silica, porous glass, aluminosilicates, borosilicates, metal oxides (e.g. aluminum oxide, iron oxide, nickel oxide) and clay containing one or more of these.
  • the solid support may be an organic substance such as a cross-linked polymer.
  • Non-limiting examples of a suitable cross-linked polymer may be selected from the group consisting of polyamide, polyether, polystyrene and mixtures thereof
  • the prefe ⁇ ed solid support for use herein is conventional and may be selected from controlled pore glass bead or polystyrene beads. Further, the support may be either in particle form (e.g., beads), three-dimensional slabs (e.g., polymeric inserts and foams) or in a flat two-dimensional like format (e.g., plastic sheets, glass chips, silicon wafers, etc.).
  • the material used for the support may also be soluble in certain solvents (e.g., liquid-phase supports), but can be precipitated or crystallized from other solvents.
  • linker arm (again, Z is a linker moiety and T is an organic radical), may then be reacted with a conventional nucleoside-linker compound to produce another linker arm according to the present invention.
  • This other linker arm has the following formula:
  • NUCLEOSIDE is a moiety selected from one of the following formulae:
  • R 6 and R 10 are the same or different and are hydrogen or a protecting group
  • R is hydrogen (for deoxy ⁇ bonucleosides or DNA) or -OR 11 (for ⁇ bonucleosides or RNA) wherein R 11 is hydrogen or a protecting group
  • the linker can be attached to either the 5'-, 3'- or (if ribose) 2'- hvdroxyl positions Indeed, for RNA sequences, it makes little difference whether the ester linker formed between the nucleoside and the linker compound is at the 2'- or 3'- hydroxyl position of the nucleoside
  • the nucleoside may be protected or blocked at the va ⁇ ous of its hydroxyl moieties
  • Non- mitmg examples of useful protecting groups may be selected from the group consisting of acetyl, chloioacetyl, methoxyacetyl, t-butyl phenoxyacetyl, phenoxyacetyl, t ⁇ tyl, methoxyt ⁇ tyl, dimethoxyt ⁇ tyl (DMT), dialkylphosphite, pivalyl-isobutyloxvcarbonyl, r-butyldimethvlsilyl, phenoxyacetal, 9-phenylxanthen-9-yl (pixyl), tetrahydropyranyl, methoxvtetrahydropyranyl, methoxymethyl, benzyloxymethyl, methoxyethoxymethyl, methylthiomethyl, dialkylphosphate, levulmyl, dimethylphenylsilyl, t ⁇ methylsilyl, isopropyldimethylsilyl, diisopropyl
  • the prefe ⁇ ed protecting group for desired 5'-hydroxyl position(s) is the acid labile dimethoxytrityl group.
  • the prefe ⁇ ed protecting groups for these positions are trialkylsilyl (i.e. t-butyldimethylsilyl) or acetyl. Additional information may be obtained from the following references:
  • a prefe ⁇ ed method for production of deoxy ⁇ bonucleosides in the context of the present invention is to use a nucleoside with a 5'-d ⁇ methoxytntyl protecting group and an appropnate exocychc amino protecting group, e g , N 6 -benzoyl-5'- d ⁇ methoxyt ⁇ tyl-2'-deoxyadenos ⁇ ne, N 4 -benzoyl-5'-d ⁇ methoxyt ⁇ tyl-2'- deoxycytidme, 5'-d ⁇ methoxytntyl-N 2 - ⁇ sobutyryl-2'-deoxyguanosme, or 5'- dimethoxyt ⁇ tylthymidine
  • a prefe ⁇ ed method for production of ⁇ bonucleosides in the context of the present invention is to use a 5'-d ⁇ methoxyt ⁇ tyl protected nucleoside, with appropnate exocychc ammo protection, and no protecting groups on either of the 2'- or 3'- hydroxyl positions
  • the linker can then react with either one of the two adjacent hydroxyl groups (it doesn't matter which) to give a mixture of 2'- and 3'- hnkages
  • the unreacted hydroxyl groups may then be acetylated by treatment of the immobilized nucleoside with acetic anhyd ⁇ de
