EP0547142A1 - Synthese sur support solide d'oligonucleotides places en queue en position 3' par l'intermediaire d'une molecule de liaison - Google Patents

Synthese sur support solide d'oligonucleotides places en queue en position 3' par l'intermediaire d'une molecule de liaison

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Publication number
EP0547142A1
EP0547142A1 EP91916634A EP91916634A EP0547142A1 EP 0547142 A1 EP0547142 A1 EP 0547142A1 EP 91916634 A EP91916634 A EP 91916634A EP 91916634 A EP91916634 A EP 91916634A EP 0547142 A1 EP0547142 A1 EP 0547142A1
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EP
European Patent Office
Prior art keywords
oligonucleotide
molecule
tail
linking molecule
terminus
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Withdrawn
Application number
EP91916634A
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German (de)
English (en)
Other versions
EP0547142A4 (fr
Inventor
Michael W. Reed
Rich B. Meyer, Jr.
Charles R. Petrie
John C. Tabone
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Nanogen Inc
Original Assignee
MicroProbe Corp
Epoch Pharmaceuticals Inc
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Application filed by MicroProbe Corp, Epoch Pharmaceuticals Inc filed Critical MicroProbe Corp
Publication of EP0547142A1 publication Critical patent/EP0547142A1/fr
Publication of EP0547142A4 publication Critical patent/EP0547142A4/en
Withdrawn legal-status Critical Current

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
    • C07D401/02—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings
    • C07D401/06—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing two hetero rings linked by a carbon chain containing only aliphatic carbon atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D207/00—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom
    • C07D207/02—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom
    • C07D207/04—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members
    • C07D207/10—Heterocyclic compounds containing five-membered rings not condensed with other rings, with one nitrogen atom as the only ring hetero atom with only hydrogen or carbon atoms directly attached to the ring nitrogen atom having no double bonds between ring members or between ring members and non-ring members with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
    • C07D207/12—Oxygen or sulfur atoms
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H21/00—Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids

Definitions

  • This invention is directed to a method of synthesis of oligonucleotides having low molecular weight tail molecules joined to the 3'-terminus of the oligonucleotide via a linking molecule, to oligonucleotides having low molecular weight tail molecules joined to their 3'-terminus and to intermediates utilized for the synthesis of such oligonucleotides.
  • Oligonucleotides have various uses including acting as primers for polymerase chain reaction synthesis of DNA. Oligodeoxynucleotides (abbreviated as ODN) are conveniently synthesized on solid phase supports using phosphite-triester synthetic methods. A detailed review of such syntheses was published in Atkinson, T. , Smith, M. (1984) in Oligonucleotide Synthesis, A Practical Approach , Gait, M.J. (ed.), IRL Press, pp. 35-81. This review gives detailed step by step conditions for the practical synthesis of oligonucleotides. Indeed, methods as outlined in this review are presently utilized in commercial oligonucleotide synthesizers available from various manufacturers.
  • ODN Oligodeoxynucleotides
  • the compounds of Letsinger et al . were prepared by first manually preparing a support bound dinucleoside hydrogen phosphonate derivative. A cholesteryl 'group was then tethered to the internucleoside phosphorus by oxidative phosphor- amidation. The oligonucleotide was elongated from the original dinucleotide on a commercial DNA synthesizer using phosphor- amidite chemistry.
  • Controlled pore glass beads for use in commercial oligonucleotide synthesis are available from CPG, Inc. , Pierce Chemical Co. and Sigma Chemical Co.
  • the controlled pore glass beads hereinafter alternately referred to as CPG's
  • CPG's are derivatized by the manufacturers with a long chain alkylamine group, such that a free amino group is available at the end of the long chain alkylamine that in turn is attached to the CPG.
  • Various amide linkages can be formed with the terminal amine of the long chain alkylamine on the CPG's for attachment of a growing oligc ⁇ cleotide during synthesis of the same.
  • An improvement of thxs synthesis was reported by Pon et al .
  • Nelson et al. Nuc. Acids Research 17(18) :7187-7194 (1989) , recently described the synthesis of an oligonucleotide incorporating a 3'-terminal substituent thereon.
  • Nelson et al derivatized the secondary hydroxyl of N-Fmoc-0-DMT-3-amino-l, 2-propanediol by treating with succinic anhydride in the presence of DMAP (dimethylaminopyridine) , and then subsequently treated with p-nitrophenol in DCC. The activated derivative was then anchored to a long chain alkylamine CPG support.
  • DMAP dimethylaminopyridine
  • the dimethoxytrityl blocking group was removed from the primary alcohol of the propanediol and an oligonucleotide was synthesized stepwise from the primary hydroxyl group while supported on the CPG support.
  • the synthetic oligonucleotide was deprotected and cleaved from the CPG support.
  • the purity of this 3•-amine-modified oligonucleotide was not demonstrated.
  • the 3'- terminal tail substituent has yet to be coupled to the oligonucleotide.
  • the crude oligonucleotide was biotinylated with a "Biotin-XX- ⁇ HS" ester. After, biotinylation, a second purification was necessary by both Sephadex and by HPLC. No yield data were given.
  • oligonucleotides can be enhanced by* including small molecular weight groups at their 3' end as, for instance, the above referred to 3'-tailed cholesterol and 3'- tailed acridine oligonucleotides of Letsinger et al . and Asseline et al .
  • the stability of 3'-tailed oligonucleotides in serum may also be enhanced. For instance, unmodified ODNs are rapidly degraded by 3'-exonucleases in serum-containing media. Certain chemical modifications of the 3'-terminal phospho- diester bond can block this degradation. Shaw et al . , Nucl . Acids Res .
  • a linking molecule bearing an appropriate small molecular weight molecule onto which an oligonucleotide can be constructed discloses a linking molecule beaming a protected amine group onto which an oligonucleotide can be constructed.
  • a third aspect of the claimed invention provides "tailing reagents" bearing an intercalating group that can be added to amine-modified oligonucleotides.
  • a fourth aspect of the invention describes a linking molecule bearing a protected alkanol group onto which an oligonucleotide can be constructed.
  • the method of the first aspect includes selecting as the linking molecule a molecule having three independent functional groups with the chemical reactivity of each of the three functional groups being independent and distinct from the reactivity of the other two of the functional groups.
  • the first functional group of the linking molecule is reacted with a low molecular weight tail molecule to join the tail molecule to the linking molecule.
  • the second functional group of the linking molecule is treated with succinic anhydride; the resulting carboxylic acid residue is reacted with a solid phase support to connect or anchor the linking molecule having the tail molecule joined thereto to the solid phase support.
  • 3*-phosphoramidite nucleotides are subsequently reacted with the 5' end of a preceding nucleotide to form a synthetic oligonucleotide attached to the linking molecule at the oligonucleotide's 3' terminus.
  • the attachment of the synthetic oligonucleotide via its 3 » terminus to the linking molecule concurrently joins the oligonucleotide to the tail molecule and to the solid phase support.
  • the growing oligonucleotide is attached to the solid phase support during the reactions of the further nucleotides with the growing oligonucleotide.
  • the oligonucleotide having the tail molecule joined to its 3' terminus via the linking molecule is then disconnected from the solid phase support by cleaving the connection between the second functional group of the linking molecule and the solid phase support.
  • the oligonucleotide having the tail molecule joined to its 3' terminus via the linking molecule can then be isolated.
  • the functional groups on the linking molecule include a primary alcohol, a secondary alcohol and an amine.
  • the tail molecule is reacted with the amine to join the tail molecule to the linking molecule
  • the solid phase support is reacted with the secondary alcohol to connect the linking molecule having the tail molecule joined thereto to the.
