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WO1998004575A2 - Analogues lipophiles d'oligonucleotides - Google Patents

Analogues lipophiles d'oligonucleotides Download PDF

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Publication number
WO1998004575A2
WO1998004575A2 PCT/US1996/012530 US9612530W WO9804575A2 WO 1998004575 A2 WO1998004575 A2 WO 1998004575A2 US 9612530 W US9612530 W US 9612530W WO 9804575 A2 WO9804575 A2 WO 9804575A2
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WIPO (PCT)
Prior art keywords
oligonucleotide
lipophilic
linkages
pseudohydrocarbyl
hydrocarbyl
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PCT/US1996/012530
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English (en)
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WO1998004575A3 (fr
Inventor
Norbert W. Bischofberger
Kenneth M. Kent
Richard W. Wagner
Chris A. Buhr
Kuei-Ying Lin
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Gilead Sciences, Inc.
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Application filed by Gilead Sciences, Inc. filed Critical Gilead Sciences, Inc.
Priority to CA002261704A priority Critical patent/CA2261704A1/fr
Priority to EP96926831A priority patent/EP0923596A2/fr
Priority to PCT/US1996/012530 priority patent/WO1998004575A2/fr
Publication of WO1998004575A2 publication Critical patent/WO1998004575A2/fr
Publication of WO1998004575A3 publication Critical patent/WO1998004575A3/fr

