JPH11502818A - Thionotriester-modified antisense oligodeoxynucleotide phosphorothioate - Google Patents

Abstract

  (57) [Summary] Antisense oligonucleotides with improved cell uptake, increased nuclease resistance, and thermodynamically more stable target binding ability have been disclosed. These new oligonucleotides are characterized by having 1 to 10 thionotriester phosphorothioates with a lipophilic moiety.

Description

DETAILED DESCRIPTION OF THE INVENTION Thionotriester-modified antisense oligodeoxynucleotide phosphorothio Eat                                Background of the Invention Field of the invention   The present invention relates to modified oligonucleotides, methods for their synthesis, and gene expression. To methods of their use for inhibiting. Summary of related technologies   Antisense oligonucleotides and their modified cognates control gene expression. To control (ZAMECNIK in Projects for Anti) Sense Nucleic Acid Therapy for Cance r and AIDS, pp. 1-6 (Wikstrom, E, Ed., Wil eye Lis, N.M. Y. Agrawal, Trends i, 1991); nBiotechnology 10, 152-158 (1992); Wicks tom, Trends in Biotechnology 10, 281-28 6 (1992); Rapport et al., Proc. Natl. Acad. Sci . USA 89, 8577-8580 (1992); (19 91) Projects for Antisense Nucleic Acid   Therapy for Cancer and AIDS, pp. 145- 158, supra)) and HIV (Agrawal et al., Proc. Natl. Acad. Sci. USA 85, 7079-7083 (1988); al et al., Proc. Natl. Acad. Sci. USA 86, 7790-77 94 (1989); Agrawal and Sar in Advanced Dr. ug Delivery Reviews, pp. 251-270 (Julia no, R .; J. , Ed. , Elsevier, Amsterdam 1991). Agrawal and Tang, Antisense Res. Dev. 4,2 61-266 (1992); Liziewize et al., Proc. Natl. Ac ad. Sci. USA 89, 11209-11213 (1992); and Liz. iewidth, Proc. Natl. Acad. Sci. USA 90,386 0-3864 (1992)), influenza virus (Leiter et al., Pr. oc. Natl. Acad. Sci. USA 87, 3430-334 (1990) )), Human papillomavirus (Vickers et al., Nucleic Acids). Res. 19, 3359-3368 (1991)), and herpes simplex virus ( Gao et al., Antimicrob. Agents Chem. 34, 808-8 12 (1990)). Was shown in tissue culture studies. Use antisense oligonucleotides as drugs To be stable under biological and physiological conditions, The ability to bind effectively and to be easily taken up by cells Necessary for development.   Significant efforts have recently been made to meet this criterion, Nates, phosphoamidates, phosphotriesters (Koziolkiewicz)   and Wilk, in Protocols for Oligonucl eotides and Analogs Vol. 20: Protocols   for Oligonucleotides and Analogs, pp . 207-224, supra, and references the oligonucleotides, including those that are non-phosphate internucleoside linkages, Structural modifications of nucleotides (Uhlmann and Peyman, Chem. R ev. 90, 543-584 (1990) and references therein; Methods i. n Molecular Biology Vol. 20: Protocols   for Oligonucleotides and Analogs (Agraw al, S.M. , Humana Press, New Jersey, 1993) and And references cited therein; Antisense Research and Appl ications (Crooke, ST and Lebleu, B., E ds. , CRC Press, Ann Arbor 1993) and references cited therein. ).   Miller et al., Biochem. 21, 5468 (1982) is internal ethyl Both isomers of the decamer with phosphotriester linkages have been disclosed. 10 mer After addition of a polyadenylic acid terminal to the 3'-terminal of E. col It was found that polymerization was inhibited by iDNA polymerase I.   Stec et al., Tetrahedron Lett. 26, 2191 (1985) ) Discloses dimers linked by isopropyl phosphorothioate triester groups did. These dimers form the 3'-O-isopropyl phosphomorpholidite intermediate It was synthesized through.   Hau et al., Tetrahedron Lett. 32, 2497 (1991) To form the corresponding neopentyl phosphorothioate triester dimer Elemental sulfur in pyridine (S₈) With O-neopentyl phosphite thiol Disclosure. The four of these dimers then go through a phosphodiester bond, Alternating phosphodiester and neopentyl phosphorothioate triester linkages. Conjugated to yield a 3'-atalysine-capped octamer with union.   Letsinger et al., Proc. Natl. Acad. Sci. USA 86 , 6553 (1989) discloses a single 3'-terminal cholesteryl phosphorothioate. Trie Oligonucleosides having interstereonucleoside linkages have been disclosed. They are cells Fusion formation and viral proteins p17, p24 and HI in MOLT-3 cells Inhibition of reverse transcription expression of VI was observed. The best results were found with the 20 mer , 10-mers and heptanucleotides were also active. These results show that the 10-mer Coupled with the observation that the activity of It implied that the mechanism was working.   Letsinger et al., Nucleic Acids Res. 14,348 7 (1986) disclose an O-phosphotrie with a C2-C4 trichloroalkyl moiety. Nucleotide dimers with ster and phosphoramidate internucleotide linkages and The trimer was synthesized. They found that these dimers and trimers were relatively stable Was found to bind to nucleotides.   Enzymatic degradation of oligonucleotide phosphorothioates is mainly from the 3 'end is there. Efforts around this problem involved oligonucleotide phosphorothioate 3 ' Various modifications at the ends were made. Koziolkiewicz et al., Supra; Temsamani et al., Antisense Res. Dev. 3,277 ( 1993); Mackelar, et al., Nucleic Acids Res. 2 0, 3411 (1992); and Tang et al., Nucleic Acid. s Res. 21. 2729 (1993).   Antecedents have developed oligonucleotides that increase their effectiveness for hybridization to target nucleic acids. He showed continued interest in emitting. Such oligonucleotides have strong cellular uptake High stability when bound to target, and high resistance to nuclease attack You.                                Summary of the Invention   The present invention provides increased cell uptake, higher stability when bound to target nucleic acids, and increased A novel class of modified antisense oligonucleotides with high resistance to ase attack Consist of types. One to ten of these novel antisense oligonucleotides Has a thionotriester phosphorothioate internucleotide linkage. Be signed. These internucleotide linkages have the following structure: Here, R represents a linear or branched alkyl group, a cholesteryl derivative, or an adamanti. Large lipophilic moieties such as thiol derivatives. One or more of these types Oligonucleotides with linkages are T4 polymerase and DNA polymerase I 2 shows an increase in resistance to degradation by. They are also oligonucleotide phosphorothio It shows an increase in melting point when paired with the target nucleic acid compared to oate.   