  • solid phase support and the first phosphoramidite nucleotide is reacted with the primary alcohol to attach that first nucleotide to the linking molecule.
  • linking molecule is (2s,4R)-4-hydroxy-2-hydroxymethylpyrrolidine (also designated as 4-hydroxy-(2s-trans)-2-pyrrolidinemethanol or trans-4-hydroxy-L-prolinol) .
  • the tail molecule can be selected as any one of a number of molecules of interest including reporter groups, intercalating groups, lipophilic groups and cleaving groups. Suitable as a lipophilic group would be cholesterol. Suitable as a reporter group would be biotin and fluorophores including acridine, fluorescein, rhodamine, Lissa ine rhodamine B, Malachite Green, erythrosin, tetramethylrhoda ine, eosin, pyrene, anthracene, 4-dimethylaminonaphthalene, 2-dimethyl- aminonaphthalene, 7-dimethylamino-4-methylcoumarin, 7-di- methylaminocoumarin, 7-hydroxy-4- th lcou arin, 7-hydroxy- coumai ,n, 7-methoxycoumarin, 7-acetoxycoumarin, 7-diethyl- amino-3-phenyl-4-met 1 -y
  • Suitable as an intercalating group would be acridine, ellipticine, methidium, ethidium, phenanthroline, 2-hydroxy- ethanethiolato-2,2' ,2' *-terpyridine-platinum(II) and quinox- aline and suitable as a cleaving group would be an EDTA ligand or porphyrin ligand for attaching Fe and a phenanthroline ligand for attaching Cu.
  • the tail molecule can be selected to include an inherent connecting group or an appendant connecting group can be attached to it via an appropriate chemical synthesis. If used, after attachment, the appendant connecting group, like an inherent connecting group, is used to link the tail molecule to the linking molecule. Whether or not an inherent or an appendant connecting group is utilized, the connecting group is such that it reacts with the linking molecule to attach the tail molecule to the linking molecule.
  • the third functional group of the linking molecule can be selectively blocked.
  • the linking molecule bearing the blocked third functional group is then attached to the solid state support via the second functional group.
  • the third functional group is then deblocked and the first phosphoramidite nucleotide is attached to the linking molecule via the deblocked third functional group.
  • a phosphate ester is located on that 3'-terminal hydrox l and is of the structure:
  • n — R or o m and m' independently are positive integers less than 11, n is 0 or 1, Q is a connecting group and R is selected from the group consisting of reporter groups, intercalating groups, lipophilic groups and cleaving groups.
  • Particularly preferred 3'-tailed oligonucleotides would include an oligonucleotide having either cholesterol or acridine joined via a linking molecule to the oligomer's 3' tail.
  • the cholesterol moiety is bonded to the linking molecule utilizing a carbamate linkage and the acridine moiety, preferably 9-ethylacridine or another 9-alkylacridine, is joined to the oligonucleotide utilizing an amide linkage (in effect z alkylamine linkage if the ethyl group of the 9-ethylacridine is considered) .
  • Other tail groups can be joined to the linking molecule via urea, thiourea or sulfonamide linkages.
  • the connecting group Q can preferably be selected from the group consisting of alkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkyl, aryl, aryloxy, aralkyl, heterocyclic, heteroaryl, substituted aryl and substituted aralkyl.
  • a compound and support for oligonucleotide synthesis comprising: a compound of the structure:
  • n and m' independently are positive integers less than 11, n is 0 or 1, Q is a connecting group, R is selected from the group consisting of reporter groups, intercalating groups, lipophilic groups and cleaving groups, Y is H or dimethoxy trityl and X is solid phase support.
  • Useful supports include controlled pore glass supports derivatized with long chained alkylamines.
  • n and m' independently are positive integers less than 11, n is 0 or 1, Q is a connecting group, R is selected from the group consisting of reporter groups, intercalating groups, lipophilic groups and cleaving groups and Y is H or dimethoxy trityl.
  • n and m 1 are positive integers less than 11, that is m and m 1 independently are 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10.
  • Particularly preferred are compounds wherein m and m' are 6 or less, that is m and m' independently are 1, 2, 3, 4, 5 or 6.
  • Another aspect of the claimed invention provides improved compounds t- id methods for synthesis of tetrafluorophenyl (TFP) esters of carboxylic acids in general.
  • An exemplary method provides improved synthesis of an activated ester derivative of an acridinyl carboxylic acid.
  • Such activated ester derivative may be advantageously used: (1) for making acridine-CPG, which in turn may be used to make 3'- tailed oligonucleotides; (2) for post-synthetic modification of 3'-amine-tailed oligonucleotides; and (3) for modification of internally amine-modified oligonucleotides.
  • Figure 1 shows the time course of swimming behavior of Paramecium after microinjection of antisense ODN 39.
  • Figure 2 presents a comparison of biological effect of antisense ODN 39 versus a random or "sense" ODN.
  • Figure 3 is a dose response curve of antisense ODN 39 in the Paramecium swimming behavior assay.
  • an oligonucleotide can be synthesized having a low molecular tail molecule joined to the 3' terminus of the oligonucleotide by use of a linking molecule.
  • the linking molecule is selected to have three chemically distinct functional groups on it. When such a linking molecule is utilized, a low molecular weight tail molecule is first joined to the linking molecule. The linking molecule with its low molecular weight tail molecule is then joined or anchored to a solid phase support.
  • the oligonucleotide is then synthesized. During its synthesis, the oligonucleotide is anchored on the linking molecule which in turn is anchored to the solid state support system.
  • the oligonucleotide is synthesized using standard phosphoramidite chemistry, either manually or on a DNA synthesizer.
  • the linking molecule having the synthesized oligonucleotide and a low molecular weight tail molecule joined to it is cleaved from the solid state support. This frees the oligonucleotide from the support system.
  • the oligonucleotide now has the small molecular weight tail molecule joined to its 3' terminus via the linking molecule.
  • oligonucleotides having low molecular weight tail molecules joined to their 3' terminus need only be subjected to a single purification step.
  • oligonucleotides having molecules of interest joined to their 3' terminus are prepared in a facile and expeditious manner.
  • the linking molecule having an appropriate low molecular weight molecule of interest attached to it can be synthesized independent of the oligonucleotide synthesis.
  • a linking molecule having a tail molecule of interest linked thereto is prepared by reacting the tail molecule with the first functional group of the linking molecule, the combination of the linking molecule and the tail molecule can be considered as a reagent suitable for use in a DNA synthesizer for preparation of numerous different oligonucleotides each of which have the low molecular weight molecule attached to their 3' terminus. This allows for large scale synthesis and storage, if desired, of the linking molecule-tail molecule combination. Additionally other combinations of the linking molecule with various other tail molecules also can be prepared in the same manner.
  • oligonucleotides that may have a common sequence for a number of nucleotides and then a divergent sequence for the remainder of the oligonucleotides but all of which have the same 3'-tail molecule of interest attached via the linking molecule, can be prepared by simply subdividing into aliquots the solid state support having a partially formed "common" oligonucleotide and its attaching linking molecule and tail molecule of interest. The synthesis of the divergent segments of the oligonucleotides is then completing by loading an individual aliquot of the solid state support having the common segment of the oligonucleotide on the DNA synthesizer and completing the desired sequence of nucleotides.
  • the compound (2s,4R)-4-hydroxy-2-hydroxymethyl- pyrrolidine, compound 2 of Scheme I, serves as a particularly useful linking molecule for attaching molecules of interest to the 3' terminus of an oligonucleotide.
  • This compound is readily prepared from commercially available N-CBZ-L-hydroxyproline (Sigma Chemical Company) . Reduction of the N-CBZ-L-hydroxyproline utilizing a procedure similar to that described by Stanfield et al . , J. Org. Chem .