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

Definitions

  • the invention is directed to oligonucleotide analogs, in particular to lipophilic oligonucleotide analogs that efficiently enter cell cytoplasm or cross membranes without the aid of either transfection compounds or other agents or techniques.
  • the invention relates to oligonucleotide analogs which are capable of passive permeation of cell membranes and synthetic intermediates therefor as well as their use in cell staining, diagnostic and therapeutic applications.
  • Workers have described oligonucleotide analogs, complexes of oligonucleotide analogs and nucleoside analogs having lipophilic or related modifications for enhancing their delivery into cells, increasing nuclease stability or other purposes (see e.g., WO 96/15778; WO 96/07392; WO 96/05298;
  • compositions or methods include one or more compounds or methods that accomplish one or more of the following objects.
  • Another object of the invention is to provide lipophilic oligonucleotide analogs that are suitable for permeation into cell cytoplasm or cell nuclei in vitro or in vivo in the presence or absence of serum or blood.
  • Another object is to provide lipophilic oligonucleotide analogs that are suitable for staining one or more subcellular organelles or compartments.
  • Another object of the invention is to provide lipophilic oligonucleotide analogs that are suitable for tagging or marking items or compounds. Another object is to provide methods to deliver lipophilic oligonucleotide analogs into cells in vitro or in vivo.
  • Another object is to provide compositions comprising lipophilic oligonucleotide analogs bonded to a detectable label.
  • the invention is directed to oligonucleotides capable of passive diffusion across mammalian cell or organelle membranes or any other cell membrane (plant, parasite, bacterial, yeast, viral, or fungal).
  • oligonucleotide analogs are characterized by oligonucleotides comprising internucleotide linkages, bases and sugars wherein the oligonucleotide has a Log value of the octanohwater partition coefficient of -0.3 to +2.5 and a solubility in water of at least 0.001 ⁇ g/mL, and the and salts, solvates and hydrates thereof.
  • invention embodiments include oligonucleotide analogs having structure (1)
  • R is OH, blocked OH, N(R 14 ) 2 , P(0)(R 15 ) 2 , or a linker;
  • R 1 is an oligonucleotide, a blocking group, OH, N(R 14 ) 2 , P(0)(R 15 ) 2 , a solid support, or a linker bonded to the 2' or 3' position of a furanose ring (or its carbocyclic analog), and the remaining 2' or 3' position is substituted with
  • each R 2 independently is an internucleotide linkage bonded to the 2' or 3' position, and the remaining 2' or 3' position is substituted with R 3 ; each R 3 independently is H, OH, F, blocked hydroxyl, N(R 14 ) , -O-alkyl (Ci- ⁇ ), -O-alkyl (Ci-s) where the alkyl group is substituted with halogen, hydroxyl or oxygen, -O-alkenyl (C3-8), -S-alkyl (Ci- ⁇ ) or a linker; each R 4 independently is O or CH 2 ; each R 5 independently is CH 2/ NR 6 , O, S, SO, S0 2 ; each R 6 independently is H, alkyl (C1.6) or alkyl (C1-6) where the alkyl group is substituted with halogen, hydroxyl or oxygen; each R 14 independently is hydrogen, a protecting group, hydrocarbyl, or pseudohydrocarbyl; each R 15 independently
  • the invention oligonucleotide analogs are useful to visualize or stain cells and are thus useful in a method comprising: contacting cells to be visualized with the oligonucleotide under conditions wherein diffusion across the cell membrane can occur so as to internalize said oligonucleotide within the cells; removing from the cells any oligonucleotide which has not diffused across the membrane and become internalized; and detecting the oligonucleotide which has been internalized in the cells to visualize the cells.
  • the invention oligonucleotide analogs are useful as agents to deliver oligonucleotides into cells and are thus useful in a method comprising: contacting a cell with an invention oligonucleotide.
  • Figure 1 shows a standard curve for the determination of partition coefficient based on retention time in RPLC.
  • Figure 2 shows the chemical structures of oligonucleotide analogs used to visualize cells.
  • Halogen means F (fluorine), Cl (chlorine), Br (bromine) or I (iodine).
  • Alkyl means linear, branched or cyclic saturated hydrocarbons.
  • Alkenyl means linear, branched or cyclic unsaturated hydrocarbons where one or more double bonds are present.
  • Alkynyl means linear, branched or cyclic unsaturated hydrocarbons where one or more triple bonds are present.
  • hydrocarbyl groups contain only carbon and hydrogen and includes alkyl, alkenyl or alkynyl groups. Hydrocarbyl groups typically contain 1, 2 ,3 ,4, 5, 6, 7 or 8 carbon atoms, but includes groups having more than 8 carbon atoms, such as groups containing 9, 10, 11, 12, 13, 14, 15, 16, 17 or 18 carbon atoms.
  • psuedohydrocarbyl groups are hydrocarbyl substituents which contain one or more heteroatoms (including those present as substituents) representing less than 50% of the total non-hydrogen atoms in the pseudohydrocarbyl substituent.
  • pseudohydrocarbyl groups bonded to invention oligonucleotide analogs include structure (1) oligonucleotides, contain 1-18 carbon atoms, usually 3-12. Pseudohydrocarbyl moieties optionally contain 1, 2, 3 or 4 heteroatoms. Heteroatoms usually found in pseudohydrocarbyl groups are O, N, S or halogen.
  • the heteroatoms may be present as an ether, ketone, hydroxyl, thiol (SH), protected thiol, primary amine, secondary amine, tertiary amine, protected primary amine, amide, thioether (-S-), carboxyl, protected carboxyl, nitro (N0 2 ), azido (N3), ester (-C(0)-OR x where R x is hydrocarbyl or pseudohydrocarbyl), carbonate (- 0-C(0)-OR x ) or carbamate (-0-C(0)-NR 14 R x ).
  • pseudohydrocarbyl substituents that facilitate passive diffusion decrease the polarity or increase the lipophiliciry of the parent oligonucleotide and do not carry any charged atoms or groups, unless more than about 14 carbon atoms are present.
  • base means protected and unprotected purine, pyrimidine heterocycles found in nucleic acids or their modified forms. Modifications include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles previously described (see, e.g., PCT US94/10539). Bases suitable for use herein include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other analogs of purine or pyrimidine bases and their aza and deaza analogs.
  • Exemplary bases include N 4 ,N -ethanocytosine, 7-deazaxanthosine, 7-deazaguanosine, 8-oxo-N 6 - methyladenine, 4-acetyicytosine, 5-(carboxyhydroxylmethyi)uracil, 5- fluorouracil, 5-bromouracil, 5-carboxymethylaminomethyl-2-thiouracil, 5- carboxymethylaminomethyl uracil, inosine, N 6 -isopentenyl-adenine, 1- methyladenine, 2-methylguanine, 5-methylcytosine, N 6 -methyladenine, 7- methylguanine, 5-methylaminomethyl uracil, 5-methoxy aminomethyl-2- thiouracil, 5-methoxyuracil, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil, 5- (l-propynyl)-4-thiouracil, 5-(l-propyny
  • bases are adenine, guanine, thymine, uracil, cytosine, 5-methylcytosine, 5-(l-propynyl)uracil, 5-(l- propynyl)cytosine, 8-oxo-N 6 -methyladenine, 7-deaza-7-methylguanine, 7- deaza-7-methyladenine, 7-deazaxanthosine, 7-deaza-7-(l-propynyl)adenine, 7- deaza-7-(l-propynyl)guanine, 7-deaza-7-(l-butynyl)adenine, 7-deaza-7-(l-butynyl)guanine, 7-deaza-7-(l- butynyl)guanine, 5-(l-butyn
  • Nucleoside “nucleotide” and “monomer” include those moieties which contain both the common purine and pyrimidine bases adenine, guanine, cytosine, thymine and uracil, and modified bases or analogs thereof, particularly either lipophilic analogs or analogs that enhance binding affinity for complementary nucleic acid sequences.
  • Monomers, nucleosides or nucleotides are bonded together to form the invention oligonucleotide analogs.
  • nucleoside is generic to ribonucleosides or ribonucleotides, deoxyribonucleosides or deoxyribonucleotides, or to any other nucleoside which is an N-glycoside or C- glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base.
  • stereochemistry of the sugar carbons may be other than that of D-ribose in one or more residues.
  • oligonucleotide-like compounds or analogs where the ribose or deoxyribose moiety is replaced by an alternate structure such as the 6-membered morpholino ring described in U.S. patent number 5,034,506 or where an acyclic structure serves as a scaffold that positions the base or base analogs in a manner that permits efficient binding to target nucleic acid sequences or other targets.
  • Oligonucleotide-like compounds with acyclic structures in place of the sugar residue and/or the linkage moiety are specifically intended to include both (i) structures that serve as a scaffold that positions bases or base analogs in a manner that permits efficient sequence-specific binding to target nucleic acid base sequences and (ii) structures that do not permit efficient binding or hybridization with complementary base sequences.
  • Elements ordinarily found in oligonucleotides, such as the furanose ring or the phosphodiester linkage may be replaced with any suitable functionally equivalent element.
  • Linkage and internucleotide linkage mean an uninterrupted chain of atoms that bond adjacent monomers or nucleotides together.
  • Linkage includes unmodified phosphodiester linkages, -0-P(0)(OH)-0-, and modified or substitute linkages.
  • Substitute linkage means a linkage other than a phosphodiester linkage that links the sugar or sugar analog of adjacent monomers or nucleotides.
  • Substitute linkages may be used at R 2 -R 5 in oligonucleotides of structure (1). Many substitute linkages are non-ionic and contribute to the desired ability of the oligomer to diffuse across membranes.
  • Substitute linkages are defined herein as conventional alternative linkages such as phosphorothioate or phosphoramidate, are synthesized as described in the generally available literature.
  • Substitute linkages groups thus include, but are not limited to linkages comprising a moiety of the formula -O- P(0)S-0-, ("thioate"), -0-P(S)S-0- ("dithioate"), -0-P(0)N(R 6 ) 2 -0-, -0-P(0)R6-0- , -0-P(0)OR 7 -0-, -O-CO-O-, or -0-CON(R 6 ) 2 -0- wherein each R 6 independently is H (or a salt) or alkyl (Ci-s) and R 7 is alkyl ( - ⁇ ).
  • alkylphosphonate linkages such as methyl-, ethyl- or propylphosphonates.
  • Substitute linkages that may be used in the oligonucleotides disclosed herein also include nonphosphorous-based internucleotide linkages such as the 3'- thioformacetal (-S-CH 2 -0-), 5'-thioformacetal (-0-CH 2 -S-), formacetal (-0-CH 2 - O-), 5' amine (-CH 2 -CH 2 -NR 13 - where R 13 is hydrogen, a protecting group or alkyl C ⁇ _ 6 , see, e.g., PCT US91/06855), and 3'-amine (-NR 13 -CH 2 -CH 2 -) internucleotide linkages.