The oligonucleotides of the present invention can be used both in vitro and in vivo. Useful for suppressing nucleic acid expression. Use of this oligonucleotide in vitro Some are identified by regulating the expression of the gene encoding the protein under study. Of proteins play a role in biological processes. Most biochemistry known today Elucidation of critical pathways achieved by isolating and studying deletion mutants in vitro Was done. However, studying deletion mutants is difficult. The claimed oligonucleotide Tide does not regulate gene expression using antisense oligonucleotides. An attractive alternative, as it is much less labor than using the deletion mutation approach I will provide a. Thus, the oligonucleotide of the present invention is useful as a research tool. It is for.   The oligonucleotides of the invention are equally suitable for in vivo applications. You. Increased stability of oligonucleotide-target pairing complex toward nuclease attack Increased resistance to and uptake of cells of the present invention A wide variety of diseases caused by pathogens, including viral and bacterial Ideal for treating infected infections. Designed for in vivo applications, The disclosed nucleotides are sufficiently complementary in sequence to the pathogen nucleic acid (target nucleic acid) region. , Both causing pairing to the target nucleic acid and suppression of its expression under intracellular conditions Both have one region.   Due to its gene regulation effectiveness, the oligonucleotides of the present invention are It is also useful for treating diseases arising from genetic abnormalities that result in insufficiency or overexpression. It is for. For diseases in which the abnormal gene is overexpressed, for example, Gonucleotides directly target the abnormal gene, or alternatively, It is designed to target genes that encode proteins that promote expression. vice versa Encodes a protein that suppresses abnormal gene expression when the abnormal gene is under-expressed Design an oligonucleotide that suppresses the expression of the gene to be expressed.   In summary, the claimed oligonucleotides need to regulate gene expression In any case essentially both in vitro and in vivo Useful.   The invention also relates to the oligonucleotides and pathogen diseases of the invention and to abnormal gene expression. To use oligonucleotides to treat other abnormal conditions arising from the present And a novel compound comprising the method of   The prior art merely summarizes certain aspects of the present invention and should not be construed as It is not intended to limit the invention in any way. All patents and patents cited in this specification And other publications are hereby incorporated by reference in their entirety.                              Brief description of figures   Figure 1. Production of oligonucleotides with S-triester internucleotide linkage 1 shows a synthetic scheme for preparing a phosphoramidite intermediate useful in the present invention.   FIG. 2 shows a modified S-triester phosphorothioate oligonucleotide modified by T4 polymerase. 1 shows an autoradiogram of the digestion of a gonucleotide.   FIG. 3 shows O-ethyl triester phosphorothioate by DNA polymerase I. 3 shows an autoradiogram of the digestion of the stomach.   FIG. 4 shows normal phosphorothioate and O-ethyltrithioate by T4 polymerase. Autoradiogram of digestion of the ester phosphorothioate oligonucleotide Show.                       Detailed Description of the Preferred Embodiment   The present invention provides, inter alia, modifications as compared to prior art antisense oligonucleotides. It consists of a new class of antisense oligonucleotides with good properties. Oligonucleotides with large lipophilic groups increase sensitivity to cell uptake , Increased resistance to exonuclease degradation, and oligonucleotides and their targets 2 shows an increase in the stability of the paired complex formed during the process. These properties allow these Ligonucleotides are used for gene expression both in vitro and in vivo. Ideal to adjust.   2 to 100 oligonucleotides according to the invention in length And any of the following nucleotides: In a preferred embodiment, the oligonucleotide is Tides may be from 13 to 50 nucleotides in length. More favorable fruit In embodiments, the oligonucleotide is 20 to 35 nucleotides in length. Will.   In one embodiment, the present invention provides an oligodeoxynucleotide having the structure Phosphorothioate (S-triester phosphorothioate) internucleotide linkage Antisense oligos having 1 to 10 thionotriesters Consists of nucleotides: Where R reduces the effectiveness of the oligonucleotide in regulating gene expression A large lipophilic group that does not substantially alter the size of the internucleotide linkages. Good In a preferred embodiment, R is C_Two~ C_{twenty two}Linear or branched alkyl group, having the following structure Cholesteryl derivatives: Where n = 2 to 12, an adamantyl derivative having the following structure: Where n = 2-12, 1,2-di-O-alkyl-rac-3 having the following structure -Glyceryl derivative: Where R₁Is C_nH_{2n + 1}And DL α-g having the following structure: Coferol (vitamin E) derivatives: Here, n = 0 to 10. An invention according to the invention having two or more S-triester internucleotide linkages. In a lignonucleotide, the R groups of the different bonds may be the same or different.   In another preferred embodiment, R is 1-adamantyl-2-ethyl, 1-hexa Groups consisting of decyl and cholesteryl-3-carboxyamino-6-hexyl Choose It is.   An oligonucleotide according to the invention is synthesized via a precursor having the following structure: Where R is as described above, B is a suitably protected base (if necessary), and X is H, F, benzyloxy, -OCH_TwoCH = CH_Two, Or linear or branched Where n = 1-5 C_nH_{2n + 1}Is an alkoxy having Figure 1 shows these intermediates 1 shows a schematic diagram for synthesizing, and a representative protocol is provided in the Examples below.   In one aspect of this embodiment, the oligonucleotide comprises 1 to 10 oligonucleotides. It has an S-triester phosphorothioate internucleotide linkage. Favorable fruit In embodiments, the oligonucleotide is at the 3 'or 5' end linkage, or 3 'and S-triester phosphorothioate internucleotide linkage at both 5 'end linkages Having. When the oligonucleotide of the invention has one or more S-triesters All of the S-triester linkages may have the same R substituent, May be different.   The number of S-triester bonds to be included in the oligonucleotide and the number of R substituents In the determination of identity, technicians found that increased lipophilicity resulted in reduced solubility in aqueous solutions. That waxing and steric interactions change conformations and may affect pairing think of.   