  • ⁇ bonucleosides which have a 5'-d ⁇ methoxyt ⁇ tyl group, appropnate exocychc amino group protection, and either a 3'-hydroxyl protecting group or a mixture of 2'- and 3'- protecting groups can be used
  • the 3'-protected compounds are generally unwanted
  • Z— O— T [SUPPORT] may be produced by a process comprising the step of reacting together the compound of Formulae I and II:
  • the reusable linker arm having the formula:
  • activating group is intended to have a broad meaning and is intended to encompass electrophilic reagents capable of activating a carboxyl moiety (e.g., on the linking compound of Formula II) by attachment of a leaving group to the acyl carbon of the carboxl moiety - see, for example, M. Bodanszky, "Principles of Peptide Synthesis", Second Edition, Springer- Verlag, Berlin (1993), the contents of which are hereby incorporated by reference.
  • the activating agent should be capable of initiating at least one of the following: (a) formation of a reactive acylating agent (this is an example of a derivate) from the carboxyl moeiy in a separate step or steps, followed by immediate treatment with the amino component (in this case, for example, an amino-terminated support) to form an amide linkage or a hydroxy component (in this case a hydroxy-terminated support or a hydroxyl group on the desired nucleoside) to form an ester linkage; (b) formation of an isolable acylating agent, separately, optionally with purification prior to treatment with the amino or hydroxy component as discussed in (a); and (c) formation of an acylating intermediate in the presence of the amino/ hydroxy component, by the addition of an activating agent to a mixture of the two components.
  • a reactive acylating agent this is an example of a derivate
  • the amino component in this case, for example, an amino-terminated support
  • a hydroxy component
  • the Letsinger method which first reacts oxalyl chloride with triazole, and then adds a nucleoside to the resulting oxalyl triazolide is an example of route (a).
  • Conversion of the carboxylic acid group into an "active" ester using either p-nitrophenol, or di-, tri-, tetra-, or penta- chlorinated or fluorinated phenols, or N-hydrosuccinimide are common examples of route (b).
  • the reaction of the compounds of Formulae I, II and III is conducted in the presence of a nucleophilic catalyst or additive (typically 4-dimethylamino pyridine (DMAP), 1-hydroxybenzotriazole (HOBt), or l-hydroxy-7-azabenzotriazole (HOAt)) to speed up the reaction and a tertiary amme base (typically tnethylamme, py ⁇ dine, or dusopropylethylamme) to ionize the carboxylic acid group
  • a nucleophilic catalyst or additive typically 4-dimethylamino pyridine (DMAP), 1-hydroxybenzotriazole (HOBt), or l-hydroxy-7-azabenzotriazole (HOAt)
  • DMAP 4-dimethylamino pyridine
  • HABt 1-hydroxybenzotriazole
  • HOAt l-hydroxy-7-azabenzotriazole
  • the precise nature of the activation agent is not particularly restncted provided, of course, that the activated carboxylic acid group is capable of initiating formation of the ester or amide linkage, as appropriate, and the activating reagent does not have any delete ⁇ ous effect on the desired nucleoside
  • an active ester I e , nitrophenyl, mtrophenylthio, tnchlorophenyl, t ⁇ fluorophenvl, pentachlorophenyl, pentafluorophenyl, or 3-hydroxv-2,3- d ⁇ hydro-4-oxo-benzot ⁇ azme esters
  • an active hydroxylamine ester (1 e , N- hydroxyphthahmide or N-hydroxysuccimmide
  • acid anhydride or mixed anhyd ⁇ de
  • Non-limiting examples of actuating agents may be selected from the group consisting of arylsulfonyl chlo ⁇ des (e g , benzenesulfonyl chlo ⁇ de (BS- Cl), mesitvlenesulfonyl chlo ⁇ de (MS-C1), t ⁇ isopropylsulfonylchlo ⁇ de (TPS-C1)), active arylsulfonyl esters (I e , lmidazole, tnazole, nitrot ⁇ azole, or tetrazole esters ofBS-Cl.