  • Compound 2 includes a primary hydroxyl group that is utilized as the foundation on which an oligonucleotide is synthesized. Further compound 2 includes a secondary alcohol group that is utilized for attachment to a solid state support and a secondary amine that is utilized for attaching a tail molecule of interest. After synthesis of the oligonucleotide is completed, the tail molecule of interest remains attached to the oligonucleotide via the linking molecule.
  • the secondary hydroxyl group of the linking molecule is essentially held rigid in space and is removed from the vicinity of the phosphate linkage that attaches the linking molecule to the first nucleoside. This results in increased stability of the bond between the linking molecule and the oligonucleotide's 3' terminal phosphate group.
  • compound 2 is a single optical isomer and is not a mixture of stereoisomers
  • the bond between the 3' terminus of the oligonucleotide and this linking molecule also yields a single isomer and not a mixture of stereoisomers that might affect the physical properties. This may be particularly importance when attaching intercalating groups or other reactive groups to the 3' terminus end of an oligonucleotide because of a potential requirement for precise geometry for optimum binding of the intercalating agent with the double stranded complex of the oligonucleotide and its complementary DNA segment.
  • linking molecules are compounds of the structure:
  • linking molecules also have an amino, a primary hydroxyl and a secondary hydroxyl functional group included in their structure.
  • Two particularly useful linking molecules of this class are 3-amino-l,2-propanediol and 4-amino-l,3-butanediol (13) (see Scheme III) .
  • Attachment of the tail compound of interest to the linking molecule is done completely independent of any oligonucleotide synthesis as described above.
  • a low molecular weight tail molecule of interest is attached to the linking molecule utilizing organic chemistry techniques and reactions.
  • the secondary amino group of linking molecule is utilized to link the low molecular weight tail molecule of interest to the linking molecule, e.g., compound 2, via any one of a number of suitable connections or linkage, as for instance an amide linkage, a carba ate linkage, an urea linkage, a thiourea linkage, or a sulfonamide linkage.
  • suitable reactions between the secondary amine of linking molecule, e.g., compound 2, and appropriate connecting or linking groups on compounds of interest also will be suggested to the art skilled.
  • linking molecule For the purposes of clarity of this specification and the claims attached hereto, to avoid confusion between the above referenced “linking molecule” and any further “linking group” that might be used to attach a tail molecule to this linking molecule, the terminology “connecting group” is utilized to indicated these connecting or linking groups. Irrespective of the name given to these groups, they link, connect or bond a tail molecule to the linking molecule.
  • the primary hydroxyl group on the linking molecule is appropriately protected, as for instance with a dimethoxytrityl group.
  • a dimethoxytrityl group is conveniently accomplished utilizing dimethoxytritylchloride (DMTrCl) in pyridine in the presence of 4-dimethylaminopyridine (DMAP) .
  • DMTrCl dimethoxytritylchloride
  • DMAP 4-dimethylaminopyridine
  • the succinic ester of the linking molecule 'bearing the low molecular weight compound of interest thereon can be coupled to a controlled pore glass support utilizing either the older p-nitrophenol-DCC method of Atkinson et al . , above, or preferably using the facilitated DEC method of Pon et al . , above.
  • DEC is less toxic than DCC, is water soluble and eliminates the necessity of treating the succinic ester with p-nitrophenol, it is the presently preferred method.
  • the dimethoxytrityl protected linking molecule bearing a low molecular weight compound of interest attached thereto is then coupled or anchored to a controlled pore glass support having a long chain alkylamine group attached thereto in the normal manner.
  • Controlled pore glass supports derivatized with long chain alkylamines are available from Pierce Chemical or from Sigma Chemical. These are preactivated utilizing the procedure of Pon et al . , above, with dichloro- acetic acid and then reacted in pyridine utilizing DEC as the coupling reagent with the dimethoxytrityl protected linking molecule having the molecule of interest attached thereto.
  • excess long chain alkylamino groups on the support are capped by acetylating the same with acetic anhydride.
  • the dimethoxytrityl group is removed from the primary alcohol of the linking molecule by treating with 3% dichloroacetic acid in dichloromethane.
  • the resulting controlled pore glass support having the low molecular weight tail molecule attached thereto via the linking molecule (and with the primary hydroxyl group of the linking molecule now deblocked) , is now ready for synthesis of the oligonucleotide thereon.
  • the solid state support loaded with the linking molecule and molecule of interest can also be prepared in bulk and then subdivided for the synthesis of multiple oligonucleotides or even stored for later use. In any event oligonucleotide synthesis is initiated from the primary hydroxyl group of the linking molecule using phosphoramidite chemistry on a DNA synthesizer, as for instance a Milligen DNA synthesizer, in a normal manner.
  • the oligonucleotide is deprotected in the standard manner for oligonucleotides synthesized on automated DNA synthesizers.
  • the oligonucleotide with the low molecular tail molecule joined to its 3' terminus via the linking molecule is then cleaved from the solid state support also in the normal manner for automated DNA synthesis utilizing concentrated ammonia at room temperature in the normal manner.
  • the low molecular weight tail molecule to be joined to the 3' terminus of the oligonucleotide can be any one of a number of molecules of biological interest. Included in this group would reporter groups, intercalating groups, lipophilic groups and cleaving groups. Particularly preferred at this time for the lipophilic group is cholesterol.
  • biotin and the fluorophores including acridine, fluorescein, rhodamine, Lissamine rhodamine B, Malachite Green, erythrosin, tetramethylrhodamine, eosin, pyrene, anthracene, 4-dimethyl- a inonaphthalene, 2-dimethylaminonaphthalene, 7-dimethyl- amino-4-methylcoumarin, 7-dimethylaminocoumarin, 7-hydroxy- 4-methylcoumarin, 7-hydroxycoumarin, 7-methoxycoumarin, 7-acetoxycoumarin, 7-diethylamino-3-phenyl-4-methylcoumarin, isoluminol, benzophenone, dansyl, dabsyl, mansyl, sulfo rho ⁇ damine, 4-acetamido-4'-stilbene-2,2'-disulfonic acid diso
  • intercalating group Particularly preferred at this time for the intercalating group are acridine, ellipticine, methidium, ethidium, phenanthroline, 2-hydroxy-ethanethiolato-2,2' , 2' '- terpyridine-platinum(II) and quinoxaline.
  • cleaving group Particularly preferred at this time for the cleaving group would be an EDTA ligand or porphyrin ligand for attaching iron and a phenan ⁇ throline ligand for attaching copper.
  • the tail molecule is reacted with an appropriate reagent to attach an appendant connecting group thereon that is capable of reacting with the amine substituent of the linking molecule.
  • an appendant connecting group thereon that is capable of reacting with the amine substituent of the linking molecule.
  • the tail molecule is reacted with the amino group of the linking molecule to attach the tail molecule to the linking molecule.
  • OH of the invention is selected from one of the structures: 0 0 0 S
  • n and 1 independently are selected to be positive integers less than 11, n is selected as 0 or 1, and Q is a connecting group.
  • the tail molecule R is selected from the group consisting of reporter groups, intercalating groups, lipophilic groups and cleaving groups and Y is H or dimethoxytrityl.
  • cholesterol chloroformate is reacted with the linking molecule to attach the cholesterol group to the linking molecule via a carbamate connecting group.
  • 9-acridinepropionic acid is reacted with the linking molecule yielding 9-ethylacridine attached linking molecule via an amide linkage (see Scheme II) .
  • n 0 thus Q is absent and Z therefore is:
  • n 1 thus Q is present and is an alkyl moiety, i.e., ethyl (Scheme II).
  • Z is:
  • suitable for use as the connecting group Q are alkyl, alkoxy, alkoxyalkyl, alkenyl, cycloalkyl, aryl, aryloxy, aralkyl, heterocyclic, heteroaryl, substituted aryl and substituted aralkyl groups.