The novel internucleotide linkages disclosed herein allow the oligonucleotides of the invention to Otides are resistant to nucleases and have bulky lipophilic groups, which Substantial increase in cellular uptake compared to inter-holothioate homologues However, it still has the ability to specifically pair to a complementary target nucleic acid under normal stringent conditions. You. 5 ° C and above melting point (T_m) Increases in the phosphorothioate homologue series 2. A 5- to 3-fold increase in cell uptake was observed with a corresponding increase. As shown below, Contrary to the expectation that these large substituents will interfere with pairing, the rise in melting point will be significant. It was found that an increase in melting point was observed even when a strong R moiety was present.   The antisense oligonucleotides of the present invention have many additional It may be designed to include features. For example, they are “self-stabilizing,” Has a first region that is sufficiently complementary to the second region to allow intramolecular pairing , Thereby providing oligonucleotides that are not susceptible to nucleolytic attack May be designed. Such oligonucleotides are available from PCT Internet ionical Application Pu replication No. WO94 / 01550. Alternatively, the oligonucleotides of the present disclosure may be PCT International   Application Publication No. WO94 / 170 91, “folded triple helix formation”, ie, complementary to the target nucleic acid, Between the first region and the double helix formed by the first region and the target nucleic acid Second region having a sequence that allows triple helix formation by Hoogsteen base pairs It may be designed to have an area.   The oligonucleotides according to the invention can be used both in vitro and in vivo. Useful for applications. In vitro application, it is used as a research tool for gene function determination. And useful. Because they can be synthesized to be complementary to specific sequences, Ligonucleotides can be used to selectively inhibit target gene expression. Book Oligonucleotides are attractive and easily used mutations (eg, deletion mutations) It provides an alternative to the labor intensive method of gene inhibition due to variability. The importance of this Realize that elucidation of all currently known biological pathways has been determined by deletion mutations Is evaluated.   Oligonucleotides of the invention may also be used for physiologically involved diseases or expression of specific genes. Useful as a therapeutic for conditions. Oligonucles useful for treating diseases or conditions Reotide is sufficiently complementary to the target nucleic acid to bind under physiological conditions Have a unique nucleotide sequence. As used herein, the terms "complementary" and "ten `` Complementary to minute '' is used interchangeably and describes the sequence of the antisense oligonucleotide. When used to describe an oligonucleotide sequence Under the conditions of interest (eg, in vitro and physiological conditions), the target nucleic acid Means to inhibit the expression of Generally, oligos according to the invention Nucleotides are nucleic acids that are essential to biological processes (eg, genes or mRNA) Has a sequence complementary to As described in detail below, such a process is And the reproductive and metabolic processes of other pathogenic infectious agents. Otherwise, biological The process may be, for example, spermatogenesis in men requiring fertility control and ovulation in women, It can be naturally occurring requiring its inhibition. Oligonucleotides of the invention May also be genes whose expression causes or is involved in a pathological or other abnormal condition in an organism. Or it may be complementary to other nucleic acids.   Due to their effectiveness in gene regulation, the claimed oligonucleotides Also useful for treating diseases caused by genetic abnormalities that cause under-expression or over-expression of genes It is. For diseases in which the abnormal gene is overexpressed, for example, Nucleotides may be used to target the abnormal gene directly, or alternatively, Designed to target genes that encode proteins that promote offspring expression Good. Conversely, if the abnormal gene is under-expressed, Oligos that suppress the expression of genes encoding proteins that suppress the expression of abnormal genes Nucleotides may be designed.   In many cases, the target nucleic acid sequence will be a viral nucleic acid sequence. Blocks various viruses The use of harmful antisense oligonucleases is well known and is described by Agrawal, Tibtech 10, 152 (1992). Effective antisense The viral nucleic acid sequence paired with the lignonucleotide is human immunodeficiency virus type 1 ( U. S. Patent No. 4,806,463), herpes simplex virus (U. S. Patent No. 4,689,320), influenza virus (U . S. Patent No. 5,194,428), and human papillomavirus ( Story et al., Nucleic Acids Res. 19: 4109-4 114 (1991)). These nuclei A sequence pair for any of the acid sequences will match a nucleic acid sequence from any other virus. Can be used so that complementary nucleotide sequences are possible. Depart from it The addition of a nucleic acid sequence that allows the synthesis of antisense oligonucleotides Additional viruses include, but are not limited to, foot-and-mouth disease virus (Robertson et al., J. Virology 54, 651 (1985); Harris et al. Vi rotry 36, 659 (1980)), yellow fever virus (Rice et al.) , Science 229, 726 (1985)), VZ virus (Dav is on and Scott, J.M. Gen. Virology 67, 2279 (1 986)), cucumber virus (Richards et al., Virology). 89, 395 (1978)), hepatitis B virus (Raney and M). cLachlen, in Molecular Biology of Hep atitis B Yirus (see CRC Press, 1991)), type C Hepatitis virus (Miller and Purcell, Proc. Natl. Acad. Sci. USA 87, 2057 (1990); Proc. Natl. Acad. Sci. USA 89, 4942 (1992); General Virology 74, 661 (1993)), and respiratory scintillation virus (Collins, in The Paramyxo Viruses, Ch) after4, pp. 103-162 (David W. Kingsbury, Ed. , 1991)).   Alternatively, the oligonucleotide of the invention is a nucleic acid complementary to the nucleic acid sequence of the pathogenic organism. Has an array. The nucleic acid sequences of many pathogen organisms are found in malaria organisms, Plasmod. ium falciparum, and many pathogenic bacteria have been described. Pathogenicity with a known nucleic acid sequence against which this oligonucleotide can be synthesized Examples of eukaryotes include, but are not limited to, Trypanosoma bruceig ambiance and Leishmania (See Campell et al., Nature 311, 350 (1984)), And Kantetsu (Zurita et al., Proc. Natl. Acad. Sci. U. SA84, 2340 (1987)). Antifungal oligonucleotides For example, a nucleotide sequence that is complementary to a nucleic acid sequence from a chitin synthase gene The antibacterial oligonucleotide according to the invention is, for example, It can be synthesized using a racemase.   