  • arylsulfonyl chlo ⁇ des e g , benzenesulfonyl chlo ⁇ de (BS- Cl)
  • MS-C1 mesitvlenesulfonyl chlo ⁇ de
  • TPS-C1 t ⁇ isopropylsulfonylchlo ⁇ de
  • the order of reaction is not particularly rest ⁇ cted
  • the compounds of Formulae I and III are initially reacted to form a conjugate which is reacted with the compound of Formula II
  • the compounds of Formulae I and II are initially reacted to form a conjugate which is reacted with the compound of Formula III
  • the capping reagent should be reversible so that the capping agent can be removed to regenerate the hydroxyl sites p ⁇ or to the next round of support de ⁇ vatization
  • Capping of the unreacted sites is conventional and can be performed by reaction with an activated carboxylic acid or anhyd ⁇ de to form an ester, or by addition of a protecting group, as desc ⁇ bed hereinabove
  • N-methyhmidazole in THF solution are useful examples of capping reagents
  • DMT dimethoxyt ⁇ tyl
  • B refers to a nucleobase as desc ⁇ bed hereinabove
  • the support is recycled after oligonucleotide cleavage and support regeneration to a point in the reaction scheme where it may again be coupled with the HQPD-nucleoside conjugate for further oligonucleotide synthesis.
  • Step #3 With further reference to "Oligo Synthesis” (Step #3) in Figure 2, once the present linker arm has been produced, it may be used in the conventional manner to synthesize an oligonucleotide - see, for example, United States patent 5,112,962 (Letsinger), incorporated by reference hereinabove. Once the oligonucleotide has been synthesized, it may be cleaved from the solid support to yield the free oligonucleotide and the support may then be regenerated - see Step #4 of Figure 2. The cleavage step comprises hydrolysis at the point of attachment of the initial nucleoside to the linking compound.
  • the regeneration of the support involves the removal of two moieties: (i) the removal of the structure represented by Formula I (above) from Formula II (above), which occurs simultaneously with the release of the oligonucleotide product, and (ii) the removal of the moiety used to protect (cap) unreacted hydroxyl sites of Formula II (above) on the support. Removal of these two moieties can occur simultaneously or separately to regenerate the support. Simultaneous removal of both moieties using only a single reagent is simpler but care should be taken to use reagents which will not deleteriously affect the oligonucleotide product.
  • a two-step regeneration involving the removal of the oligonucleotide using one reagent (typically ammonium hydroxide) and then treatment of the support with a second reagent (which may be faster but otherwise damaging to the oligonucleotide product thereby necessitating use of a two-step regeneration) allows flexibility in the choice of capping and regeneration reagents.
  • the reagent used to effect cleavage is not particularly restricted and is within the purview of a person skilled in the art.
  • the reagent is a base mild enough not to damage the oligonucleotide product but sufficiently strong to effect rapid cleavage.
  • Non-limiting examples of suitable reagents for this purpose may be selected from the group consisting of ammonium hydroxide, ammonium hydroxide/methanol, ammonia/methanol, ammonium hydroxide/methylamine, potassium carbonate/methanol, t-butylamine, ethylenediamine, mefhylamine, dimethylamine, trimethylamine/water and the like. Cleavage may also be performed under neutral conditions using fluoride ion (i.e. 1M tetrabutylammonium fluoride/THF or triethylamine trihydro fluoride).