  • sulfonyl halide precursors tail molecules are available from Molecular Probes, Inc., Eugene, Oregon.
  • Such sulfonyl halides are aromatic sulfonyl halides wherein the sulfonyl halide moiety is present as an inherent connecting moiety on one of the rings of a tail molecule of interest, as for instance a rhodamine, a naphthalene, a pyrene, or an anthracene ring.
  • n is 0 and the connecting group Q is therefore absent.
  • the halide ion is chlorine or fluorine however bromine and iodine might also be useful.
  • Useful sulfonyl halides include sulforhodamine, sold by Molecular Probes, Inc. under the tradename "Texas Red.” Further would be Lissamine rhodamine B sulfonyl chloride, Lissamine rhodamine B sulfonyl fluoride, 5-dimethylamino- naphthalene-1-sulfonyl chloride (dansyl chloride) , 2-dimethyl- aminonaphthalene-5-sulfonyl chloride, 2-dimethylamino- naphthalene-6-sulfonyl chloride, 6-(N-methylanilino)- naphthalene-2-sulfonyl chloride (mansyl chloride) , 1-pyrenesulfonyl chloride, 2-anthracenesulfonyl chloride, 5-dimethylaminonaphthalene-l-sulfonyl fluoride
  • isothiocyanates precursor tail molecules are useful for preparing thiourea linkages between the tail molecule and the linking molecule.
  • isothiocyanates is present as an inherent connecting moiety on an aromatic ring of the tail molecule, and as such, in the above formula, n is also 0 and therefore Q would be absent.
  • isothiocyanates are also available from Molecular Probes, Inc.
  • Suitable isothiocyanates for'reacting with the linking molecule include fluorescein-5-isothiocyanate, fluorescein-6- isothiocyanate, tetramethylrhodamine- 5-(and-6)-isothiocyanate, Rhodamine X isothiocyanate, Malachite Green isothiocyanate, eosin-5-isothiocyanate, erythrosin- 5-isothiocyanate, 7-diethylamino-3-(4*-isothiocyanatophenyl)- 4-methylcoumarin, p-(5-dimethylaminonaphthalene-l-sulfonyl)- aminophenylisothiocyanate, N-(4-(6-dimethylamino- 2-benzofuranyl)phenylisothiocyanate hydrochloride, 1-pyreneisothiocyanate, 2-anthraceneisothiocyanate, 4-di
  • tetrafluorophenyl (TFP) esters include tetrafluorophenyl (TFP) esters, 5-(and 6-)carboxyfluorescein diacetate succinimidyl ester, 7-dimethylaminocoumarin-4-acetic acid, 7-amino- 4-methylcoumarin-3-acetic acid, 7-diethylaminocoumarin- 3-carboxylic acid, 7-hydroxycoumarin-4-acetic acid, 7-hydroxy- 4-methylcoumarin-3-acetic acid, 7-hydroxycoumarin-3-carboxylic acid, 7-methoxycoumarin-3-carboxylic acid, 7-carboxymethoxy-4- methylcoumarin, 7-acetoxycoumarin-3-carboxylic acid, acridone- 2-acetic acid, acridone-10-acetic acid, 9-anthracenepropionic acid, 1-pyrenebutanoic acid (pyrenebutyric acid) and N
  • EDTA* Fe(II) has been used as a cleaving group in conjunction with an oligonucleotide.
  • the EDTA molecule was attached to the base of a uridine nucleoside.
  • a carboxyl terminated chain was extended from the uracil moiety and the EDTA attached to it. While this approach yields a nucleoside having EDTA attached to it, any oligonucleotide that incorporated such a nucleoside might suffer from the EDTA molecule interfering with initial base pairing between the oligomer and its complementary DNA stand, since the EDTA is on the base.
  • an EDTA moiety can be extended from the linking molecule, away from the nucleotide's base and thus in a more non-interfering position for initial base pairing with a complementary stand of DNA.
  • Reaction of the linking molecule with an alkyl isocyanate as for instance ethyl isocyanatoacetate, followed by treatment with ethylenediamine and EDTA-trie nylester- N-hydroxysuccinimide ester, would serve to attach the EDTA moiety to the linking molecule via amide linkages.
  • an appendant connecting group is utilized to form the attaching bonds of the EDTA cleaving group with the linking molecule.
  • Cleavage reaction conditions are initiated in aqueous solution by adding Fe(II) and an appropriate oxidant, such as dithiothreitol, in a manner as is set forth in Dreyer et al . , Proc. Natl . Acad. Sci . 82:968-972 (1985).
  • phen- anthroline-Cu(I) complex 1,10-phenanthroline can be aminated at the 5 or 6 position.
  • the amine can then be succinylated.
  • the resulting terminal carboxylate would then be activated, as for instance by converting to an N-hydroxysuccinimide ester, for reaction with the amine of the linking molecule or with a further appendant connecting group that in turn is attached to the linking molecule.
  • Cleavage with this reagent is initiated by the addition of cupric sulfate and mercaptopropionic acid in a manner similar to Francois et al . , Biochemistry 27:2272-2276 (1988) .
  • the secondary amino substituent of ellipticine might be directly succinylated with succinic anhydride in pyridine and then activated to the N-hydroxy succinimide ester (an NHS ester) with DCC in THF for attachment to the linking molecule.
  • Quinoxaline requires amination of its ring, in a manner as per phenanthroline, prior to succinylation and activation.
  • Biotin having a long chain spacer is commercially available as a succinimidyl ester, also from Molecular Probes, Inc.
  • This product, 6-(6-(biotinoylamino)hexanoylamino)hexanoic acid succinimidyl ester is also referenced as biotin-XX- succinimidyl ester.
  • phenanthroline succinimidyl ester it is reacted with the amino group of the linking molecule to join the biotin tail to the linking molecule.
  • Dervan et al. J. Am . Chem . Soc.
  • the 9-acridinylalkanoic acids bind strongly to DNA (S. Takenaka et al., Anal . Sci . 4:481 (1988)), and can be prepared with a variety of different chain lengths.
  • a ⁇ ridine- modified ODNs have strict geometric requirements for efficient intercalation of the acridine molecule between the base pairs of a DNA duplex, but these geometric requirements are difficult to predict.
  • a variety of linker arm lengths can be evaluated with respect to binding strength. The strength of binding of an oligonucleotide (ODN) to its complementary nucleic acid strand is readily determined through thermal denaturation (T m ) studies.
  • the 9-acridinylalkanoic acids may be prepared with a variety of alkyl chain lengths by heating the corresponding aliphatic diacid with diphenylamine and zinc chloride, using a modification of the method of H. Jensen and L. Howland, J. Am . Chem . Soc. 48:1926 (1989). For example, 45 gm (16% yield) of 5-(9-acridinyl)pentanoic acid were obtained from the condensation reaction with adipic acid. Two alkyl chain lengths used for preparation of a ⁇ ridine-CPG were selected to approximate the length previously reported to give optimal T m for other 3'-acridine tailed ODNs (U. Asseline et al., Proc. Natl . Acad. Sci . USA 81:3297 (1984)).
  • One embodiment of this aspect of the present invention provides an improved method for synthesis of acridine-CPG.
  • This improved method uses an activated ester derivative of an acridinyl carboxylic acid as a precursor molecule.
  • a variety of activated esters of acridinyl carboxylic acids were evaluated as potential precursors to acridine-CPG (10, 23) .
  • N-hydroxysuccinimide (NHS) esters and p-nitrophenyl esters were prepared by "activating" a carboxylic acid with dicyclohexylcarbodiimide (DCC) , and then reacting with N-hydroxysuccinimide or p-nitrophenol.