  • the phosphodiester or substitute linkages herein are used to bond the 2' or 3' carbon atoms of ribose or ribose analogs to the 5' carbon atoms of the adjacent ribose or ribose analog.
  • the linkages in oligonucleotides are used to bond the 3' atom of the 5' terminal oligonucleotide to the 5' carbon atom of the next 3'-adjacent nucleotide or its analog.
  • sugar includes furanose moieties usually found in nucleic acids and their isomers, e.g., arabinose, as well as other sugars, hexoses such as glucose. Sugar also includes carbocyclic analogs of these sugars.
  • Sugars optionally comprise modification of the 2' or 3' position by a O-hydrocarbyl, NH-hydrocarbyl, S-hydrocarbyl group, O-pseudohydrocarbyl, NR 14 - pseudohydrocarbyl, or S-pseudohydrocarbyl group, including 2'- or 3'-0- methyl, O-ethyl, O-propyl, O-isopropyl, O-butyl, O-isobutyl, O-propenyl or O- allyl, which are used due to their increased lipophilicity compared to the 2'- hydrogen or 2'-hydroxyl found in unmodified DNA or RNA.
  • S-alkyl or NH-alkyl substituents may also be utilized. Modifications such as 2'-0-alkyl C 1 - 4 , 2'-0-haloalkyl C 1 - 4 and 2'-fluoro are generally suitable for binding competent oligonucleotides and may thus be used to prepare the oligonucleotides. One may modify the 2' or 3' position using pseudohydrocarbyl groups.
  • Such groups will typically contain about 1-6 carbon atoms and include substituents such as 2'- or 3'-0-(CH 2 ) ⁇ - 3 -0-(CH 2 )i- 3 -R n where R 11 is a halogen, hydrogen, hydroxyl or NHR 12 and R 12 is hydrogen or a protecting group, and substituents such as 2'- or 3'-0-(CH 2 )i- 2 -0-(CH 2 ) ⁇ - -0- (CH 2 )i- -R 11 . Workers have described such modifications (see, e.g., U. S.
  • oligonucleotide or “oligomer” is generic to polydeoxyribonucleotides (containing 2'-deoxy-D-ribose or modified forms thereof such as arabinose or carbocyclic analogs of ribose), i.e., DNA, to polyribonucleotides (containing D-ribose or modified forms thereof), i.e., RNA, and to any other type of polynucleotide which is an N-glycoside or C- glycoside of a purine or pyrimidine base, or modified purine or pyrimidine base.
  • Oligonucleotide or oligomer is intended to include (i) compounds that have one or more furanose moieties that are replaced by furanose derivatives or by any structure, usually cyclic, that may be used as a point of covalent attachment for the base moiety, (ii) compounds that have one or more phosphodiester linkages that are either modified, as in the case of phosphoramidate or thioate linkages, or where the phosphorus atom is completely replaced by a suitable linking moiety as in the case of, e.g., formacetal linkages, and /or (iii) compounds that have one or more bonded furanose-phosphodiester linkage moieties replaced by any structure, cyclic or acyclic, that may be used as a point of covalent attachment for the base.
  • Invention oligonucleotides are of any convenient length and generally comprise 2-50 bonded monomers, often 3-20, usually 4-15. Oligonucleotide also includes short molecules such as dimers, trimers and tetramers having a Log value of the octanohwater partition coefficient of -0.3 to +2.5 and a solubility in water of at least 0.001 ⁇ g/mL, which are useful as synthetic intermediates.
  • a standard oligonucleotide dimer with two linkage groups has a molecular weight of about 650 Daltons.
  • the corresponding invention oligonucleotides optionally have molecular weights of 1,500 or more, e.g., 1,500-6,000, or 6,000-10,000.
  • lipophilic oligonucleotide As used herein, the terms “lipophilic oligonucleotide,” “lipophilic linkage,” lipophilic base,” lipophilic sugar,” “lipophilic modification,” and “lipophilic substitution” mean an oligonucleotide, or a linkage, base, sugar, modification or substitution, respectively, that makes the modified or substituted molecule more lipophilic than the corresponding unmodified molecule at pH 7.4 in water or low ionic strength buffer.
  • An unmodified oligonucleotide means one that is composed of ribose, or 2'-deoxyribose, phosphodiester linkages and the bases guanine, adenine, cytosine , thymine and/or uracil, i.e., DNA or mRNA.
  • each modified linkage, base or sugar optionally comprises a single lipophilic modification, although more than one may be present, particularly at a linkage or base.
  • a linkage such as -0-CH 2 -0- has a single lipophilic modification, i.e., -CH 2 - replaces -P(0)(OH)- in the 0-P(0)(OH)-0- linkage.
  • a linkage such as -CH 2 -CH 2 -CH 2 - would have 3 lipophilic modifications compared to the normal phosphodiester linkage in DNA or RNA.
  • a -CH 2 -CH 2 -CH 2 - linkage is considered to be a single modification for the purpose of determining the % of modifications in an oligonucleotide.
  • an oligonucleotide having 6 bonded monomers with only -CH 2 -CH 2 -CH - linkages would have 100% of the linkages modified and not 300%.
  • An invention oligonucleotide, such as a structure (1) oligonucleotide, containing modifications at each linkage, base and sugar would have modifications that sum to 300%, i.e., 100% of linkages plus 100% of sugars plus 100% of bases.
  • Oligonucleotide uptake into cells We have discovered lipophilic oligonucleotide analogs that enter cells by passive diffusion across cell, endosome and/or organelle membranes. The oligonucleotide analogs may enter cells by multiple mechanisms, e.g., pinocytosis, receptor-mediated uptake, phagocytosis as well as by passive diffusion. It has been generally assumed that oligomers containing the native phosphodiester linkages enter cells by receptor-mediated endocytosis (Loke, S.L., et al., Proc. Natl. Acad. Sci.
  • the partition coefficient for native DNA or RNA is relatively low with a log value of the octanohwater partition coefficient being less than -4 (Dagle, J.M., et al., Nucl. Acids Res. 12:1805-1810 1991).
  • DNA modified by synthesis of 2-methoxyethylphosphoramidite internucleoside linkages in place of the phosphodiester linkage eliminates the negative charge associated with the internucleotide linkage, which increases the hydrophobicity of DNA.
  • the log value of the octanohwater partition coefficient (Log Poet) remains less than -2 (Dagle, supra).
  • oligonucleotides can be modified by appropriate design of their molecular features so as to permit their passive diffusion across cellular membranes, despite the high molecular weights inherent in these molecules. Because of the high molecular weights of the invention oligonucleotide analogs, the relevant factor generated in determining distribution between membrane and aqueous medium is very small, which indicates that such a molecule is essentially impermeable to cell membranes.
  • the dimers and higher molecular weight oligonucleotides of this invention are, however, capable of passive diffusion into cells, and are thus not sequestered exclusively in endosomes in the same manner as described for previously known oligonucleotide analogs (Fisher et al., Nucl. Acids Res. 21:3857-3865 1993).
  • oligonucleotide analogs is our finding that when one introduces lipophilic modifications into multiple sites on the oligonucleotide, the oligonucleotide usually will passively diffuse into cells. We believe this may be due, at least in part, to the use of multiple, relatively small lipophilic modifications, such as hydrocarbyl or pseudohydrocarbyl moieties containing about 1-8 carbon atoms, at several locations on the oligonucleotide, rather than one or several large lipophilic moieties such as cholesteryl groups (see, e.g., Kabanov et al., FEBS 252:327-330 1990, Severin et al., Adv. Enz. Regul.
  • hydrocarbyl or pseudohydrocarbyl moieties containing about 1-8 carbon atoms
  • Oligonucleotide synthesis Oligonucleotides and the nucleotide synthons therefor are conventionally synthesized. Methods for such synthesis are found, for example, in Froehler, B., et al., Nucleic Acids Res. 14:5399-5467 1986; Nucleic Acids Res. 1 ⁇ :4831-4839 1988; Nucleosides and Nucleotides £:287-291 1987; Froehler, B., Tetrahedron Letters 22:5575-5578 1986. Amine, carboxyl and hydroxyl groups present anywhere on the molecule may be protected during oligonucleotide synthesis using standard protecting groups.
  • R or R 1 optionally are oligonucleotide coupling groups.
  • "Coupling group” as used herein means any group suitable for generating a linkage or phosphodiester substitute linkage between nucleotide bases or their analogs
  • These coupling groups are conventional and well-known for the preparation of oligonucleotides, and are prepared and used in the same fashion here.
  • each compound of structure (1) will contain two blocking groups: R or R 1 , but with only one of them being a coupling group.
  • the coupling groups are used as intermediates in the preparation of 3'-5' 5'-3', 5'-2' and 2'-5' internucleotide linkages in accord with known methods.
  • the "blocking group" of R or R 1 refers to a substituent other than OH that is conventionally coupled to oligomers or nucleosides, either as a protecting group, an activated group for synthesis or other conventional conjugate partner such as a solid support, label, immunological carrier and the like.
  • Suitable protecting groups are, for example, hydroxyl protecting groups such as DMT, MMT or FMOC, suitable activated groups are, for example, H-phosphonate, methyl phosphonate, methylphosphoramidite or ⁇ -cyanoethylphosphoramidite.
  • R or R 1 may also comprise a solid support.
  • the nucleosides and oligomers of the invention may be de ⁇ vatized to such "blocking groups" as indicated in the relevant formulas.
  • Suitable coupling groups for phosphodiester linkages include OH, H- phosphonate; (for amidite chemistries) alkylphosphonamidi.es or phosphoramidites such as beta-cyanoethylphosphoramidite, N, N- diisopropylamino-beta-cyanoethoxyphosphine, N,N-dusopropylam ⁇ no- methoxyphosphine, N,N-d ⁇ ethylam ⁇ no-methoxyphosph ⁇ ne, N,N- diethylamino-beta-cyanoethoxyphosphine, N-morphohno-beta- cyanoethoxyphosphine, N-morphohno methoxyphosphine, bis-morpho no- phosphine, N,N-d ⁇ methylam ⁇ no-beta-cyanoethylmercapto-phosph ⁇ ne, N,N- dimethylam ⁇ n
  • R 1 is a coupling group then R typically will be hydroxyl blocked with a group suitable for ensuring that the monomer is added to the oligomer rather than dimerizing.
  • groups are well known and include DMT, MMT, FMOC (9-fluorenylmethoxycarbonyl), PAC (phenoxyacetyl), a silyl ether such as TBDMS (t-butyldiphenylsilyl) and TMS (trimethylsilyl).
  • R is DMT
  • R 1 is located on the 3' carbon
  • the remaining R 1 is H
  • the R 1 groups are in the alpha anomer conformation.