  • the reagent used to remove the capping reagent from unreacted sites may consist of the above reagents or other stronger bases such as sodium or potassium hydroxide.
  • ammonium hydroxide can be used to cleave the oligonucleotide product from the support, remove the HQPD linker arm, and cleave chloroacetyl protected hydroxyl groups in a single regeneration step.
  • the prefe ⁇ ed temperature for the cleavage and regeneration is room temperature, but higher or lower temperatures can be employed, subject to the limitations of the apparatus used.
  • LCAA Long chain alkylamine
  • Gly glycerol
  • CPG controlled pore glass
  • Ammonium hydroxide solutions 28-30% and solvents were obtained from VWR Canlab (Edmonton, Alberta, Canada);
  • Capping solutions were formulated as either Cap A (acetic anhydride/2, 6-lutidine/THF in a volume ratio of 1:1:8) and Cap B (N-methylimidazole and THF in a volume ratio of 16:84) or Cap A (chloroacetic anhydride and THF, 17% by weight) and Cap B (2, 6-lutidine and N-methylimidazole in THF in a volume ratio of 12:16:72); 7. Anhydrous pyridine and acetonitrile, distilled from CaH 2 ;
  • DMAP 4-dimethylaminopyridine, reagent grade
  • DEC l-(3-dimethylaminopropyl)-ethylcarbodiimide, reagent grade
  • nucleoside (loading) on the insoluble supports was determined by spectrophotometric trityl analysis. In this procedure, a sample of support (4-5 mg) was accurately weighed directly into a 10 mL volumetric flask. A solution of dichloroacetic acid in 1 ,2-dichloroethane in a volume ration of 5:95 was then added to fill the flask. The contents were then thoroughly mixed and the absorbance of the orange coloured solution was measured at 503 nm using a Philips UV/Vis spectrophotometer. The nucleoside loading (in ⁇ mol/g of CPG) was then calculated as:
  • a 503 absorbance at 503 nm
  • Vol solution volume in mL
  • Wt amount of CPG tested in mg. The accuracy of the trityl determination was approximately ⁇ 2-3%.
  • Example 1 SYNTHESIS OF NUCLEOSIDE-3'-0-HODA HEMIESTERS 5'-Dimethoxytrityl-N-protected deoxyribonucleoside (10 mmol), hydroquinone-O, O'-diacetic acid (15 mmol, 3.39 g), 4-dimethylaminopyridine ( 1 mmol, 122 mg), and l-(3-dimethylaminopropyl)-3-ethylcarbodiimide hydrochloride ( 15 mmol, 2.88 g) were combined in a 100 mL round bottom flask equipped with a magnetic stir bar. Triethylamine (0.8 mL) and anhydrous pyridine (50 mL) were added to the flask and the contents were sti ⁇ ed at room temperature overnight.
  • Triethylamine 0.8 mL
  • anhydrous pyridine 50 mL
  • the reaction was checked by TLC (5% methanol/chloroform). If more than a trace of starting nucleoside was visible, more l-(3-dimethylaminopropyl)- 3-ethylcarbodiimide hydrochloride (2-5 mmol) was added to the reaction and stirring was continued for another day. When TLC showed complete disappearance of the starting nucleoside, the solution was concentrated by evaporation until a thick oil was formed. The oil was redissolved in chloroform ( ⁇ 200 mL) and transfer to a separatory funnel. The chloroform solution was washed with aqueous sodium bicarbonate ( ⁇ 100 mL x 2) and then water ( ⁇ 100 mL x 3).
  • the funnel was slowly inverted to mix the two phases.
  • the chloroform phase was collected and the aqueous phase was discarded. If an inseparable emulsion was formed, then either centrifugation (for small volumes) or (for large volumes) precipitation by addition of hexanes followed by filtration and redissolving the sticky precipitate back into chloroform can be performed.
  • the chloroform solution was added to anhydrous magnesium sulfate and mixed to remove residual moisture from the solution.