  • DCC dicyclohexylcarbodiimide
  • TFP (tetrafluorophenyl) esters are suitably reactive with the nucleophilic amino group in trans- 4-hydroxy-L-prolinol 2, yet stable enough to allow purification by flash chromatography.
  • TFP ester 19 or 20 is prepared by first “activating” the carboxylic acid with 2-fluoro-methyl- pyridinium tosylate (FMPT) , thereby forming an unstable intermediate, and then reacting this unstable intermediate with 2,3,5,6-tetrafluorophenol to provide the stable product, as shown in Scheme IV, Method a.
  • FMPT 2-fluoro-methyl- pyridinium tosylate
  • No insoluble DCU is generated by this reaction, and the insoluble acridinyl carboxylic acid becomes soluble as the reaction proceeds.
  • the resulting homogeneous mixture is stripped of solvent and purified by flash chromatography.
  • FMPT is a particularly preferred activator for carboxylic acids that are not appreciably soluble in polar, aprotic organic solvents (such as ether, THF, DMF and acetonitrile) .
  • the present invention also describes an improved reagent for preparation of TFP esters. Briefly, 2,3,5,6- tetrafluorophenol is treated with trifluoroacetic anhydride to provide TFP trifluoroacetate 18. This improved reagent 18 reacts with carboxylic acids in the presence of triethylamine to yield TFP esters, as shown in Scheme IV, Method b. For instance, 9-acridinylpropanoic acid may be treated with TFP trifluoroacetate 18 and triethylamine in methylene chloride to produce the desired acridinyl TFP ester 19.
  • carboxylic acids are suitable for reaction with TFP trifluoroacetate 18.
  • exemplary carboxylic acids in this regard include N-CBZ-L-phenylalanylglycine, protoporphyrin IX, 3-amino-9-ethylcarbazole succinamide and the like.
  • Advantages of the disclosed reagent 18 and its method of use include: (1) TFP trifluoroacetate is readily prepared from inexpensive starting materials; (2) expensive condensing reagents are not required for production of TFP esters using this method of synthesis; and (3) improved purification of TFP ester product, since trifluoroacetate is the only by-product of the described reaction.
  • acridine-CPG supports (10 and 23) are prepared from trans-4-hydroxy-L-prolinol 2 and the appropriate acridinyl- propionic or -pentanoic acid TFP ester (19 or 20) , as shown in Scheme V. Purified TFP ester 19 or 20 is reacted with aminodiol 2 to give quantitative yield of the key intermediate amide product 7 or 21.
  • This method (Method 2) provides more reproducible results than preparation of the diol amide 7 directly from acridine carboxylic acid via the intermediate N- methylpyridinium ester (Example XI; Method 1) (see Scheme II) .
  • the primary hydroxyl group in 7 or 21 is selectively protected as the DMTr ether using standard conditions and good yields of 8 or 22 are provided.
  • the remaining secondary hydroxyl group is succinylated and the resulting carboxylic acids are immobilized on a long chain alkyl amine-controlled pore glass support (LCAA-CPG) to produce the desired Acr-CPG (10 or 23) .
  • the DMTr loading for these CPGs is 18.5 ⁇ mol/g and 20.6 ⁇ mol/g, respectively.
  • 3'-Tailed oligonucleotides may be directly synthesized from a solid support having a 3'-tail covalently attached thereto (Method A, as described in Sections A.l. and A.2).
  • 3'-tailed OD ⁇ s may be synthesized using a specially prepared solid support that incorporates a protected nucleophilic amino group or thiol group.
  • a conjugate group is then introduced into such 3'-tailed OD ⁇ s by postsynthetic treatment of the deprotected OD ⁇ with a suitable electrophile.
  • post-synthetic modification according to Method B is advantageously used to introduce sensitive 3'-tail molecules that cannot survive the synthesis conditions required for Method A.
  • Method B is advantageously used for preparation of small quantities of modified ODNs.
  • the two methods for preparing 3•-tailed ODNs disclosed herein were compared by examining the reaction of acridine TFP esters 19 and 20 with 3'-amine-tailed ODNs. More specifically, 3'-amine-tailed 11-mer ODNs having a sequence complementary to the initiation codon region of mRNA corresponding to Hepatitis B surface antigen protein were prepared. Such 3'-tailed ODNs that possess improved target nucleic acid binding properties may be advantageously used as "antisense" oligonucleotides.
  • a further aspect of the present invention provides an improved solid support for synthesis of 3'-amine-tailed ODNs, as shown in Scheme VI.
  • a particularly preferred support in this regard is aminohexyl-modified CPG (AH-CPG) , prepared using 6-aminohexan-l-ol.
  • modification of the 3*-terminus of ODNs may be performed using a commercially available support (for instance, "Amine-ON-CPG" by ClonTech Laboratories, Inc., Palo Alto, CA) .
  • a commercially available support for instance, "Amine-ON-CPG" by ClonTech Laboratories, Inc., Palo Alto, CA
  • Such supports . (corresponding to the support described by Nelson et al. , supra) have significant attendant disadvantages, including production of at least two distinct 3'-tailed ODN products ( « 1:1) by PAGE analysis.
  • derivatization of the 3'- amine tail with various active esters consistently results in poor yields, and HPLC analysis indicates that less than 50% of the starting 3'-amine-tailed ODN reacts.
  • the suggested mixed products may be a result of: (1) O to N migration of the phosphate moiety of the first nucleotide during synthesis of ODN on the commercial solid support; (2) deprotection of the FMOC moiety of the tail and subsequent capping by acetic anhydride during ODN synthesis; and/or (3) formation of a cyclic phosphate between the primary and secondary hydroxyl group of the tail with concomitant loss of the ODN.
  • the resultant 3'-tailed ODN would be subsequently unreact' a with active esters and would produce distinct species upon PAGE analysis.
  • the modified solid support herein overcomes these disadvantages associated with commercially available solid supports for 3'-terminus modification of ODNs. Additional advantageous characteristics provided by the claimed modified solid supports include: (1) a unique amine-protecting group that also functions as the site of CPG attachment; and (2). a dimethoxytrityl-protected hydroxyl group. Moreover, these modified supports are compatible with all procedures used with commercially available DNA synthesizers.
  • the 3•amine-tailed ODN is preferably removed from the CPG support off the DNA synthesizer. Since the claimed supports do not possess a vicinal diol, release of the 3'-amine tailed ODN from the support with ammonium hydroxide is compatible with preservation of the aminohexyl group at the 3• terminus of the ODN. In contrast, amine-ON-CPG contains a vicinal diol, and release of ODN from this support using ammonium hydroxide results in significant loss of tail molecules from the ODN.
  • 6-aminohexan-l-ol reagent for making the claimed solid support
  • other aminoalkanols are commercially available and suitable for production of modified CPG according to the present invention.
  • aminoalkanols By substituting aminoalkanols with various alkyl chain lengths for 6-aminohexan-l-ol, ODNs having 3'-amine tails of various lengths may be synthesized by the methods of the disclosure herein.
  • aminodiols of varying lengths are not generally commercially available, and thus solid supports having a range of alkyl chain lengths cannot be readily produced according to the method and support of Nelson et al.
  • R is alkyl, aryl, arylalkyl, heteroalkyl or heteroaryl
  • R is (CH 2 ) 6 .
  • Oligonucleotides having a 3•-amine tail can be efficiently synthesized using the modified solid support of the present invention.
  • Such 3 '-amine-tailed oligonucleotides were compared to analogous 3'-tailed ODNs prepared using the commercially available amine-ON CPG support.