  • oligonucleotides may also be partially or fully synthesized using solution phase methods such as triester synthesis. These methods are workable, but in general, less efficient for oligonucleotides of any substantial length.
  • the starting materials in Table A generally possess a ribose or a ribose analog comprising a 5' hydroxyl group and a 3' or 2' hydroxyl group, prepared as described herein or in the citations, with the substitute linkage being substituted for the phosphodiester linkages in unmodified nucleic acids.
  • Sequentially useful starting materials are designated by an arrow. Bracketed monomers are reacted to form dinucleotide analogs having the R 2 -R 5 substitute linkage. The reactions are repeated or ganged with phosphodiester linkages in order to produce trimers, tetramers or larger oligomers.
  • blocking group refers to a substituent other than H that is conventionally attached to oligomers or nucleotide monomers, either as a protecting group, a coupling group for synthesis, PO3" 2 , or other conventional conjugate such as a solid support.
  • blocking group is not intended to be construed solely as a nucleotide protecting group, but also includes, for example, coupling groups such as hydrogen phosphonate, phosphoramidite and others as set forth herein.
  • blocking groups are species of the genus of "protecting groups” which as used herein means any group capable of preventing the O-atom or N-atom to which it is attached from participating in a reaction involving an intermediate compound of structure (1) or otherwise forming an undesired covalent bond.
  • protecting groups for O- and N- atoms in nucleotide monomers or nucleoside monomers are described and methods for their introduction are conventionally known in the art.
  • Protecting groups also are useful to prevent reactions and bonding at carboxylic acids, thiols and the like as will be appreciated by those skilled in the art.
  • the invention oligonucleotides will have at least 60%, often at least 80%, of their internucleotide linkages as lipophilic modifications or at least 60%, often at least 80%, of their bases will contain a lipophilic substitution, or at least 60%, often at least 80%, of their sugars will contain a lipophilic substitution, or wherein the percent non-ionic nucleotide linkages, the percent bases containing a lipophilic substitution and the percent sugars containing a lipophilic substitution sums to at least 60%, often at least 80%.
  • oligonucleotides are present as ionic linkages, non-ionic linkages or as a mixture of ionic and non-ionic linkages. Lipophilic modifications for the oligonucleotides are independently chosen for each modification of the molecule. Generally, the log value of the partition coefficient between octanol and water will be 0.0-2.5, typically 0.2-2.3, usually 0.6-2.1. Invention embodiments include structure (1) oligonucleotide analogs wherein the linker at R, R 1 or R 3 optionally has 1-10 carbon, oxygen, sulfur and/or nitrogen atoms bonded together in an uninterrupted chain.
  • the linker which may be bonded to any invention oligonucleotide analog, including structure (1) oligonucleotides, will either connect the oligonucleotide analog to a detectable moiety or is bonded to the oligonucleotide analog at one end of the linker and, at the other end, has a reactive group available for bonding with a detectable moiety, e.g., amine, carboxyl or hydroxyl group.
  • the carbon or other atoms that comprise the uninterrupted linker chain may be substituted with one or more substituents that do not interfere with the function of the linker or the oligonucleotide analog, e.g., hydrocarbyl containing 1, 2, 3, 4, 5 or 6 carbon atoms or pseudohydrocarbyl containing 1, 2, 3, 4, 5 or 6 carbon atoms and one or more of the substitutions described above.
  • substituents that do not interfere with the function of the linker or the oligonucleotide analog, e.g., hydrocarbyl containing 1, 2, 3, 4, 5 or 6 carbon atoms or pseudohydrocarbyl containing 1, 2, 3, 4, 5 or 6 carbon atoms and one or more of the substitutions described above.
  • Invention oligonucleotide analogs, such as structure (1) oligonucleotides optionally have no linkers, but when a linker is present there is usually only 1 linker which, for structure (1) is located at R, R 1 , R 3 or a base,
  • the oligonucleotides include structure (1) oligonucleotides, contain 2, 3 or more linkers, which are usually linkers having 1-10 bonded atoms that form an uninterrupted chain.
  • each R 2 independently is a phosphodiester linkage or another linkage bonded to the 2' or 3' position and usually any linkage containing a phosphorus atom is bonded to the 3' position.
  • each R 3 independently is in the ribo or ara configuration and independently is H, OH, F, blocked hydroxyl, N(R 14 ) 2 , O-hydrocarbyl including -O-alkyl (C ⁇ -8 ) and -O-alkenyl (C3-8), and O- pseudohydrocarbyl including -O-alkyl (Ci-s) where the alkyl group is substituted with halogen, hydroxyl, NO2/ N3, carboxyl, ester, amide or oxygen as a keto or ether moiety, -S-alkyl (C 1 -8) or a linker, wherein the linker optionally has 1-10 carbon atoms, usually each R 3 independently is H or -O- alkyl (C1-3).
  • R 3 When R 3 is in the ara configuration it is usually H or F. Usually R 4 is O. Usually R5 is CH 2 . Usually R 6 is H or CH3. Generally one R 14 is hydrogen, C1-6 hydrocarbyl or Ci-8 pseudohydrocarbyl, and usually the other R 14 is hydrogen or a protecting group. Usually n is 2-13.
  • oligonucleotide analogs including structure (1) oligonucleotides, each B independently is a base or an analog thereof in the ⁇ or ⁇ anomeric configuration, usually in the ⁇ configuration, wherein base analogs are optionally lipophilic analogs that comprise a Ci-8 alkyl, C5-8 cycloalkyl, C6-8 aryl, C3-8 heteroaryl, C 2 -8 alkenyl or C 2 -8 alkynyl moiety or the analog contains a linker having 1-10 carbon atoms, wherein the oligonucleotide has a log value of the octanohwater partition coefficient of about -0.3 to +2.5, generally 0.0-2.5, typically 0.2-2.3, usually 0.6-2.1 and having a solubility in water of at least 0.001 ⁇ g/mL, usually at least 0.01 ⁇ g/mL.
  • base analogs are optionally lipophilic analogs that comprise a Ci-8 alkyl, C5-8 cycloalkyl,
  • Oligonucleotides having nucleosides containing bases in the ⁇ anomeric configuration binds to duplexes in a manner similar to that for the ⁇ anomers, and one or more nucleosides may contain a base in the ⁇ anomeric configuration, or more typically a domain thereof.
  • the linkage (R 2_ R 5 ) will generally be a linkage moiety that is 3-5 atoms in length, usually 4.
  • the linkages typically comprise bonded carbon, oxygen, nitrogen, sulfur and /or phosphorus atoms in an uninterrupted chain.
  • invention embodiments include oligonucleotide analogs of structure (1) wherein each lipophilic substitution at substituted bases independently is a hydrocarbyl group, typically a C ⁇ - $ hydrocarbyl group or a pseudohydrocarbyl group that is substituted with one or more heteroatoms selected from the group consisting of nitrogen, oxygen and sulfur, typically a C1-8 pseudohydrocarbyl group.
  • Exemplary lipophilic substitutions include ones wherein the C1-8 hydrocarbyl or C1-8 pseudohydrocarbyl group is bonded to a purine or pyrimidine base position selected from the group consisting of a C5 position of pyrimidines, the 04 position of thymine, the N6 position of adenine, the C8 position of adenine, the N2 position of guanine, the C8 position of guanine, the N4 position of cytosine and the C7 position of 7- deazapurines.
  • Any phosphate present at the 5' or 3' terminus of the oligonucleotide is optionally derivatized, for example, by further esterification to a lipophilic group containing about 3-8 carbon atoms.
  • a particularly useful derivatizing group may contain a linker and a label, for example, a fluorescent label such as fluorescein, rhodamine, or dansyl.
  • useful derivatizing groups include Fl-CONH (CH 2 ) 2 -8- and Rh-CONH (CH 2 ) 2 -8-, wherein FI and Rh signify fluorescein and rhodamine, respectively.
  • Invention oligonucleotides are optionally coupled to a label such as a fluorescent moiety and usually the label is coupled through a linker.
  • a linker is a moiety that contains an uninterrupted chain of atoms, e.g., 1-25 bonded (or linked) atoms, that connect the label and the oligonucleotide analog. Generally the linker contains 1-10 bonded atoms in such a chain.
  • the invention oligonucleotides are optionally able to bind single or double- stranded nucleic acid in a sequence-specific manner when the lipophilic base, sugar and linkage modifications do not completely destroy the oligonucleotide's binding capacity.
  • Such modifications include, e.g., (a) an alkynyl group such as 1-propynyl or 1-butynyl present at the 5 position of pyrimidine bases or pyrimidine base analogs, (b) an alkynyl group such as 1-propynyl or 1-butynyl present at the 7 position of 7- deazapurine bases or 7-deazapurine base analogs, (c) non-ionic linkages such as formacetal linkages or (d) sugar analogs where R 3 is -O-alkyl (C 1 -3) or F.
  • the oligonucleotide may be delivered into cells in tissue culture medium containing serum or lacking serum, or the oligonucleotide may be in an aqueous buffer or solution such as PBS.
  • aqueous buffer or solution such as PBS.
  • Invention embodiments include a method to visualize cells, usually viable cells which are optionally mammalian cells in tissue culture or in vivo, which method comprises: (a) contacting the cells to be visualized with an invention oligonucleotide containing a detectable moiety, e.g., a fluorescent moiety or radioactive moiety, under conditions wherein passive diffusion across the cell membrane can occur so as to internalize the oligonucleotide; (b) washing the cells to remove any oligonucleotide which has not passively diffused across the membrane and become internalized; (c) and detecting the internalized oligonucleotide to visualize the cells.
  • a detectable moiety e.g., a fluorescent moiety or radioactive moiety
  • the inventors have found that some of the invention oligonucleotides are suitable for staining or visualizing a subcellular compartment of the viable cell.
  • oligonucleotides of structure (1) when at least 80% of the linkages are modified, trimers have 2 modified internucleotide linkages and tetramers have 3, and so forth. Thus, all internucleotide linkages must be converted to lipophilic forms for oligomers which are less than hexamers. For hexamers, having 5 internucleotide linkages, only 4 of these need to be modified if only linkages, but not any bases or sugars, are modified.
  • oligonucleotide analogs relatively large lipophilic moieties, e.g., 1, 2, 3 or 4 C 9 - 16 hydrocarbyl substituents, or 1, 2, 3, or 4 C14- 1 8 pseudohydrocarbyl substituents.