In yet another embodiment, the oligonucleotide is a cellular gene or gene transcription, Has a nucleotide sequence complementary to the product that results in abnormal expression or disease state You. The nucleic acid sequence of some of these cellular genes is the prion protein (Stahl) and Prusiner, FASEB J. et al. 5, 2799 (1991)), A Amyloid-like protein associated with lzheimer's disease (U.S. Patent N o. 5,015,570) and c-myb, c-myc, c-abl and n -Including various well-known oncogenes and proto-oncogenes such as ras Was.   In addition, it may affect spermatogenesis, sperm motility, sperm binding to eggs or sperm viability. In other stages, the synthesis of structural proteins or enzymes that are mainly or exclusively included Inhibiting oligonucleotides are used as male fertility controls. Similarly, women Of fertility is regulated by the hormones involved in ovulation, fertilization, implantation, or in those processes. Biosynthetic smell Oligonucleotides that inhibit the production of proteins or enzymes contained therein. High blood Pressure is an angiotease that converts enzymes or enzymes related to the renin / angiotensin form Can be controlled by oligonucleotides that inhibit synthin synthesis; Use in cardiovascular and cerebral circulatory insufficiency, infarction, arteriosclerosis, embolism and thrombosis Can be controlled by inhibiting the synthesis of enzymes required for the synthesis of thromboxane A2; Deposition of cholesterol on the vascular wall can be controlled by inhibiting the synthesis of fatty acyl coenzyme A Cholesterol acyltransferase in arteriosclerosis; Inhibition of the synthesis of sufotransferase may be useful for hypolipidemia. Orient of the present invention Many gods that gonucleotides can use to reduce or eliminate the malignant effects of a disorder I have a transitory disorder. For example, inhibition of monoamine oxidase synthesis is described by Parkins can be used for on-disease; inhibition of catechol O-methyltransferase is Can be used for treatment; and inhibition of indole N-methyltransferase is Can be used to treat schizophrenia. Arachidonic acid cascade (prostaglandin and Inhibition of selected enzymes in leukotrienes) can lead to platelet aggregation, allergy , Inflammation, pain, and control of asthma. For development of resistance to various anticancer drugs And the major obstacle in chemotherapy, the multidrug resistance (mdr) gene Suppression of the expressed protein would prove beneficial in treating cancer. This From any of these genes A nucleotide sequence complementary to a nucleic acid sequence can be any cellular gene or gene transfer. Oligonucleotide sequences complementary to transcription, aberrant expression or production resulting in disease state As can be used for oligonucleotides according to the invention.   Antisense regulation of gene expression in plant cells is described in U.S. Pat. S. Patent N o. 5, 107, 065.   The nucleotide sequence of the oligonucleotide is essentially the Wat The oligonucleotides of the invention can be adapted to form oson-Crick base pairs. The therapeutic spectrum of tides should be very broad. Certain diseases are of particular interest. You. For example, various viral diseases include AIDS, ARC, oral or genital herpes. , Papilloma warts, influenza, foot and mouth disease, yellow fever, chickenpox, shingles, HTLV leukemia One or more S-triester phosphorothioates, including disease and hepatitis Treated with an oligonucleotide having an internucleotide linkage. Ori according to the invention Among the fungal diseases that can be treated with gonucleotides, candidiasis, histoplasmosis, Cryptococcosis, Bacillus subtilis, Aspergillosis, Sporotorrosis, Chr omomycosis, Dematophytosis, and Coccidio Idomycosis. Methods include Rickett's disease (eg, typhus, Ro) cky Mountain Spotted Fever) To treat sexually transmitted diseases caused by trachoma and lymphogranulomas Also used for Various parasitosis include amoebiasis, Chegas disease, toxopra Zuma disease, Pneumocytosis, Gyardiassis, Crypto sporodiosis, trichomoniasis, and carinii pneumonia; Can be treated with oligonucleotides. Ascariasis, filariasis, trichinosis, schistosomiasis Also includes worms and parasites such as nematodes and tapeworm infections (parasitic diseases). Malaria is described in f alciparum, p. vivax, P.E. orale, or P.O. malari It can be treated with the oligonucleotides of the invention, whether or not they occur in ae. Up The infectious diseases identified in the above are all known as infectious agents for these diseases, Oligonucleotides having or more S-triester phosphorothioate linkages Can be treated with otide, and thus the oligonucleotides according to the invention are essentially Nucleic acid sequences that are essential nucleic acid sequences for transmission of infectious agents, such as genetic genes. It can be synthesized with nucleotide sequences that match. As used here, A child or nucleic acid is required for a biological process, without which no biological process occurs It is.   The following examples are provided for illustrative purposes only and limit the invention in any way As such, they are not intended and should not be construed.                                  Example   Unless otherwise indicated, the following applies to each of the embodiments shown below. Anhydrous Cetonitrile, tetrahydrofuran, dichloromethane, ethyl alcohol, 2- Propanol, pentane, triethylamine, 1-hexadecanol, tetrazo Cholesteryl chloroformate, 6-amino-1-hexanol, bis (diiso Propylamino) chlorophosphine and 1-adamantaneethanol are Aldr ich (Milwaukee, WI). Hexane, ethyl acetate, and And methanol are described in J. Am. T. Baker Inc. (Phillipsburg, N . J. ) Purchased from. 5'-DMT-deoxyadenosine (tBA) cyanoa Tyl phosphoramidite, 5'-DMT-deoxycytidine (tBA) cyanoe Tyl phosphoramidite, 5'-DMT-deoxyguanosine (tBA) cyano Ethyl phosphoramidite, 5 'DMT-thymidine cyanoethyl phosphoramida , Cap A, Cap B, activator and C-18 SEP-PAK cartridge Ledges were purchased from Millipore (Bedford, Mass.). BEAUC age reagent (3H-1,2-benzodithiol-3-one-1,1-dioxide ) Is R. I. Chemical (Orange, CA). Fluore CEJN-ON phosphoramidite is CLONTECH Laboratorie s, Inc. (Palo Alto, CA). Used for PACE Reagent (Sequegel sequence Stem; concentrates, diluents and buffers) are from National Diagnostics cs (Atlanta, GA).³¹P NMR spectrum (121 . 