  • An exemplary modified solid support of the claimed invention provided enhanced yield of 3'-amine-tailed ODN as a single product by both HPLC and PAGE. Subsequent derivatization of a 3'-amine- tailed ODN made from the claimed modified solid support proceeded rapidly to completion, and provided quantitative yields of 3'-modified ODN without the need for HPLC purification.
  • the present invention provides means to introduce multiple internal intercalating groups into ODNs.
  • Such multiple internal intercalating groups may provide enhanced binding of ODN to target nucleic acid strand(s) , but they may. have strict topological requirements for effective intercalation.
  • the predicted increase in nucleic acid binding affinity attributable to the intercalating groups may be neutralized or overwhelmed by steric effects that disturb the normal Watson-Crick base pairing in the duplex.
  • the claimed methods include use of acridine TFP esters to prepare bis-modified ODNs that "sandwich" either one or two base pairs in the mini duplex formed between ODN and target.
  • internally modi ad ODNs may be designed with linking arms that optimize interaction between intercalating groups and the target nucleic acid strand.
  • 5-aminopropyl-deoxyuridine groups have been selectively introduced into ODNs, and the effects on T m of one and two internal acridine modifications examined.
  • ODNs were reacted with acridine TFP esters 19 or 20 to determine the effect of chain length on binding efficiency (T m ) .
  • An acridine-modified ODN • with a 5 carbon length linking arm increased the T m by 5.9°C, as compared to an unmodified control, while a 3 carbon length linking arm resulted in a 1.1°C increase, as compared to the same control.
  • T m When two internal acridine modifications were examined, an increase in T m from 45.5°C (unmodified ODN) to 56.2°C (bis intercalated ODN) was observed with optimized linker arm lengths.
  • multiple internal acridine modifications may improve cellular uptake of ODNs and stability of ODNs to nuclease digestion.
  • Unmodified ODNs are rapidly degraded by 3'- exonucleases in serum-containing media.
  • Certain chemical modifications of the 3 '-terminal phosphodiester bond i.e., changing the last two internucleotidic phosphodiester bonds to phosphorothioates, phosphoroamidates, or inverted linkages
  • Other chemical linkages may provide enhanced nuclease stability, such as ⁇ -deoxynucleotide derivatives (C. Cazenave et al., Nucl . Acids Res . 15:10507 (1987)) and methylphosphonate derivatives.
  • intercalating groups are bulky 3'-substituents, they may also protect the terminal phosphodiester linkage from 3'- exonucleases (E. Uhlmann and A. Peyman, Chem . Rev. 90:543 (1990)).
  • 3'-cholesterol-, 3'-acridine-, and 3'- hexylamine-tailed ODNs have been examined in cell culture assay and demonstrated increased stability in comparison to unmodified ODNs.
  • a method for synthesizing oligonucleotides that resist degradation by 3'-exonucleases using a modified solid support is described.
  • This modified solid support may be advantageously used to synthesize ODNs that do not contain other 3'-modifications and to synthesize ODNs useful for in vivo evaluation of structure- activity relationships of other 3'-modifications, such as cholesterol or acridine. Since 3'-modified ODNs made according to this method do not interfere with hybridization, the modified solid support herein described is a suitable substitute for immobilized DMTr-protected nucleosides that are currently used for ODN synthesis. Disadvantages associated with these immobilized nucleosides include expense and the need to have four types of CPG available.
  • the modified solid support may be used as a universal reagent for preparation of any ODN sequence.
  • the DMTr-protected hydroxyl on the arm protruding from the solid support is unhindered, in contrast to the DMTr- protected 5'-hydroxyl of the immobilized nucleoside. Therefore, the DMTr-protected hydroxyl of the modified solid ' support displays enhanced accessibility to bulky phosphoramidite reagents and may provide higher yields of ODN product.
  • the modified solid support of this aspect of the invention features an "R" group (i.e., alkyl, aryl, arylalkyl, heteroalkyl, or heteroaryl) that connects support and DMTr- protected hydroxyl.
  • R is (CH 2 ) 6 .
  • Schemes I-VIII The following illustrative examples correlate to the reaction sequences of Schemes I-VIII.
  • the identifier "chol" indicates a cholesteryl moiety.
  • Scheme I shows synthesis of cholesterol-CPG 6;
  • Scheme II describes synthesis of acridine-CPG 10;
  • Scheme III illustrates synthesis of cholesterol-CPG 17;
  • Scheme IV depicts synthesis of acridine TFP esters 19 and 20;
  • Scheme V shows an improved method of synthesizing acridine-CPG 10 and 23;
  • Scheme VI describes synthesis of aminohexyl-CPG 30;
  • Scheme VII illustrates synthesis of hexanol-CPG 38;
  • Scheme VIII depicts structures of 3'-acridine tails.
  • the solid residue was purified by flash chromatography (4 x 15cm silica) using a gradient of methanol in l:l/hexanes:ethyl acetate. The product eluted with 10% methanol. The fractions containing pure product were stripped of solvent to give 1.42g (81% yield) of 3 as a white solid.
  • the succinylated cholesterol derivative 5 was immobilized to long chain alkyl amine-controlled pore glass support (LCAA-CPG, Sigma) using a published procedure (R.T. Pon et al., Biotechn . 6:768 (1988)).
  • LCAA-CPG (5.0g) was stirred for 3h with lOOmL of 3% dichloroacetic acid in methylene chloride.
  • the CPG was filtered on a 30mL sintered glass funnel and washed with 150mL of chloroform and 150mL of ether.
  • the solid was dried under vacuum and combined in a 250mL round bottom flask with 50mL of dry pyridine, 932mg (1 mmole)- of the succinylated cholesterol derivative 5, 0.4mL of triethylamine, 1.92g (10 mmoles) of l-ethyl-3-(3-dimethylaminopropyl)- carbodiimide, and 60mg of 4-dimethylaminopyridine.
  • the mixture was swirled on an orbital mixer at lOOrpm for 38h.
  • the CPG was filtered on a 30mL sintered glass funnel and washed with 50mL pyridine, lOOmL of methanol, 50mL of chloroform and 50mL of ether, then dried under vacuum. Residual amine groups on the CPG were capped by swirling the support in 15mL of dry pyridine and 2.0mL of acetic anhydride. After 2h, the CPG was filtered and washed as described above and dried under vacuum to give 5.0g of the product 6. This material was analyzed for dimethoxytrityl content according to the published procedure (T. Atkinson and M. Smith, in Oligonucleotide Synthesis, a Practical Approach, Gait, M.J.
  • An oligonucleotide with the base sequence CTCCATGTTCGTCACA was prepared on a Milligen DNA synthesizer using standard phosphoramidite chemistry. The 5'-DMTr protecting group was left on. The oligonucleotide was cleaved from the CPG and deprotected by treatment with 2mL of concentrated ammonia at room temperature for 3 days.
  • the supernatant was injected directly on a PRP-1 reverse phase HPLC column (elution with a gradient of 20% acetonitrile to 100% acetonitrile in pH 7.5 triethylammonium acetate) .
  • the product was collected in one fraction and lyophilized to give the 5'-DMTr protected product.
  • the DMTr group was removed by treatment with 80% acetic acid (16h at room temperature) and repurified by HPLC.
  • the collected product was analyzed by UV at 260nm and found to contain 0.54mg of product. In an alternate iteration of this procedure, the DMTr group was removed while on the synthesizer with 3% DCA to give an increased yield of the oligonucleotide.
  • EXAMPLE XI Acridine-CPG support 10
  • the succinylated acridine derivative 9 was immobilized to a long chain alkyl amine-controlled pore glass support using the procedure described above for the cholesterol-CPG support 6.
  • Acid washed LCAA-CPG (0.85g) was combined in a round bottom flask with 8.5mL of dry pyridine, 128mg (0.170 mmoles) of the succinylated acridine derivative 9, 0.068mL of triethylamine, 325mg (1.7 mmoles) of l-ethyl-3- (3-dimethylaminopropyl)-carbodiimide, and 10.2mg of 4-dimethylaminopyridine.