  • oligonucleotide analogs will generally contain either (1) a domain or structural feature in the large hydrocarbyl or pseudohydrocarbyl substituent that limits membrane binding, e.g., branched, polar or charged groups or regions or (2) there will usually only be 1 or 2 of such large groups, if they are compatible with membrane binding.
  • such large hydrocarbyl or pseudohydrocarbyl groups particularly if they are compatible with membrane binding, will not be located on adjacent monomers, i.e., there will be 1, 2, 3 or more monomers that contain a smaller lipophilic group or no lipophilic group interspersed between the monomers containing the larger groups.
  • the invention oligonucleotide analogs will generally contain a lipophilic modification at least at 60%, often at least 80%, of either their internucleotide linkages, sugars and/or their bases.
  • invention embodiments include oligonucleotide analogs where no sugar contains a lipophilic modification and at least 60%, often at least 80%, of the internucleotide linkages are non-ionic or at least 60%, often at least 80%, of the bases contain a lipophilic modification.
  • oligonucleotide analogs where no base contains a lipophilic modification and at least 60%, often at least 80%, of the internucleotide linkages are non-ionic or at least 60%, often at least 80%, of the sugars contain a lipophilic modification.
  • Other embodiments include oligonucleotide analogs where no linkage is non-ionic and at least 60%, often at least 80%, of the bases contain a lipophilic modification or at least 60%, often at least 80%, of the sugars contain a lipophilic modification.
  • the oligonucleotide will have 6 modified or substituted linkages, bases or sugars, or two each may be modified or substituted or the oligonucleotide will have some combination that results in 6 modifications or substitutions at the linkages, sugars or bases.
  • hydrocarbyl groups e.g., ones containing 9-18 carbon atoms will generally contribute more lipophilic character to a nucleoside, i.e., a base, sugar or linkage, than is required for permeation competence.
  • nucleoside i.e., a base, sugar or linkage
  • the presence of several of the larger groups such as ones containing 16-20 carbon atoms may result in anchoring of the oligonucleotide in cell membranes.
  • Ci-8 hydrocarbyl groups and/or C ⁇ -8 pseudohydrocarbyl groups to obtain a sufficiently lipophilic monomer.
  • oligonucleotides By the use of appropriate larger lipophilic substituents, such as moieties containing about 10-16 carbon atoms, the proportion of either bases, sugars and /or linkages that must be modified for permeation competence can be reduced to about 40-60% or less.
  • substituents such as moieties containing about 10-16 carbon atoms
  • Invention oligonucleotides have a log value of the partition coefficient between octanol and water of -0.3 to +2.5, generally 0.0-2.5, typically 0.2-2.3, usually 0.6-2.1. These oligonucleotides optionally contain Ci-8 hydrocarbyl substituents, generally C ⁇ .(, hydrocarbyl substituents, or - 14 pseudohydrocarbyl substituents, generally C 2 - 8 psudohydrocarbyl substituents. Such oligonucleotides optionally contain 1 or 2 C 9 - ⁇ 2 hydrocarbyl substituents or they optionally contain 1 or 2 C 1 5- 18 pseudohydrocarbyl substituents.
  • Such oligonucleotides typically contain 3-30 monomers, usually 4-15.
  • the hydrocarbyl or pseudohydrocarbyl substituents are generally located at least at 60%, usually at least at 80% of the linkages, bases or sugars, or the oligonucleotides have these substituents at a combination of the linkages, bases and sugars that sums to at least 60% or at least 80%.
  • Exemplary embodiments include oligonucleotides wherein the hydrocarbyl or pseudohydrocarbyl substituents are located on 60-90%, usually at 80-120% of the linkages, bases or sugars.
  • the oligonucleotides have these substituents on a combination of the linkages, bases and sugars that sum to 60- 90% or 80-120%.
  • exemplary embodiments include oligonucleotides having branched or unbranched alkyl, alkenyl or alkynyl hydrocarbyl groups (Ci- ⁇ ) or branched or unbranched alkyl, alkenyl or alkynyl pseudohydrocarbyl groups (Ci-14) at the linkages, bases and/or sugars.
  • oligonucleotides having cyclic alkyl or alkenyl hydrocarbyl groups (C5-8) or cyclic alkyl or alkenyl pseudohydrocarbyl groups (C3-8) at the linkages, bases and /or sugars.
  • the target may be a single-chain or duplex nucleic acid.
  • Appropriate substitutions for binding competent modified oligomers with target nucleic acids refer to substitutions at base, linkage and/or sugar positions that do not completely disrupt the o gonucleotide's capacity to hydrogen bond with complementary nucleic acids.
  • Those positions on bases include the N6 or C8 of adenine, the N2 or C8 of guanine, the C5 of pyrimidines, N4 of cytosine and C7 of 7-deazapu ⁇ nes Synthesis of such modified bases is described in the art, as are methods for incorporation of such bases into oligonucleotides by solid-phase or solution-phase methods (Uhlmann, E., et al., Chemical Reviews, 2Q_:543-584 1990, and references cited therein, U.
  • oligonucleotides designed to bind single-stranded or double-stranded nucleic acid targets care must be taken to place the lipophilic substituents in such a way so as to avoid disruption of binding to the target
  • oligonucleotide analogs that do not involve binding to complementary sequences by base pairing e.g., staining cells or subcellular components
  • the bases, sugars or linkages may have modifications that are not compatible with oligonucleotide binding competence or with base pairing.
  • Representative lipophilic substituents at the base residues include saturated and unsaturated straight-chain, branched-chain, or cyclic hydrocarbyl groups, such as an alkane Ci-8 (usually C 2 -4), alkene Ci-8 (usually C2-4), or an alkyne C1-8 (usually C2-4), including ethynyl, vinyl, isopropyl, isobutyl, butynyl, butenyl, pentyl, pentenyl, isopentyl, phenethyl, methyl, ethyl, propyl, propynyl, phenyl, phenylvinyl, propenyl, butyl, pentynyl and their stereoisomers and positional isomers substituted at appropriate positions on the base.
  • an alkane Ci-8 usually C 2 -4
  • alkene Ci-8 usually C2-4
  • alkyne C1-8 usually C2-4
  • Exemplary base substituted nucleosides include 5-ethynyl-dU (5- ethynyl-2'-deoxyuridine), 5-ethynyl-dC, 8-ethynyl-dG, 5-vinyl-dU, 5-ethyl-dU, 8-ethynyl-dA, 8-propynyl-dG, 8-propynyl-dA, 5-pentyl-dU, 5-pentynyl-dU, 5- phenethyl-dU, 5-pentyl-U, 5-pentynyl-U, 5-benzyl-dC, N6-methyl-8-oxo-2'- deoxy-A, 4-O-butyl-T, 5-propynyl-dC and 5-propynyl-dU.
  • oligonucleotide analogs are optionally labeled using a detectable moiety, such as a fluorescent label, radiolabel or enzyme label.
  • a detectable moiety such as a fluorescent label, radiolabel or enzyme label.
  • One links detectable moieties to the oligonucleotide analog using a linker.
  • linkers A wide range of linkers are known and may be used by known methods. Exemplary linkers include linkers that having 2-10 carbon atoms and contain reactive groups that are convenient for linking to oligonucleotides, e.g., amines, hydroxyls, thiols or carboxylic acids.
  • Exemplary linkers include ones having a structure such as H 2 N-(CH 2 ) 2 . ⁇ -NH 2 or H 2 N-(CH 2 ) 2 -5-R 5 -(CH 2 ) 2 -5-NH 2 , where R 5 is O, C(O), NR 1() , S, SO or S0 2 where R 10 is hydrogen, alkyl C 1 - 4 or a protecting group.
  • Such linkers would link an invention oligonucleotide, at e.g., the R, R 1 or R 3 position of structure (1) oligonucleotides, to a detectable moiety.
  • Many linkers are available commercially.
  • Linkers can also comprise a chelating agent that binds to a detectable atom (see, e.g., U.S. Patent No. 5,534,497).
  • Therapeutic methods which utilize oligonucleotides as active agents are based on a number of end strategies.
  • PCR polymerase chain reaction
  • the antisense approach permits targeting of any desired nucleic acid sequence, e.g., mRNA, by the properly selected oligonucleotide.
  • mRNA nucleic acid sequence
  • the ability to obtain specifically binding oligonucleotides in this way has expanded the possibilities for oligonucleotide therapy because one can design oligonucleotides to target substances that reside at the cellular surface or at intracellular locations such as in the cytoplasm or nucleus.
  • RNA antisense sequences generated in situ that are complementary to a target sequence or in cell-free in vitro systems with exogenously added oligomers (Oeller, P.W., et al., Science 251:437-439 1991; Joshi, S., et al., /. Virol. West:5524- 5530 1991; Haeuptle, M-T., et al., Nucl. Acids Res. 14:1427-1448 1986).
  • the oligonucleotides of the invention are characterized by having a minimum solubility in water or aqueous media of at least 10 nM, usually 50 nM.
  • the minimum solubility requirement is based on the minimum concentration of fluor required by current fluorescent microscopes for visualizing the label.
  • oligonucleotides of the invention when utilized as (i) diagnostic or therapeutic agents that bind to intracellular or extracellular structures such as proteins or nucleic acids, or (ii) labeled compounds to detect or visualize complementary nucleic acid sequences, or cells, cell membranes or subcellular components in tissue samples, intact cells or in cell lysates or fractions, are characterized by a minimum solubility in water or aqueous media of at least about 0.001 ⁇ g/mL.
  • oligonucleotides of the invention were found to bind to specific subcellular components such as endoplasmic reticulum or mitochondria. Because of this, permeation-competent oligonucleotides that are fluorescently labeled can be used to directly visualize live cells or cell components in cell lysates. The aspects of the compounds that confer subcellular component-specific binding on the oligonucleotides of the invention are believed not to reside in the fluorescent moiety that is attached to the compound. However, the same oligonucleotides, either containing the fluorescent label or without the label can be synthesized utilizing, say, 32 P or 14 C instead of the normal nonradioactive phosphorus or carbon isotope.
  • radiolabeled oligonucleotides would retain their cell component-specific binding properties, but need not be directly visualized. In this case, cells or cell lysates can be specifically bound by the oligonucleotide followed by detection of bound oligonucleotide as a means to measure the presence or amount of bound material. Radiolabeled oligonucleotides used in this manner would have a minimum solubility requirement in water or aqueous media of about 0.001 ⁇ g/mL in order to be conveniently detected or quantitated by conventional methods such as scintillation counting.