65Mhz) is Varian (Palo Alto, CA) UNITY300 (Chemical shift is 85% H_ThreePO_FourCorrected for). Thermal melting The data was obtained from a GBC920 UV-Vis spectrophotometer (Dandenong, Victor) ria3175, Australia). Oligonucleotide synthesis Was performed on an 8909 Expedite DNA synthesizer (Millipore). PAGE is an S2 type array gel electrophoresis apparatus (LIFE TECHNOLOGY, G Aethersburg, MD) and EC600-90 power supply (E-CAPPAR) ATUS CORPORATION, St. Petersburg, FL) Was done. Fluorescence was measured using a spectrofluorometer (PTI Technology, South) Brunswick, N .; J. ). UV absorption is UV-160AUV Measured on a spectrophotometer (SHIMADZU, Clumbia, MD). flow Cytometry data is available from EpicsXL (Coulter, Hialeah, F L). Preparative HPLC detects DYNAMX Model UV-C absorption (Dynamax Instrument Co., Woburn, MA) Waters (Milford, Mass.) 650E HPLC system Was. Flash column chromatography Is silica gel 60 F₂₅₄(Merck, Gibbstown, NJ) and Ricagel 60 F₂₅₄T. l. c. Was performed using Product at 264 nm Absorption or 5% in 5% sulfuric acid (NH_Four)₆Mo₇O_{twenty four}And 0.2% Ce (S O_Four)_Two(Dark blue in the case of an oxidizing compound) t. l. c. Visualize on Was done.                                 Example 1 Synthesis of cholesteryl-3-carboxyamino-6-hexanol   CH of cholesteryl chloroformate_TwoCl_Two(60 ml) solution was stirred with 6-amido No-1-hexanol (2.40 g, 20.5 mmol) and triethylamine (3.6 ml, 2.6 g, 25.6 mmol) CH_TwoCl_Two(60ml) solution At 0 ° C. The mixture was stirred overnight at room temperature. The reaction mixture removes the resulting salt Filtered to get rid. Then 100ml CH_TwoCl_TwoIs added to the mixture Was. The solution is NaHCO_ThreeAnd saturated NaCl solution, anhydrous Na_TwoSO_FourUp And filtered. The solvent was removed under reduced pressure to obtain a pale yellow solid crude product . The crude product was purified by flash silica gel chromatography (eluent: CH_TwoC l_Two/ CH_ThreeOH98: 2) and purified as a white solid as cholesteryl-3-ca. Obtained ruboxamino-6-hexanol; TLC R_f 0.23 (CH_TwoCl_Two / CH_ThreeOH 95: 5).                                 Example 2 N, N, N ', N'-tetraisopropyl-O-ethyl-phosphorodiamidite Synthesis of   Absolute ethanol (1.1 ml, 0.86 g, 18.7 mmol) in THF (4 m l) The solution was bis (diisopropylamino) chlorophosphine (5.0 g, 18 . 7 mmol) and triethylamine (3.92 ml, 2.84 g, 28.1 m) mol) in a stirred THF (19 ml) solution at 0 ° C. 1 hour at room temperature After stirring, the reaction mixture was filtered and the filtrate was concentrated to give an oil. Oil is anhydrous pentane And filtered. Solvent was removed under reduced pressure. The mixture is again Dissolved in 15 ml of anhydrous pentane and filtered to remove residual precipitate . The solvent was evaporated under reduced pressure to afford O 2 O as a colorless oil (4.83 g, 93.3% yield). -Ethyl-phosphordiamidite was obtained;³¹P NMR (CDCl_Three) 137. 79.                                 Example 3 N, N, N ', N'-tetraisopropyl-O-isopropyl-phosphordiami Synthesis of Dite   As shown in Example 2, O-isopropyl-phosphordiamidite was (Diisopropylamino) chlorophosphine (5.0 g, 18.7 mmol) , Triethylamine (3.92 ml, 2.84 g, 28.1 m) mol), anhydrous 2-propanol (1.43 ml, 1.13 g, 18.74 mm) ol) and THF (23 ml) using a colorless oil (4.00 g, 73.4% yield). );³¹P NMR (CDCl_Three) Δ 126.95.                                 Example 4 N, N, N ', N'-tetraisopropyl-O- (1-adamantyl-2-ethyl ) -Synthesis of phosphordiamidite   As described in Example 2, O- (1-adamantyl-2-ethyl) -phos Hordiamidite is bis (diisopropylamino) chlorophosphine (5.0 g, 18.7 mmol), triethylamine (3.92 ml, 2.84 g, 28 . 1 mmol) 1-adamantane ethanol (3.38 g, 18.74 mmol) l) and a white solid (6.60 g, 86.3% yield) using THF (31 ml). Obtained as³¹P NMR (CDCl_Three) Δ136.68.                                 Example 5 N, N, N ', N'-tetraisopropyl-O- (cholesteryl-3-carboxy Synthesis of (Cyamino-6-hexyl) -phosphordiamidite   As described in Example 2, O- (cholesteryl-3-carboxyamino- 6-hexyl) -phosphordia Mididite was bis (diisopropylamino) chlorophosphine (1.56 g, 5 . 84 mmol), triethylamine (1.22 ml, 0.89 g, 8.76 m) mol), cholesteryl-3-carboxyamino-6-hexanol (3.10 g, 5.84 mmol) and THF (3.6 ml) using a colorless viscous oil (4. 38 g, 98.4% yield).³¹P NMR (CDCl_Three) 136 , 17.                                 Example 6 N, N, N ', N'-tetraisopropyl-O- (1-hexadecyl) -phospho Synthesis of ludiamidite   As described in Example 2, O- (1-hexadecyl) -phosphorodiamidi Is bis (diisopropylamino) chlorophosphine (5.0 g, 18.74). mmol), triethylamine (3.92 ml, 2.84 g, 28.1 mmol) ), 1-hexadecanol (3.38 g, 18.74 mmol) and THF (2 9 ml) as a colorless oil (7.82 g, 88.1% yield);³¹P  NMR (CDCl_Three) Δ136.28.                                 Example 7 5'-dimethoxytrityl-2'-deoxythymidine-N, NO-ethylphos Synthesis of foramidite (5'-DMT-dT-O-ethyl-phosphoramidite) Success   O-ethyl-phosphordiamidite (1.02 g, 3.67 mmol) is CH_Two Cl_TwoOf 5'-DMT-T (1.0 g, 1.84 mmol) in (5.0 ml) The stirred suspension was added in one portion under nitrogen. Acetonitrile (4.7 ml )) Was added dropwise to a solution of tetrazole (0.129 g, 1.84 mmol) in A compound was produced. After the mixture was stirred at room temperature for 5 hours, the solvent was removed under reduced pressure To give a foamy solid. The crude product was purified by flash column chromatography (eluent: CH_TwoCl_Two/ EtOAc / Et_ThreeN70: 20: 10) and purified at -78 ° C. Precipitated from hexane, 5'-DM as a white foam (0.62 g, 47.0% yield) T-dTO-ethyl-phosphoramidite was obtained; TLC R_f0.66 (CH_Two Cl_Two/ EtOAc / NEt_Three  45:45:10);³¹P NMR (CDCl_Three ) Δ157.73, 158.52.                                 Example 8 (5'-Dimethoxytrityl-N '-(4-tert-butylphenoxy) ace Tyl-2'-deoxycytidine-N, N-diisopropyl-O-ethyl-phospho Luamidite (5'-DMT-dC^tBA-O-ethyl-phosphoramidite) Synthesis   CH_TwoCl_TwoO-ethyl-phosphordiamidite (1.71) in (9.7 ml). g, 4.64 mmol) in CH_TwoCl_Two5'-DMT-d in (11.3 ml) C^tBA (2.25 g, 3.08 mmol) to a stirred pale yellow suspension under nitrogen. Was. Tetrazole (0.216 g, 3.0) in acetonitrile (9.7 ml). (8 mmol) was added dropwise to the resulting mixture. The mixture is stirred at room temperature for 5 hours. After evaporation, the solvent was removed under reduced pressure to give a foamy solid. Crude product is flash color Chromatography (eluent: CH_TwoCl_Two/ EtOAc / Et_ThreeN₇0:20: Purified in 10) and precipitated from hexane at -78 ° C to give a white foam (1.63 g, 5 5'-DMT-dC as 8.5% yield)^tBA-O-Ethyl-phosphoramidite TLC R_f 0.80 (CH_TwoCl_Two/ EtOAc / NEt_Three= 45: 45:10);³¹P NMR (CDCl_Three) 158.20, 159.08.                                 Example 9   5'-dimethoxytrityl-2'-deoxythymidine-N, N-diisopropyl Le-O-isopropyl-phosphoramidite (5'-DMT-dT-O-isoprop Synthesis of ropyl phosphoramidite)   CH_TwoCl_TwoO-isopropyl-phosphordiamidite (0. . 80 g, 2.8 mmol) in CH_TwoCl_Two5'-DMT- in (5.0 ml) To a stirred suspension of T (1.0 g, 1.9 mmol) was added under nitrogen. A Tetrazole (0.129 g, 1.84 mmol) in setonitrile (5.7 ml) l) solution is raw Was added to the mixture. After the mixture was stirred at room temperature for 5 hours, the solvent was removed under reduced pressure. Removed to give a foamy solid. The crude product is purified by flash column chromatography ( Eluent: CH_TwoCl_Two/ E_tOA_c/ Et_ThreeN 70:20:10). Precipitated from hexane at 8 ° C. as a white foam (0.66 g, 48.7% yield) 5 ′ -DMT-dT-O-isopropyl-phosphoramidite was obtained;³¹P NMR (CDCl_Three) Δ156.63, 157.14.                                