  • the mixture was stirred under argon for 19h.
  • the CPG was filtered off and washed with pyridine, methanol, chloroform and ether, then dried under vacuum. Residual amine groups on the CPG were capped by stirring the support in 2.5mL of dry pyridine and 0.34mL of acetic anhydride. After 2h, the CPG was filtered and washed as described above and dried under vacuum to give 0.85g of the product 10. This material was analyzed for dimethoxytrityl content found to have a loading of 18.5 micromoles/gram of CPG support.
  • a quantity of acridine-CPG 10 corresponding to 1 micromole of dimethoxytrityl content was packed into an oligonucleotide synthesis column.
  • An oligonucleotide with the base sequence 5•-CTCTCCATCTTCGTCACA was prepared on a Milligen DNA synthesizer using standard phosphoramidite chemistry. The 5'-DMTr protecting group was removed on the synthesizer.
  • the oligonucleotide was cleaved from the CPG and deprotected by treatment with 2mL of concentrated ammonia at 40°C for 24h.
  • the supernatant was injected directly on a PRP-1 reverse phase HPLC column (elution with a gradient of 20% acetonitrile to 100% acetonitrile in pH 7.5 triethylam onium acetate).
  • the product 10' was collected in one fraction and lyophilized to give 1.99mg (determined by UV at 260nm) of the fluorescent oligonucleotide product as a pale yellow solid.
  • Dicyclo- hexylcarbodiimide (52mg, 0.25 mmoles) was added and the mixture was stirred for 15min at room temperature and cooled in a refrigerator for 16h.
  • the crude p-nitrophenyl ester solution was filtered through a small pad of Celite to remove DCU and the filtrate was added directly to 0.50g of LCAA-CPG in 1.5mL of DMF.
  • O.lmL of ethyl diisopropylamine was added and the mixture was stirred for 18h under argon.
  • the CPG was filtered on a sintered glass funnel and washed with 3 x lOmL of DMF, 3 x lOmL of methanol and 3 x lOmL of ether.
  • the derivatized CPG was dried on a vacuum pump and "capped" by treatment with 1.5mL of dry pyridine and 0.2mL of acetic anhydride. After stirring for 3h under argon, the CPG was filtered and washed with 3 x lOmL of methanol and 3 x lOmL of ether. Drying on a vacuum pump gave 0.46g of cholesterol-CPG 17.
  • the CPG was analyzed for DMTr content according to the protocol described in Gait and found to have a loading of 24 micromoles/gram of CPG support.
  • a single thymidine residue was added to 41.6mg (1 micromole) of the solid support.
  • a Milligen DNA synthesizer was used along with standard phosphoramidite coupling chemistry.
  • the 5'-dimethoxytrityl protecting group on the thymidine was not removed in order to aid in isolation and characterization of the product.
  • the CPG was washed well with acetonitrile (8mL) in order to eliminate trace amounts of unreacted DMTr-thymidine phosphoramidite and other non- covalently attached impurities.
  • the CPG was dried on a vacuum pump and analyzed for DMTr content as described above, and found to have a loading of 27 micromoles/gram.
  • the 3*- cholesterol tailed thymidine was cleaved from 20mg of the support by 24h treatment with lOmL of concentrated ammonia at 44°C in a 5mL Reactivial (Teflon liner).
  • the 3'-cholesterol tailed thymidine was isolated by removing the supernatant (pasteur pipet) and washing the support with 3 x 2mL of methanol.
  • the presence of the acridine intercalating agent raised the Tm approximately 4°C, in comparison to a similar oligonucleotide that did not bear a 3'-tailed acridine molecule.
  • Appropriate reporter groups attached to the 3' tail of appropriate oligonucleotides are useful for identifying the presence of the oligonucleotide. Fluorescence, chemilu- minescence or other properties of such reporter groups serve to nonradioactively "tag" these nucleotides.
  • the nucleotides can then be identified in a sample of interest, as for instance a biological sample, by the presence of fluorescence or other like property.
  • a lipophilic tail group, as for instance a . cholesterol tailed 3' oligonucleotide assists in the transfer of the oligonucleotide across the cell membrane.
  • a cleaving group on the 3* tail of the oligonucleotide can assist in site specific cleavage of DNA bearing the oligonucleotide's complementary sequence after binding of the oligonucleotide to such complementary sequence. Such use might be implicated in gene identification, isolation and the like.
  • tail molecules or “conjugates” might be selected based on other properties both biological and physical.
  • Such other biological tail molecules might include appropriately blocked synthetic peptides, puromycin, digoxigenin and the like.
  • Other tail molecules having useful physical properties might include spin-labeled compounds, DTPA chelating agents, phospholipids, di- and trinitrophenyl groups and cross-linking agents including alkylating agents, azidobenzenes, psoralen, iodoacetamide, azidoproflavin and azidouracil.
  • Trifluoroa ⁇ etic anhydride 28 mL, 0.2 mol was added dropwise with stirring to 27.1 g (0.163 mol) of 2,3,5,6- tetrafluorophenol.
  • Boron trifluoride etherate (0.2 mL) was added and the mixture was refluxed overnight.
  • the residual solution was distilled at atmospheric pressure to remove trifluoroacetic anhydride and trifluoroacetic acid.
  • Method a To a solution of 400 mg (1.59 mmol) of 3- (9-acridinyl)propionic acid and 0.22 mL of triethylamine in 20 mL of methylene chloride was added 496 mg (1.75 mmol) of 2- fluoromethylpyridinium tosylate (FMPT) . The mixture was stirred at room temperature for 15 min. 317 mg (1.91 mmol) of 2,3,5,6-tetrafluorophenol and 0.22 mL of triethylamine were added and the mixture was stirred for 15 min.
  • FMPT 2- fluoromethylpyridinium tosylate
  • Method b To a stirred slurry of 251 mg (1 mmol) of 3-(9-acridinyl)propionic acid in 10 mL of methylene chloride was added 200 ⁇ L (1.4 mmol) of triethylamine and 200 ⁇ L of TFP trifluoroacetate 18. After stirring the mixture under argon for two days, the heterogeneous mixture was filtered through Celite and the filtrate was evaporated to dryness. The residue was purified by flash chromatography (2 x 25 cm silica) using 1:1 hexanes:ethyl acetate. The fractions containing product were combined and evaporated to dryness.
  • 3'-Tailed oligonucleotides having a sequence complementary to the initiation codon region of mRNA corresponding to Hepatitis B surface antigen protein (5'-TCCATGTTCGT) were synthesized using CPG supports 6, 10 and 23. Such 3*-tailed ODNs with improved binding properties may be advantageously used as "antisense" oligonucleotides.
  • ODNs were prepared from two different CPG supports on a 1 ⁇ mole scale using standard /3-cyanoethyl phosphoramidite coupling chemistry (Atkinson and Smith, supra).
  • AH-CPG 30 prepared as described above in Example XXIX, was used to synthesize 32.
  • the residue was detritylated in 80% acetic acid (500 ⁇ L, 28°C, 70 min) , precipitated with 100 ⁇ L of 3 M sodium acetate and 4 mL of 1-butanol, centrifuged, washed with 1 mL of ethanol, centrifuged, evaporated to dryness, and reconstituted with 1 mL of sterile distilled water.
  • the concentration of ODN 32 or 33 was determined from the UV absorbance at 260 nm. All ODN concentrations were measured in pH 7.2 PBS (9.2 mM disodium phosphate, 0.8 mM monosodium phosphate, 0.131 M sodium chloride) .