  • the distribution coefficient need not be determined directly; that is, the distribution of the material obtained by mixing it with octanol and water and then effecting equilibrium distribution need not be evaluated, see e.g., Dagle et al., Nucl Acids Res. 12:1805-1810 1991. Alternate ways to measure these values take advantage of simpler techniques such as reverse-phase liquid chromatography, wherein retention times can be correlated to partition coefficient (Veith, G.D., et al., Water Research 12:43-47 1979), as described in Example 1 below.
  • the oligonucleotides of the invention are capable of passive diffusion across cell membranes they can be used to visualize and label cells and intracellular organelles or other structures.
  • the oligonucleotides of the invention are provided with a detectable label, such as a radiolabel, fluorescent label, chromogenic label, or enzyme label, and are contacted with cells to be visualized. After a suitable incubation period of about 15 minutes to 2 hours, usually at about 25 to 35°C, the solution containing the labeled oligonucleotides is removed and the cells are washed to remove any unincorporated oligonucleotide.
  • the cells are then prepared for visualization by fluorescence microscopy and detected by visualization of the labeled oligonucleotide.
  • the cells can be plated on a microscope slide and visualized directly.
  • the oligonucleotides of the invention are useful in therapy and diagnosis.
  • Those oligonucleotides that are capable of significant single-stranded or double-stranded target nucleic acid binding activity to form duplexes, triplexes or other forms of stable association, or which bind specific target substances, such as proteins, are useful in diagnosis and therapy of conditions that are associated with these targets.
  • one or more genes associated with viral infections due to say, HIV, HCMV, HSV or HPV may be targeted.
  • oligomers may employ the oligomers to specifically inhibit the expression of genes that are associated with the establishment or maintenance of a pathological condition, such as those for adhesion molecules, receptor molecules or oncogenes that may be associated with inflammatory conditions, immune reactions or cancer respectively. Diagnostic applications for the oligomers include their use as probes for detection of specific sequences by any standard method. In therapeutic applications, the oligomers are utilized in a manner appropriate for treatment of, for example, viral infections or malignant conditions. For such therapy, the oligomers can be formulated for a variety of modes of administration, including systemic, topical or localized administration. Techniques and formulations generally may be found in Remington's Pharmaceutical Sciences, Mack Publishing Co., Easton, PA, latest edition.
  • the oligomer active ingredient is generally combined with a carrier such as a diluent or excipient which may include fillers, extenders, binders, wetting agents, disintegrants, surface-active agents, or lubricants, depending on the nature of the mode of administration and dosage forms.
  • a carrier such as a diluent or excipient which may include fillers, extenders, binders, wetting agents, disintegrants, surface-active agents, or lubricants, depending on the nature of the mode of administration and dosage forms.
  • Typical dosage forms include tablets, powders, liquid preparations including suspensions, emulsions and solutions, granules, capsules and suppositories, as well as liquid preparations for injections, including liposome preparations.
  • injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous.
  • the oligomers of the invention are formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or Ringer's solution.
  • the oligomers may be formulated in solid form and redissolved or suspended immediately prior to use. Lyophilized forms are also included.
  • Dosages that may be used for systemic administration preferably range from about 0.01 mg/Kg to 50 mg/Kg administered once or twice per day. However, different dosing schedules may be utilized depending on (i) the potency of an individual oligomer at inhibiting the activity of its target gene, (ii) the severity or extent of a pathological disease state associated with a given target gene, or (iii) the pharmacokinetic behavior of a given oligomer.
  • Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally.
  • penetrants appropriate to the barrier to be permeated are used in the formulation.
  • penetrants are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmucosal administration.
  • detergents may be used to facilitate permeation.
  • Transmucosal administration may be through use of nasal sprays, for example, or suppositories.
  • the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics.
  • the oligomers of the invention are formulated into ointments, salves, gels, or creams, as is generally known in the art.
  • the oligomers of the invention may be used as diagnostic reagents to detect the presence or absence of the target substances to which they specifically bind. Such diagnostic tests are conducted by complexation with the target which complex is then detected by conventional means.
  • the oligomers may be labeled using radioactive, fluorescent, or chromogenic labels and the presence of label bound to solid support detected.
  • the presence of complexes may be detected by antibodies which specifically recognize them. Means for conducting assays using such oligomers as probes are generally known.
  • the invention oligonucleotides may be used to mark or "tag" various items such as plastics, chemicals (e.g., fertilizers, explosives, gunpowders and fuels), oils and emulsions (e.g., crude or refined oils, mineral oils and cosmetics), and fibers (e.g., synthetic or natural clothing fabrics).
  • chemicals e.g., fertilizers, explosives, gunpowders and fuels
  • oils and emulsions e.g., crude or refined oils, mineral oils and cosmetics
  • fibers e.g., synthetic or natural clothing fabrics.
  • PCR techniques are known for amplifying nucleic acid analogs, or their derivatives, that one normally can not amplify by standard techniques (see, e.g., PCT US93/07130).
  • PCR When one elects to use PCR to detect invention oligonucleotides using PCR, one will generally use base analogs that retain at least some of their base pairing capacity, i.e., base pairing capacity sufficient to facilitate performing the PCR procedure.
  • base pairing capacity sufficient to facilitate performing the PCR procedure.
  • the ability some of the oligomers to inhibit gene expression can be verified in in vitro systems by measuring the levels of expression in recombinant systems using described methods (see, e.g., Lewis et al., Proc. Natl. Acad. Sci. (U.S.A.) 22:3176-3181 1996; Wagner et al., Science 26 ⁇ :1510-1513 1993).
  • the partition coefficients for compounds with unknown Log Poet may be determined by comparison of retention times of the desired compounds with compounds of known Log Poet.
  • the resulting plot was fit to a third degree polynomial curve using Cricket Graph software.
  • the column used was a Hamilton PRP-1, 10 micron, 150 x 4.6 mm ID column.
  • a flow rate of 1 mL/min was used.
  • a linear gradient was used that went from 0% to 100% solution B in 30 min. Detection was monitored at 254 and 500 nanometers.
  • DMEM tissue culture medium at decreasing concentrations. The microscope was then used to analyze the solution for fine particles, micelles, etc. Solubility was detected at a minimum oligomer concentration of 50 nM. This lower solubility limit was determined by the sensitivity of the fluorescent microscope. This value can be extended to a 10 nM concentration using more sensitive apparatus.
  • fluorescein This dye is itself permeant to most of the cell types tested, giving a total cellular fluorescence. Within 15 min after washing the dye away from the exterior of the cells, the intracellular pool of the dye is pumped out, either by an organic anion pump mechanism or by diffusion. Fluorescein was conjugated to all of the linkers (without oligonucleotide) used and these conjugates were shown to retain the same biological properties. This fluor is very fluorescent, it does however quench rapidly.
  • BODIPY which has desirable molecular characteristics such as a neutral charge at cellular pH ranges, lower molecular weight than fluorescein and a greater quantum yield than fluorescein is also a preferred fluor.
  • Fluorescent measurements were made using a Zeiss Axiovert 10 microscope equipped with a 50W mercury arc lamp and outfitted with a set of fluorescent filters available from Omega Optical (Burlingtion, VT, USA). Observations were made from live cells with a 63x or lOOx objective (culture chamber and conditions described below). Photographs were taken with Tri-X / ASA 400 Kodak film and developed with Diafine developer (ASA rating 1600). Exposure time was fixed at 15 to 60s to enable direct comparison.
  • Fluorescent measurements were also made using a Nikon Diaphot inverted microscope equipped with a phase 4 long working distance condensor, 100W mercury arc lamp, Omega optical fluorescent filters, 40x, 60x and lOOx PlanApochromat phase/oil-immersion objectives, and 100% transmission to the video port.
  • a Quantex high-intensity /intensified CCD camera was used to digitize the fluorescent information. This information was sent to a Data Translations FrameGrabber board mounted on a Macintosh II CPU. The Macintosh II was equipped with 8MB RAM and had attached to it a 330MB hard drive. Images were recorded using public domain NIH software "IMAGE". Linearity of information was established using a series of neutral density filters. Relative fluorescent intensity was compared between samples using the same camera settings and variable neutral density filters.
  • Optimal fluorescence measurements were made using a confocal microscope imaging system which optically slices "sections" through a cell.
  • a Noran real-time confocal imaging optical path equipped with a 3-line (457nm, 488nm, 529nm) laser which is hooked up to the Zeiss Axiovert 10 inverted microscope described above was used.
  • the imaging system was the Macintosh II system described above.
  • the cell staining assay utilized various cell lines and included P388D1 (mouse macrophage), HEPG2 (human liver), CV1 (monkey epithelial), ccd50sk (untransformed human fibroblast), Rat2 (rat fibroblast), MDCK (kidney cells), L6 (rat myoblast), L cells (mouse fibroblast), HeLa (human adenocarcinoma), skov3 (human ovarian adenocarcinoma), and skbr3 (human breast adenocarcinoma) cells.
  • Jurkat human T cell
  • H9 human T cell
  • NIH3T3 human fibroblast
  • HL60 human T cell
  • H4 rat liver