Example 10 5'-Dimethoxytriyl-N '-(4-tert-butylphenoxy) acetyl 2'-deoxycytidine-N, N-diisopropyl-O-isopropyl-e Suphoramidite (5'-DMT-dC^tBA-O-isopropyl phosphoramida Synthesis   O-isopropyl-phosphordiamidite (0.60 g, 2.06 mmol) CH_TwoCl_Two(2.0 ml) solution under nitrogen with 5'-DMT-dC^tBA(1.0 g, 1.37 mmol) CH_TwoCl_Two(5.0ml) Stirred pale yellow tendon of solution The suspension was added. To the resulting mixture was added tetrazole (0.096 g, 1.37). mmol) in acetonitrile (4.3 ml) was added dropwise. The mixture is at room temperature After stirring for 5 hours, the solvent was removed under reduced pressure to give a foamy solid. The crude product is Flash column chromatography (eluent: CH_TwoCl_Two/ EtOAc / Et_Three N70: 20: 10) and precipitated from hexane at -78 ° C to give a white foam ( 0.84 g, 67.1% yield) as 5'-DMT-dC^tBA-O-Isopropyl Le-phosphoramidite was obtained; TLCR_f0.75 (CH_TwoCl_Two/ EtOAc / Et_ThreeN45: 45: 10);³¹P NMR (CDCl_Three) 157.24, 1 57.56.                                Example 11 5'-dimethoxytriyl-2'-deoxythymidine-N, N-diisopropyl -O- (1-adamantyl-2-ethyl) -phosphoramidite (5'-DMT Synthesis of (-dT-O-1-adamantyl-2-ethyl-phosphoramidite)   O- (1-adamantyl-2-ethyl) -phosphordiamidite (1.14 g , 2.76 mmol) CH_TwoCl_Two(5.7 ml) solution is 5'-DMT-dT (1.0 g, 1.8 mmol) CH_TwoCl_Two(5.0 ml) stirred solution The suspension was added under nitrogen. Tetrazole (0.129 g, 1.84 mmol) A solution of in acetonitrile (5.7 ml) was added dropwise to the resulting mixture. The mixture is After stirring at room temperature for 5 hours, the solvent was removed under reduced pressure to give a foamy solid. Crude The product was purified by flash column chromatography (eluent: CH_TwoCl_Two/ EtOAc / Et_ThreeN 70:20:10) and precipitated from hexane at -78 ° C. White foam (0.86 g, 54. 5'-DMT-dT-O- (1-adamantyl-2-ethyl) as 7% yield) -Phosphoramidite obtained; TLC R_f 0.65 (CH_TwoCl_Two/ EtOAc / NEt_Three45:45:10);³¹P NMR (CDCl_Three) 157.93, 1 58.31.                                Example 12 5'-dimethoxytrityl-N '-(4-tert-butylphenoxy) acetyl Ru-2'-deoxycytidine-N, N-diisopropyl-O- (1-adamanti Ru-2-ethyl) -phosphoramidite (5'-DMT-dC^tBA-O- (1- Synthesis of adamantyl-2-ethyl) -phosphoramidite   O- (1-adamantyl-2-ethyl) -phosphordiamidite (0.85 g , 2.96 mmol) CH_TwoCl_Two(4.3 ml) solution is 5'-DMT-dC^tBA (1.0 g, 1.4 mmol) CH_TwoCl_Two(5.0 ml) of the solution The light yellow suspension was added under nitrogen. Tetrazole (0.096 g, 1. 37 mmol) in acetonitrile (4.3 ml) was added dropwise to the resulting mixture. Was. After the mixture has been stirred at room temperature for 5 hours, the solvent is removed under reduced pressure and the solid foam is removed. I got a body. The crude product was purified by flash column chromatography (eluent: CH_TwoC l_Two/ EtOAc / Et_ThreeN70: 20: 10) and purified at 78 ° C in hexane From white foam (0.83 g, 5 5'-DMT-dC as 8.6% yield)^tBA-O- (1-adamantyl-2- Ethyl) -phosphoramidite was obtained; TLC R_f 0.63 (CH_TwoCl_Two/ E tOAc / NEt_Three45:45:10);³¹P NMR (CDCl_Three) 158. 35, 158.67.                                Example 13 5'-dimethoxytrityl-2'-deoxythymidine-N, N-diisopropyl -O- (Cholesteryl-carboxyamino-6-hexyl) -phosphoramidi To (5'-DMT-dT-O- (cholesteryl-3-carboxyamino-6- Synthesis of xyl) -phosphoramidite)   O- (cholesteryl-3-carboxyamino-6-hexyl) -phosphordia CH2 of midite (2/1 g, 2.76 mmol)_TwoCl_Two(11.9 ml) solution , 5'-DMT-T (1.0 g, 1.84 mmol) CH_TwoCl_Two(5.0ml ) The solution was added to the stirred suspension under nitrogen. Tetrazole (0.129 g, 1.84 mmol) in acetonitrile (7.5 ml). Was dropped. After the mixture was stirred at room temperature for 5 hours, the solvent was removed under reduced pressure, A white foamy solid was obtained. The crude product was purified by flash column chromatography (eluent : CH_TwoCl_Two/ EtOAc / Et_ThreeN70: 20: 10) and a white foam ( 2.11 g, 95.2% yield) To 5'-DMT-dT-O- (cholesteryl-3-carboxyamino-6- Xyl) -phosphoramidite was obtained; TLC R_f 0.60 (CH_TwoCl_Two/ E tOAc / NEt_Three45:45:10);³¹P NMR (CDCl_Three) 158. 60, 158.82.                                Example 14 5'-dimethoxytrityl-N '-(4-tert-butylphenoxy) -ace Tyl-2'-deoxycytidine-N, N-diisopropyl-O- (cholesteryl -3-carboxyamino-6-hexyl) -phosphoramidite (5'-DMT -DC^tBA-O- (Cholesteryl-3-carboxyamino-6-hexyl) -e Synthesis of suformamidite   O- (cholesteryl-3-carboxyamino-6-hexyl) -phosphordia CH3 of midite (2.35 g, 3.09 mmol)_TwoCl_Two(13.1 ml) solution Is 5'-DMT-dC^tBA(1.5 g, 2.06 mmol) CH_TwoCl_Two(7 . 5 ml) To the stirred pale yellow suspension of the solution was added under nitrogen. Acetonit Dissolution of tetrazole (0.144 g, 2.06 mmol) in ril (8.4 ml) Liquid was added dropwise to the resulting mixture. After the mixture is stirred at room temperature for 5 hours, the solvent is Removed under reduced pressure to give a foamy solid. Crude product is flash column chromatography Raffy (eluent: CH_TwoCl_Two/ EtOAc / Et_ThreeN 70:20:10) as a white foam (2.58 g, 90.4% yield). 5'-DMT-dC^tBA-O- (Cholesteryl-3-carboxyamino-6-he Xyl) -phosphoramidite was obtained; TLCR_f0.63 (CH_TwoCl_Two/ Et OAc / NEt_Three45:45:10);³¹P NMR (CDCl_Three) 158.9 3, 159.25.                                Example 15 5'-dimethoxytrityl-2'-deoxythymidine-N, N-diisopropyl -O- (1-hexadecyl) -phosphoramidite (5'-DMT-dT-O- Synthesis of (1-hexadecyl) -phosphoramidite   O- (1-hexadecyl) -phosphorodiamidite (1.31 g, 2.76 m mol) CH_TwoCl_Two(5.7 ml) solution was added to 5 'DMT-T (1.0 g, 1. 84 mmol) CH_TwoCl_Two(5.0 ml) The solution was added to the stirred suspension under nitrogen. Was added. Acetonitrile of tetrazole (0.129 g, 1.84 mmol) A ril (5.7 ml) solution was added dropwise to the resulting mixture. Mixture for 5 hours at room temperature After stirring, the solvent was removed under reduced pressure to give a foamy solid. Crude product Column chromatography (eluent: CH_TwoCl_Two/ EtOAc / Et_ThreeN 7 0:20:10) and a colorless viscous oil (1.65 g, 98.0% yield). As 5'-DMT-dTO- (1-hexadecyl) -phosphoramidite Obtained; TLC R_f 0.63 (CH_TwoCl_Two/ EtOAc / NEt_Three45:45: 10);³¹P NMR (CDCl_Three) Δ159.00, 158.32.                                