  • the reaction of the amine-tailed ODN 32 with acridine TFP ester 19 was complete in less than one hour.
  • the crude reaction mixture was pre-purified by adding 1.5 L of water and centrifuging through a 3000 MW cutoff ultrafiltration membrane (Centricon-3 microconcentrator; Amicon) to a final volume of «100 ⁇ L.
  • the product was collected in one fraction and concentrated on the Speed-Vac.
  • the residue was reconstituted with 100 ⁇ L of sterile distilled water and the concentration of acridine-modified ODN 32a was determined from the A 260 measurement.
  • Theoretical yield for 29 n ole is 108 ⁇ g; actual yield was 39 ⁇ g.
  • the purity of the ODN product 32a was determined by HPLC and PAGE.
  • the fractions containing pure product were combined and concentrated on a Speed-Vac.
  • the pale yellow solid residue 32a was reconstituted with 200 ⁇ L of water and analyzed by UV at 260 nm. The concentration was determined to be 108 ⁇ g/200 ⁇ L. Theoretical yield is 108 ⁇ g.
  • the ODN 5'-TCCATGTTCGT was internally modified at positions a, b and/or c, as illustrated below: a be 5'-TCC ATG TTC GT
  • EXAMPLE XXXV Thermal Denaturation studies To examine the effects of the 3'-tail on binding affinity, T m studies were performed using various 3'-modified ODNs described above. Thermal dissociation curves were obtained by following changes in A 260 of aqueous solutions containing equimolar amounts of a selected 3'-tailed ODN and an appropriate complementary, unmodified 20-mer target ODN (5'-GTGACGAACATGGAGAACAT) .
  • the 20-mer ODN target provides nucleotide overhangs that may influence interactions with the 3'-modification of the ODN; such interactions likely will be important in the actual biological target.
  • the target was DNA and not the likely biological target, RNA.
  • T m of an acridine-modified ODN-target duplex will increase if the acridine efficiently intercalates between the base pairs of the mini duplex upon hybridization with the target strand.
  • An unmodified 11-mer ODN was used as a control in each T m study.
  • ODNs were prepared as 2 ⁇ M solutions in pH 7.2 PBS.
  • ODNs 25 and 26 showed comparable binding affinity for the target ODN (i.e., comparable T m s) . While no dramatic difference was observed between the T m s of ODNs 25 and 26 (i.e., 3- and 5-carbon linker arm lengths), ODNs 33a and 32a resulted in slightly smaller increases in T m , as compared to ⁇ m s for ODNs 25, 26 and 33b. These data suggest that linker arm length, rigidity of the linker arm, stereochemical fidelity and/or sequence-dependent intercalation effects may influence
  • Table II presents T m data obtained with unmodified and internally modified ODNs (both single and double internal modifications) .
  • Target sequence 3'- TAC AAG AGG TAC AAG CAG TG -5' position of U a b c
  • Antisense 11-mer 5'- TCC AUG UUC GT -3'
  • the T m of unmodified and internally modified ODNs was determined, and the difference between unmodified ODN T m and the T m for each internally modified ODN was calculated ( ⁇ m (°C)).
  • the calculated T m differences ranged from -2.1 to +10.7°C. While 5-carbon linker arm lengths resulted in larger T m increases (i.e., 34b, 35b, 36b), such large T m increases are not readily predictable.
  • the inclusion of a second inter ⁇ calating acridine group resulted in an additive effect on T m increase.
  • the CPG was filtered, washed with methanol, chloroform and ether, then dried under vacuum to give 1.0 g of the product 38.
  • the CPG was 'analyzed for DMTr content and found to have a loading of 26.2 ⁇ mol/g.
  • the ODN was cleaved from the solid support and rotected by treatment with 2 mL of 30% ammonium hydroxide at 40-44°C for 24 h.
  • the product was collected in one fraction and concentrated on a Speed-Vac to give the 5'- DMTr protected product.
  • the DMTr group was removed by treatment with 300 ⁇ L of 80% acetic acid (80 min at 28°C) , and the 3'-hexanol-tailed ODN was precipitated with 100 ⁇ L of 2.4 M sodium chloride and 4 mL of 1-butanol.
  • the mixture was centrifuged, washed with 1 mL of ethanol, centrifuged, evaporated to dryness.
  • the white solid residue was reconstituted with 1 mL of sterile distilled water and filtered through a 0.2 ⁇ m filter.
  • the concentration of ODN 39 was 3.75 mg/mL, as determined from UV absorbance at 260 nm.
  • Theoretical yield for 1 ⁇ mole is 7.49 mg.
  • the 3'-hexanol-tailed antisense ODN 39 was used to inhibit the expression of the calmodulin gene in Paramecium.
  • This antisense ODN 39 was microinjected into the cytoplasm of the cell and the behavior of the cell was monitored over time. The injected cells were assayed by incubation in a solution that tests the function of Na + channels. Wild-type cells swim backwards for «15 sec in such solution; individual cells injected with the antisense ODN 39 displayed a significantly reduced behavioral response. After 15 to 20 h, backwards swimming time of the injected cells dropped to a minimum of «2 sec ( Figure 1) .
  • the 3•-hexanol-tailed calmodulin antisense ODN 39 was effective at concentrations as low as 0.42 mg/mL (56 ⁇ M) ( Figure 3) .
  • a calmodulin antisense ODN that had no 3 '-modification showed no effect at concentrations as high as 6.1 mg/mL (835 ⁇ M) .
  • the 3'-tailed antisense ODN 39 demonstrated a potency at least 15-fold greater than that of the analogous unmodified ODN. This increase in potency may be attributable to improved stability of the 3'-tailed ODN to 3'-exonuclease degradation. Assuming an injection volume of 10 pL and a Paramecium volume of 200 pL, the minimum cytoplasmic concentration of ODN 39 for an observable biological effect was calculated to be 2.8 ⁇ M.

Abstract

Oligonucléotides ayant une molécule de queue de faible masse moléculaire jointe à la terminaison 3' de l'oligonucléotide par l'intermédiaire d'une molécule de liaison de la structure (I) ou (II) dans lesquelles Z représente (III), (IV), (V), (VI) ou (VII), m et m' représentent des nombres entiers positifs inférieurs à 11, n représente 0 ou 1 et Q représente un groupe de liaison, synthétisés par réaction sélective de trois groupes fonctionnels indépendants se trouvant sur la molécule de liaison, c'est-à-dire, une amine, un hydroxyle primaire et un hydroxyle secondaire, par étapes. On relie premièrement une molécule de queue R à la fonctionalité amino de la molécule de liaison. Ensuite, la combinaison molécule de liaison-molécule de queue est fixée à un support solide par l'intermédiaire du groupe hydroxyle secondaire. L'oligonucléotide est systématiquement synthétisé par étapes en commençant par le groupe hydroxyle primaire puis par libération de l'oligonucléotide de faible masse moléculaire placé en queue, joint à sa terminaison 3' à partir du support solide.
EP91916634A 1990-08-28 1991-08-28 Synthese sur support solide d'oligonucleotides places en queue en position 3' par l'intermediaire d'une molecule de liaison Withdrawn EP0547142A1 (fr)

Applications Claiming Priority (5)

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US57434890A 1990-08-28 1990-08-28
US574348 1990-08-28
US71414291A 1991-06-10 1991-06-10
US714142 1991-06-10
CA002089588A CA2089588A1 (fr) 1990-08-28 1993-02-16 Synthese sur un support solide d'oligonucleotides avec groupe terminal en 3', par l'intermediaire d'une molecule de liaison

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WO1992003464A1 (fr) 1992-03-05
EP0547142A4 (fr) 1995-01-18
JPH06500556A (ja) 1994-01-20
CA2089588A1 (fr) 1994-08-17

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