  • All cell lines are commercially available from the American Type Culture Collection, Rockville, MD. Cells were grown on 25mm-#l coverslips in media containing 25mM
  • HEPES pH 7.3
  • phenol red which can lead to high background fluorescence when working with living cells.
  • Coverslips were used so that the high numerical aperture oil-immersion lenses on the microscope could be used.
  • the coverslips were mounted onto "viewing chambers": 6-well petri dishes which have 22mm holes drilled into the bottom.
  • the slides were mounted with silicon vacuum grease which was shown to be non-toxic to the cells.
  • 12x12 mm glass rasching rings (Stanford Glassblowing Laboratory, Stanford, CA) were mounted directly onto the coverslip using paraffin wax.
  • the chamber permitted the use of incubation volumes less than 200 ⁇ L.
  • Fluorescent oligonucleotide conjugates were added at concentrations ranging from 0.1 to 150 ⁇ M.
  • oligonucleotides Stock concentrations of oligonucleotides were prepared in 25mM HEPES, pH 7.3. Oligonucleotides were added to media with or without 10% 4hr-heat inactivated (567C) fetal bovine serum. Incubation times ranged from 15 minutes to 24 hours. 2 hour incubations were generally utilized for cell staining. Cells were then extensively washed to remove extracellular oligomer using media and observed at room temperature. Slides were optionally replaced in the incubator and were observed over the following 48-72 hours.
  • 567C 4hr-heat inactivated
  • EXAMPLE 4 Subcellular Compartment Staining. Fluorescent oligomer compounds were placed on fibroblasts, hepatocytes, muscle and carcinoma cell lines at 50 ⁇ M for 2 hours at 37°C; the cells were washed with cell media and live cells were visualized for cellular staining using fluorescent confocal microscopy. The results obtained for representative compounds were:
  • HTEA+ hydrogentriethylammonium
  • 5-(l-Pentynyl)-2'-deoxyuridine (1) was prepared by the same procedure that Hobbs, F.W.J., /. Org. Chem. 54:3420-3422 1989, used for the preparation of other alkynyl substituted nucleosides.
  • a mixture of 30.0 tg (84.7 mmol) of 5-iodo-2'deoxyuridine (purchased from Sigma), 23.6 mL of 1-pentyne (Aldrich), 9.79 g of tetrakis (triphenylphosphine) palladium (0) (Aldrich), and 3.23 g of copper (I) iodide were stirred at room temperature for 26 h.
  • the triazole intermediate (2.1 g; 3.5 mmole) was dissolved in anhydrous n-butanol (12 mL) and treated with DBU (1.0 g; 7.0 mmole). After one h, the reaction mixture was concentrated to dryness. The residue was dissolved in CH 2 C1 2 , washed with 10% aqueous citric acid, dried over Na 2 S ⁇ 4 , and filtered. The residue was purified by column chromatography on silica gel, affording 1.0 g of product.
  • This compound was prepared from 5'- 0-([5'-0-(4,4'-dimethoxytrityl)-5-pentyl-2'-deoxyuridin-3'-0-yl]-methyl-5- pentyl-2 '-deoxy uridine using the same procedure described for the preparation of 5'-0-(4,4'-dimethoxytrityl)-5-(l-pentynyl)-2'-deoxyuridin-3'-0-yl- hydrogenphosphonate hydrogentriethylammonium salt.
  • This compound was prepared from 5'-0-([5'-0-(4,4'- dimethoxytrityl)-thymidin-3'-0-yl]-methyl)-thymidine by the same procedure used for the preparation of 4-0-butyl-5'-0-(4,4'-dimethoxytrityl)-thymidine. Column chromatography afforded a 52% yield of product.
  • This compound was prepared from 5'-0-([4-0-butyl-5'-0-(4,4'- dimethoxytrityl)-thymidin-3'-0-yl]-methyl-4-0-butylthymidine by the same procedure used for the preparation of 5'-0-(4,4'-dimethoxytrityl)-5-(l-pentynyl- 2'-deoxyurdin-3'-0-yl-hydrogenphosphonate hydrogentriethylammonium salt.
  • nucleoside such as 5'-0-(4,4'-dimethoxytrityl)-5-(l-pentynyI)-2'-deoxyuridine, 5'-0-(4,4'-dimethoxytrityl)-5-pentyl-2'-deoxyuridine, 4-0-butyl-5'-0-(4,4'- dimethoxytrityl)- thymidine, 5'-0-([5'-0-(4,4'-dimethoxytrityl)
  • Nucleoside substitutions (loadings) achieved were typically between 20 and 40 ⁇ mole of nucleoside per gram of functionalized support.
  • the unreacted succinic acid sites on the solid support were capped by adding 134 mg of pentachlorophenol and shaking the mixture for 16 h. This formed the corresponding ester.
  • the mixture was filtered and the support was sequentially washed with pyridine, dichloromethane, and then diethylether.
  • the support was then shaken with 10 mL of anhydrous piperidine in a 25 mL round bottomed flask for 5 min. The mixture was filtered and the support washed with dichloromethane and then diethylether.
  • the support was then added to an anhydrous solution containing 2.5 mL of acetic anhydride, 10.0 mL of pyridine, and 10 mg of DMAP.
  • the solution was placed under argon, capped, and shaken for 4 h.
  • the mixture was filtered, and the functionalized CPG was washed sequentially with pyridine, dichloromethane, methanol and diethylether.
  • the CPG was dried in vacuum and was then ready for solid phase oligonucleotide synthesis.
  • the oligonucleotide H- phosphonate having the following structures were prepared according to the following procedure.
  • the functionalized (A for sequence A, B for sequence B) solid support was placed in a reactor vessel (column) and was washed with dichloromethane. Then, a 2.5% solution of dichloroacetic acid (DCA) in dichloromethane was introduced to remove the 5' protecting group of the support-bound nucleoside. After the deprotection step, the solid support was washed with dichloromethane, and then anhydrous pyridine /acetonitrile (1/1, by volume).
  • DCA dichloroacetic acid
  • the first coupling cycle was initiated by the addition of a 1.5% solution of pivaloyl chloride in anhydrous pyridine/acetonitrile, 1/1) and ten equivalents (based on the amount of loading of the support bound nucleotide) of the appropriate protected nucleoside hydrogenphosphonate in anhydrous pyridine/acetonitrile (1/1) in alternating pulses.
  • the reagents were allowed to react for 3.5 min.
  • the oligonucleotide could be further extended by repeating the sequence of DCA deprotection and pivaloyl chloride coupling until the desired length and sequence of bases was attained.
  • the linkage or linkages could be oxidized to the thiophosphate, phosphodiester or the phosphoramidate.
  • the final coupling for fluorescent labelling utilizes coupling of 6-N-(4- methoxytrityl)-aminohexan-l-0-yl)-hydrogenphosphonate hydrogentriethylammonium salt.
  • the coupling of this hydrogenphosphonate was identical to the other hydrogenphosphonate couplings.
  • the monomethoxytrityl protecting group was removed from the amine in a similar fashion as described above.
  • the DNA H-phosphonate, prepared above, was converted directly to the thiophosphate, preferably while the DNA was still bound to the solid support, by the addition to the reactor vessel of 1 mL of an oxidizing mixture comprised of a 2.5% solution (by weight) of elemental sulfur (sublimed sulfur powder available from Aldrich Chemical Company, Milwaukee, Wisconsin, USA, Cat No. 21,523-6) in anhydrous pyridine/ carbon disulfide (1/1, v/v). The contents of the reactor were mixed for 20 min., and then the reagents were removed.
  • an oxidizing mixture comprised of a 2.5% solution (by weight) of elemental sulfur (sublimed sulfur powder available from Aldrich Chemical Company, Milwaukee, Wisconsin, USA, Cat No. 21,523-6) in anhydrous pyridine/ carbon disulfide (1/1, v/v).
  • This oxidation cycle was carried out a second time using 1 mL of an oxidizing solution comprising equal volumes of a 2.5 wt% solution of elemental sulfur in anhydrous pyridine /carbon disulfide (1/1, v/v) and 10% by volume diisopropylethylamine in anhydrous pyridine. Finally, the oxidized copolymer-bound oligonucleotide was washed with anhydrous pyridine/acetonitrile (1/1, v/v), followed by anhydrous dichloromethane.
  • Method A To the solid support, obtained from the process outlined above, was added 1 mL of an oxidizing solvent mixture comprised of 0.1 M I 2 in water /pyridine (2/98, v/v). The resulting mixture was agitated for 15 min., and then the reagents were removed. Afterwards, 1 mL of a second oxidizing solvent mixture made from equal volumes of 0.1 M I 2 in water/pyridine (2/98, v/v) and 0.1 M triethyl ammonium bicarbonate in water/pyridine (1/9, v/v) was added to the solid support. After mixing the contents of the reactor for 5 min., the reagents were removed. Finally, the oxidized copolymer-bound product was washed with anhydrous pyridine/acetonitrile (1/1, v/v) and then anhydrous dichloromethane.
  • Method B Alternatively, the oligonucleotide H-phosphonate was oxidized to the phosphoramidate analog by the following procedure: To the solid support, obtained from the procedure outlined above, was added 18 mL of an oxidizing solvent mixture made from 10% by volume of the desired amine in anhydrous/pyridine/carbon tetrachloride (1 /1, v/v). The resulting mixture was agitated for 15 min., after which time the spent oxidizing solvent mixture was discarded. Finally, the oxidized copolymer-bound product was washed with anhydrous pyridine/acetonitrile (1/1, by volume), and then anhydrous dichloromethane.
  • oligonucleotide H-phosphonates could be oxidized or converted to a number of other linkage derivatives, such as phosphoric acid triesters, dithiophosphoric acids, their corresponding esters and amidates, and other which are desirable to and which are within the skill of those knowledgeable in the art.
  • linkage derivatives such as phosphoric acid triesters, dithiophosphoric acids, their corresponding esters and amidates, and other which are desirable to and which are within the skill of those knowledgeable in the art.
  • Related oxidation procedures are described, for example, in application no. EP 0 219 342, the complete disclosure of which is incorporated herein by reference.
  • oligonucleotides having a variety of linkages derived from phosphoric acid such as phosphoric acid diesters, phosphoric acid triesters, thiophosphoric acid, dithiophosphoric acid, phosphoric acid thioesters, phosphoric acid dithioesters, phosphoric acid amidates, or thiophosphoric acid amidates, can be readily obtained from the methods described above.
  • HPLC Purification was effected by reverse-phase HPLC, under the conditions described further below.
  • HPLC Purification of the Fluorescently Labeled Oligonucleotide A crude sample containing approximately 10 Tmole of the fluorescently labeled oligonucleotide, prepared by the methods described above, and dissolved in a solvent mixture of 1/1 (v/v) methanol/water (10 mL) was concentrated under vacuum. The oligonucleotide was resuspended in 1 L of methanol and then diluted with 100 mM aqueous triethylammonium acetate (TEAA, pH 7.0) and 5% (by volume) aqueous acetonitrile to a final volume of 10 mL.
  • TEAA triethylammonium acetate