Example 16 5'-dimethoxytrityl-N '-(4-tert-butylphenoxy) -ace Tyl-2'-deoxycytidine-N, N-diisopropyl-O- (1-hexade Sil) -phosphoramidite (5'-DMT-dC^tBA-O- (1-hexadeci Synthesis of le) -phosphoramidite   O- (1-hexadecyl) -phosphordiamidite (0.97 g, 2.06 m mol) CH_TwoCl_Two(4.3 ml) solution is 5'-DMT-dC^tBA(1.0 g, 1.37 mmol) CH_TwoCl_Two(5.0 ml) Light yellow ken with stirring solution The suspension was added under nitrogen. Tetrazole (0.096 g, 1, 37 mmol )) In acetonitrile (4.3 ml) was added dropwise to the resulting mixture. blend After stirring at room temperature for 5 hours, the solvent was removed under reduced pressure to give a foamy solid. Crude The product was purified by flash column chromatography (eluent: CH_TwoCl_Two/ EtOAc / Et_ThreeN 70:20:10) and a pale yellow viscous oil (1.41 g, 93) 5'-DMT-dC^tBA-O- (1-hexadecyl) -phos Foramidite obtained; TLC R_f 0.62 (CH_TwoCl_Two/ EtOAc / NE t_Three 45:45:10);³¹P NMR (C DCI_Three) 158.71, 159.47.                                Example 17 Synthesis of O-alkyl-phosphoramidites   Methyl, ethyl, trifluoroethyl, isopropyl, and neopentyl derivatives The synthesis of a number of O-alkyl-phosphoramidites, including isomers, has been reported; Ko ziolkiewicz and Wilk in Protocols fo r Oligonucleotides and Analogs, supra Pp. 207-224 and cited examples. In a similar manner, the O-alkyl-phospho Ludiamidite is bis (diisopropylamino) chlorophosphine and alcohol It was synthesized by the following reaction. The purification of O-alkyl phosphoramidites requires Instead of vacuum distillation at fairly high temperatures, a corresponding The pentane solution was repeatedly filtered. The resulting O-alkyl-phosphordiamidi O-alkyl- with good and excellent yield by phosphidation of nucleosides A phosphoramidite was obtained. Figure 1 shows the synthesis of O-alkyl-phosphoramidite Show.                                Example 18 S-triester-phosphorothioate and O-triester-phosphotries Synthesis of telloligonucleotides   S-triester-phosphorothioate and O-tri Ester-phosphodiester oligonucleotides are conjugated with exocyclic amines (dA, dC , And dG) at the labile tert-butylphenoxyacetyl (tBA) group. Synthesized using the phosphoramidite of Example 17 in which the group was protected. Standard ben Increased instability of the tBA group over zoyl and isobutyryl protection increases base deprotection Use of mild conditions and solid support with ammonium hydroxide (room temperature, 2 hours) Allows release from the pair, which is the combination of O-ethyl, including oligonucleotides. It is of decisive importance. Oligonucleotides can be reverse phase HPLC and / or PAG Purified by E. S-triester-phosphorothioate compound no. 1.1 of³¹ P NMR spectra were obtained for S-triester-phosphorothioate and S-phos. The corresponding peaks for the holothioate internucleotide linkage are 62 and 53 p, respectively. pm. Ratio between S-triester and phosphorothioate Was 19.8: 100.0 (calculated value is 20: 100). O-phosphodies The content of tellurium bonds was less than 2.5%.                                Example 19 Oligonucleotide synthesis and purification   Unmodified or triester containing phosphorothioate and phosphodiester oligos All of the nucleotides were supplied by an automated synthesizer (Millipore 8909 Exped item, Bedford, MA) according to standard protocols on a 1 μmol scale. Was made. Modified phosphoramidite, 5'-DMT-dT-O-ethyl-phospho Luamidite, 5'-DMT-dC^tBA-O-ethyl-phosphoramidite, 5 ‘-DMT-dT-O-isopropyl-phosphoramidite, and 5 'DMT- dC^tBA-O-isopropyl-phosphoramidite is dried at a concentration of 50 mg / ml Dissolve in dry acetonitrile and add 5'-DMT-dT-O- (1-adam -Nethyl-2-ethyl) phosphoramidite, 5'-DMT-dC^tAB-Adaman Tyl-2-ethyl) -phosphoramidite, 5'-DMT-dTO- (choles Teryl-3-carboxyamino-6-hexyl) -phosphoramidite, 5'- DMT-dC^tBA-O- (Cholesteryl-3-carboxyamino-6-hexyl ) -Phosphoramidite, 5'-DMT-dT-O- (1-hexadecyl) -e Suformamidite and 5'-DMT-dC^tBA-O- (1-hexadecyl)- The phosphoramidite is dissolved in dichloromethane to a final concentration of 50 mg / ml. Dilute acetonitrile-dichloromethane with acetonitrile until complete (1: 1 ). For phosphorothioate oligonucleotides, iodo acid or step 3H-1, 2-benzodithiol-3-one-1,1-dioxide (Beaucage reagent ). J. Iyer et al. Org. Chem. 55. 4693 (1990). Ammonium chloride at room temperature Treatment for 2 hours with ammonium cleaves the oligomers from the support counterparts and removes the nucleotide bases. Made to deprotect. Oligonucleotides can be reverse phase HPLC and / or P Purified by AGE and desalted using a C-18 SEP-PAK cartridge. Fluorescein labeling of oligonucleotides   Fluorescein is FLUORESCEIN-ON^TMUsing phosphoramidites At the 5 'end of the oligonucleotide, either by an automated DNA synthesizer or by a manual method Composited. The effectiveness of fluorescein labeling was determined by spectrofluorometer (excitation 488 nm, emission (520 nm light).                                Example 20 Exonuclease resistance of triester-containing oligonucleotides   Sensitivity to 3 'exonuclease degradation was due to T4 DNA polymerase and / or Was determined by digestion with DNA polymerase I. Oligonucleotides tested Are listed in Table 1.   To study the resistance of T4 DNA polymerase, 5'-³²P-labeled oligo Nucleotides (45 pmole) were mixed with 30 μl of buffer (50 mM Tris, p H8.0, MgCl_TwoDissolved in 5 mM DTT, 0.05% BSA) at 37 ° C. With T4 DNA polymerase (7.5 units). Stop solution (95% Formamide, 10 mM EDTA, 0.05% bromophenol blue, 0.0 Inhibition with 6 μl of 5% xylene cyanol), aliquots (5 μl) were 0 and 6 Removed in 0 minutes, PAGE (20% polyacrylamide containing 8.3 M urea) And were followed by autoradiography. The results are shown in FIG.   To study the resistance of DNA polymerase I, 5'-³²P-labeled oligonucleotide Otide (30 pmole) was added to 20 μl of buffer (50 mM Tris, pH 8.0). 0, MgCl_Two, 5 mM DTT, 0.05% BSA) and DN at 37 ° C. Incubated with A polymerase I (5.0 units). 6 μl of stop solution (95% Formamide, 10 mM EDTA, 0.05% bromophenol blue, 0. Aliquots (5 μl) inhibited by 0, 60, 12 Removed at 0, 180 and 240 minutes, PAGE (20% po containing 8.3M urea) Acrylamide) and were followed by autoradiography. Typical The results are shown in FIG.   The result is that the modified S-triester-phosphorothioate is unmodified S-phosphorothioate It was clearly shown to be much more stable than Eat. Figure 4 Other modified S-trier Stele-phosphorothioate and O-triester-phosphodiester oligonucleotides Exonuclease resistance of nucleotides was also studied using T4 polymerase (Figure 2) Was done. O-triestes as well as modified S-triester-phosphorothioates Le-phosphodiester oligonucleotides also increase exonuclease resistance You can see that. S-triester-phosphorothioate is O-tries in cells May convert to ter-phosphodiesters (Wyrzykiewicz and Cole, Nucleic Acids Res. 22, 2667 (1 994) so this is important and increases the resistance of the O-triester-phosphodiester Provides a long-lasting in vivo effect.                                