  • the magnesium sulfate was filtered off, the filtrated was washed with a small amount of chloroform and then the chloroform solution was evaporated to dryness.
  • the hemiester sodium salt was converted into a more soluble pyridinium salt by dissolving the foam in pyridine ( ⁇ 50-100 mL) and then adding AG 50W- X4 W cation exchange resin (2 eq.). The mixture was sti ⁇ ed for approximately 5 minutes and then the ion exchange resin was filtered off. The pyridine solution was evaporated to dryness. A light brown foam formed and solidified. The sold was dried under vacuum overnight to remove excess pyridine.
  • This example describes the synthesis of a C 12 linker arm within the scope of the present invention and how it can be used to convert commercially available amino-derivatized supports into reusable hydroxyl-derivatized supports.
  • Example 3 - DERIVATIZATION OF TOYOPEARL HW-65F SUPPORT WITH 1.4-BUTANEDIOL DIGLYCIDYL ETHER This Example describes how hydroxyl surface groups on commercially available Toyopearl HW65 supports are extended with a butane diglycidyl linker to create a reusable support.
  • Toyopearl HW-65F vinyl alcohol/methacrylic acid copolymer was obtained as a slurry in 500 ml 20% ethanol/water. This slurry was evaporated to dryness to yield of 90 g of dry support. The hydroxyl content of the dry support was determined, in triplicate, by derivatization with dimethoxytrityl chloride/tetrabutylammonium perchlorate and trityl analysis, to be 1 ,095 ⁇ mol/g.
  • the epoxide loading was estimated to be 193 ⁇ mol/g.
  • the epoxide denvatized support 25 g
  • benzoic anhyd ⁇ de 51 g
  • 4- dimethylammopyndine 6.6 g
  • anhydrous pyndine 180 mL
  • the support was filtered off, washed (methanol, then chloroform), and dned.
  • Ports #1-4 dA Bz , dG lBu , dC Bz , and T phosphoramidites (0.2 M solutions).
  • Port #10 28% Ammonium hydroxide.
  • Port #11 1 M Chloroacetic anhydride in THF (Cap A reagent).
  • Port #12 1 M 2,6-Lutidine and 2 M N-methylimidazole in THF (Cap B reagent).
  • the synthesizer was then programmed to automatically execute the following steps:
  • a "Begin" procedure consisting of a column wash, nucleoside coupling to the support by simultaneous addition (4.0 sec) of nucleoside hemiester (port #7) and coupling reagent (port #8) and a 600 sec wait, column wash, capping of unreacted hydroxyl sites (Cap A + B reagents, 300 sec), column wash, and priming of ports #1 , 2, 3, 4, and 9.
  • the columns were removed from the synthesizer, manually treated with 0.05 M potassium carbonate/methanol solution (5 min), rinsed with methanol, dried by aspiration (5 min), re-installed on the synthesizer, and rinsed with anhydrous acetonitrile.
  • the automated synthesis was then repeated (i.e., Steps 1, 2, and 3 above) using the same synthesis column a total of twelve times.
  • the automated DNA synthesizer was set-up with reagents, as described in Example 4, with the exception of the Cap A and B reagents, which were as follows:
  • Port #12 1 M N-Methylimidazole in acetonitrile (Cap B).
  • the amount of trityl color released after the first detritylation step was collected and quantitated to determine the amount of nucleoside added to the support - the results are reported in Table 6.
  • the released oligonucleotide solution was deprotected (55°C, 16 h), evaporated to removed ammonia, and quantitated by UV at 260 nm - the results are reported in Table 7.
  • the composition of the products obtained in Table 7 was examined by gel electrophoresis and the expected products were obtained in each case. This indicated that methoxyacetic anhydride could also be used as a satisfactory capping reagent during the support recycling.