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Abstract

Analogues lipophiles d'oligonucléotides capables de se diffuser passivement et efficacement à travers les membranes cellulaires. Ces oligonucléotides contiennent au moins deux résidus de nucléotides et ont un coefficient de partage octanol:eau allant de -0,3 à +2,5 environ et une solubilité dans l'eau d'au moins 0,001 νg/mL. L'invention porte notamment sur des analogues lipophiles d'oligonucléotides dont au moins 60 % des liaisons inter-nucléotides sont lipophiles, ou dont au moins 60 % des bases contiennent des substitutions lipophiles, ou dont au moins 60 % des glucides contiennent des substitutions lipophiles, ou qui présentent une combinaison de ceux-ci à raison de 60 %. Ces oligonucléotides peuvent être conjugués pour former un marqueur et utilisés pour visualiser des cellules ou des compartiments infracellulaires.
PCT/US1996/012530 1996-07-31 1996-07-31 Analogues lipophiles d'oligonucleotides WO1998004575A2 (fr)

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CA002261704A CA2261704A1 (fr) 1996-07-31 1996-07-31 Analogues lipophiles d'oligonucleotides
EP96926831A EP0923596A2 (fr) 1996-07-31 1996-07-31 Analogues lipophiles d'oligonucleotides
PCT/US1996/012530 WO1998004575A2 (fr) 1996-07-31 1996-07-31 Analogues lipophiles d'oligonucleotides

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CA002261704A CA2261704A1 (fr) 1996-07-31 1996-07-31 Analogues lipophiles d'oligonucleotides
PCT/US1996/012530 WO1998004575A2 (fr) 1996-07-31 1996-07-31 Analogues lipophiles d'oligonucleotides

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WO1998004575A3 WO1998004575A3 (fr) 1998-03-19

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US7704965B2 (en) 2002-06-26 2010-04-27 The Penn State Research Foundation Methods and materials for treating human papillomavirus infections
EP3211126B1 (fr) 2009-02-13 2020-10-21 X-Chem, Inc. Procédés de création et de criblage de bibliothèques de codage adn
EP3000898B1 (fr) 2013-05-21 2020-11-18 Hitgen Inc. Procédé de capture de cibles de médicaments
EP3914704B1 (fr) 2019-01-22 2023-06-07 Vipergen APS Procédé de criblage d'une bibliothèque d'affichage in vitro dans une cellule

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AU778737B2 (en) 1999-04-14 2004-12-16 Musc Foundation For Research Development Tissue-specific and pathogen-specific toxic agents and ribozymes

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US5484908A (en) 1991-11-26 1996-01-16 Gilead Sciences, Inc. Oligonucleotides containing 5-propynyl pyrimidines

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US7704965B2 (en) 2002-06-26 2010-04-27 The Penn State Research Foundation Methods and materials for treating human papillomavirus infections
EP3211126B1 (fr) 2009-02-13 2020-10-21 X-Chem, Inc. Procédés de création et de criblage de bibliothèques de codage adn
US11168321B2 (en) 2009-02-13 2021-11-09 X-Chem, Inc. Methods of creating and screening DNA-encoded libraries
EP3000898B1 (fr) 2013-05-21 2020-11-18 Hitgen Inc. Procédé de capture de cibles de médicaments
EP3914704B1 (fr) 2019-01-22 2023-06-07 Vipergen APS Procédé de criblage d'une bibliothèque d'affichage in vitro dans une cellule

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WO1998004575A3 (fr) 1998-03-19
CA2261704A1 (fr) 1998-02-05

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