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Abstract

L'invention concerne un bras de liaison réutilisable pour la synthèse d'oligonucléotides sur support solide, ledit bras étant représenté par la formule (a), dans laquelle Z est un fragment de liaison et T un radical organique. Un procédé d'addition d'un ou plusieurs nucléosides sur le bras de liaison est également décrit.
PCT/CA1999/000600 1998-07-02 1999-06-30 Support solide reutilisable pour la synthese d'oligonucleotides Ceased WO2000001711A1 (fr)

Priority Applications (4)

Application Number Priority Date Filing Date Title
JP2000558112A JP2002519433A (ja) 1998-07-02 1999-06-30 オリゴヌクレオチド合成用の再使用可能な固相支持体
EP99927625A EP1091972A1 (fr) 1998-07-02 1999-06-30 Support solide reutilisable pour la synthese d'oligonucleotides
AU44940/99A AU4494099A (en) 1998-07-02 1999-06-30 Reusable solid support for oligonucleotide synthesis
US09/720,907 US7135564B1 (en) 1998-07-02 1999-06-30 Reusable solid support for oligonucleotide synthesis

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US9168398P 1998-07-02 1998-07-02
CA 2242649 CA2242649A1 (fr) 1998-07-02 1998-07-02 Support solide reutilisable pour synthese oligonucleotide, processus de production du support et processus d'utilisation connexe
US60/091,683 1998-07-02
CA2,242,649 1998-07-02

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WO2000001711A1 true WO2000001711A1 (fr) 2000-01-13

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1347297A4 (fr) * 2000-12-28 2007-06-06 Toyo Kohan Co Ltd Proc d de r utilisation d'un substrat d'immobilisation de l'adn

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992006103A1 (fr) * 1990-10-04 1992-04-16 Imperial Chemical Industries Plc Synthese d'oligonucleotides
WO1993007883A1 (fr) * 1991-10-24 1993-04-29 Isis Pharmaceuticals, Inc. Oligonucleotides derives presentant diverses qualites dont une meilleure facilite d'absorption
US5624711A (en) * 1995-04-27 1997-04-29 Affymax Technologies, N.V. Derivatization of solid supports and methods for oligomer synthesis
WO1997023496A1 (fr) * 1995-12-22 1997-07-03 University Technologies International Inc. Support solide reutilisable, destine a la synthese d'oligonucleotides, son procede de fabrication et son utilisation

Patent Citations (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO1992006103A1 (fr) * 1990-10-04 1992-04-16 Imperial Chemical Industries Plc Synthese d'oligonucleotides
WO1993007883A1 (fr) * 1991-10-24 1993-04-29 Isis Pharmaceuticals, Inc. Oligonucleotides derives presentant diverses qualites dont une meilleure facilite d'absorption
US5624711A (en) * 1995-04-27 1997-04-29 Affymax Technologies, N.V. Derivatization of solid supports and methods for oligomer synthesis
WO1997023496A1 (fr) * 1995-12-22 1997-07-03 University Technologies International Inc. Support solide reutilisable, destine a la synthese d'oligonucleotides, son procede de fabrication et son utilisation
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

Non-Patent Citations (3)

* Cited by examiner, † Cited by third party
Title
JAMES I W: "Linkers for Solid Phase Organic Synthesis", TETRAHEDRON, vol. 55, no. 16, 16 April 1999 (1999-04-16), pages 4855-4946, XP004161079, ISSN: 0040-4020 *
PON R T ET AL: "Hydroquinone-O,O@?-Diacetic Acid As A More Labile Replacement For Succinic Acid Linkers in Solid-Phase Oligonucleotide Synthesis", TETRAHEDRON LETTERS, vol. 38, no. 19, 12 May 1997 (1997-05-12), pages 3327-3330, XP004061417, ISSN: 0040-4039 *
PON R T ET AL: "Rapid Automated Derivatization of Solid-Phase Supports For Oligonucleotide Synthesis Using Uronium or Phosphonium Coupling Reagents", TETRAHEDRON LETTERS, vol. 38, no. 19, 12 May 1997 (1997-05-12), pages 3331-3334, XP004061418, ISSN: 0040-4039 *

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP1347297A4 (fr) * 2000-12-28 2007-06-06 Toyo Kohan Co Ltd Proc d de r utilisation d'un substrat d'immobilisation de l'adn

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