Example 21 Melting temperature (T_m)   Melting temperature is determined by the double helix of unmodified and modified oligonucleotides and complementary DNA Was decided. Each oligonucleotide (0.2A₂₆₀Unit) and its complementary DNA 1 ml buffer (10 mM Na_TwoHPO_Four, PH 7.4, 0.1 M NaCl) Heated to 80 ° C, slowly cooled and re-paired, then cooled to 40 ° C at a rate of 2 ° C / min Was. Mixture At a rate of 1 ° C./min to 80 ° C.₂₆₀Was recorded continuously. Oh Melting cross sections were obtained for the lignonucleotides.   The results are shown in Table 2. Changes in affinity can be directly attributed to modifications. S- Of a triester bond at the 3 'and / or 5' end of the phosphorothioate Modification reduces the total negative charge of the oligonucleotide, which may indicate enhanced pairing . As Table 2 shows, almost all of the modified S-triester-phosphorothioate Higher T compared to unmodified phosphorothioate (compound no. 1.0)_mWith I do. The only exception is four ethyl triester linkages at both the 3 'and 5' ends. Are chimeric oligomers (compound no. 1.2). High T_mIs nu This indicates that a change in the shape of the internucleotide bond has occurred. O-triester-phospho The melting temperature of the diester is that of the corresponding S-triester-phosphorothioate About 10 degrees higher, which is consistent with the results obtained from the unmodified oligonucleotide You. Uhlmann and Peyman, supra; and Metho ds in Molecular Biology 20: Protocols for Oligonucleotides and Analogs, sup ra.                                Example 22 Cell uptake   Cell culture: Human T cells and leukemia cell line H9 were used for the study. Those Is 10% fetal bovine serum (up to 56 ° C for 30 minutes to inactivate nucleases) Heat-inactivated), 2 mM glutamine, 100 μl streptomycin, 100 U / ml penicillin and 6 × 10^-FiveR reinforced with M 2-mercaptoethanol Air incubator (37 ° C, 5% CO2) in PMI medium_Two-95% O_TwoHumidified in) Cultured.   Cell uptake: Of fluorescein-labeled and unlabeled oligonucleotides in the sample Concentrations are measured by spectrofluorometer and UV spectroscopy, and the corresponding unlabeled oligonucleotide The same was adjusted by adding otide. Oligonucleotide complex (0.2 OD / 1 00 μl) of cells (5 × 10⁶Cells / ml, 0.5 ml), and Was set. After 4 hours of culture, aliquots of the cell doubling mixture are removed and washed. Hank equilibrium, supplemented with 0.1% BSA and 0.1% sodium azide Suspended in saline solution (HBSS). Propidium iodide (final concentration 10μ 1 / ml) was used to distinguish live cells from dead cells. 5,000 pieces Flow cytometry data for surviving cells from EpicsXL (Coul ter, Hialeah, FL) in list mode and the data is After gating for viable cells by side scatter versus side scatter and propidium iodide staining Analyzed with EpicsXL (version 1.5 software).   The results are shown in Table 3. Large lipophilic groups (eg, 1-adamantyl-2-e Chill, cholesteryl-3-carboxyamino-6-hexyl and 1-hexadecyl ) Modified S-triester-phosphorothioate enhances cell uptake Having.                                Example 23 Inhibition of CMV gene expression in rod culture   A series of modified CMV oligonucleotides have been synthesized according to the invention. These ori The gonucleotides are shown in Table 4.   Sequence shown in Table 4; 5'-TGGGGGCTTACCTTGCGAACA-3 '(UL36ANTI) is the UL36 / 37 intron-exon boundary and control. Direction oligonucleotide 5'-TGTTCGCAAGGGTAAGCCCCA- Complementary to 3 ', it is homologous to UL36 mRNA. These oligonucleotides Otides correspond to HCMV genome coordinates 49,565 to 49,584. Smi th and Pari, J.M. Virology 69, 1925 (1995). In addition, two other compounds were used to control the following experiment: 5'-TCTGGG TAATTACACGCAAGC-3 ', irrelevant, non-specific control oligonucleotide Tide and 5'-ACAAGCGTTCCATTCGGGGGT-3 ', meaningless system Control oligonucleotide, identical to UL36ANTI except in reverse order Column.   Human foreskin fibroblasts (HFF) were incubated for 24 hours in a 96-well microtiter plate prior to treatment. Wells on ital boards (Falcon, Franklin Lakes, NJ) Plated at a density of 5,000 cells per. Cells were prepared in CCM5 medium (Hyclon). e, Logan, Utah) at various specified concentrations. 15 with C Pre-processed until time. The growth medium is removed in pairs and cells are removed from any remaining oligos. Wash three times with Dulbecco's phosphate buffered saline to also remove nucleotides Was. Cells were infected with HCMV strain AD169 (AT) at a multiplicity of infection of 0.1 for 1 hour at 37 ° C. CC VR-538). Cells were washed again and used in preculture Fresh growth medium containing serial dilutions of antisense oligonucleotide at the same concentration as Was returned to Five to six days after infection (pi), cells were fixed (100% ethanol). Noll), HCMV UL44 gene product (Advanced Biotec) (Hnology, Rivers Park, IL) did. The cells were then labeled with horseradish peroxidase (kerkgaa). rd and Perry, Gaithersburg, MD) conjugated to a secondary antibody Reacts with various anti-mouse immunoglobulin G, develops with TMB substrate, Density plate reader (Ceres900, Biotek, Winooski) , VT).   These ELISA experiments showed that some of these modified oligonucleotides . When used at a low concentration of 08 μM, almost completely replicates HCMV DNA. Inhibitory. The results are shown in Table 5.

────────────────────────────────────────────────── ─── Continuation of front page    (81) Designated countries EP (AT, BE, CH, DE, DK, ES, FI, FR, GB, GR, IE, IT, L U, MC, NL, PT, SE), OA (BF, BJ, CF) , CG, CI, CM, GA, GN, ML, MR, NE, SN, TD, TG), AP (KE, LS, MW, SD, S Z, UG), AL, AM, AT, AU, AZ, BB, B G, BR, BY, CA, CH, CN, CZ, DE, DK , EE, ES, FI, GB, GE, HU, IS, JP, KE, KG, KP, KR, KZ, LK, LR, LT, L U, LV, MD, MG, MK, MN, MW, MX, NO , NZ, PL, PT, RO, RU, SD, SE, SG, SI, SK, TJ, TM, TR, TT, UA, UG, U S, UZ, VN (72) Inventor Tan, Jin Yang             United States 01545 Massachusetts             County Shrewsbury Sheridan             Live 19

Claims (1)

[Claims] 1. Has the following structure: Here, the R group of each thiono-triester phosphorothioate internucleotide linkage is independently a C ₂ -C ₂₂ linear or branched alkyl group, a cholesteryl derivative having the following structure: Where n = 2 to 12, an adamantyl derivative having the following structure: Where n = 2 to 12, a 1,2-di-O-alkyl-rac-3-glyceryl derivative having the following structure: Wherein R ₁ is C _n H _{2n + 1} and n = 2 to 22, or a DLα-tocopherol derivative having the following structure: Oligonucleotides consisting of 1 to about 10 thiono-triester phosphorothioate internucleotide linkages, where n = 0-10. 2. An oligonucleotide according to claim 1 having from 2 to 100 nucleotides. 3. An oligonucleotide according to claim 1 having from 13 to 50 nucleotides. 4. An oligonucleotide according to claim 1, having from 20 to 35 nucleotides. 5. 4. The oligonucleotide according to claim 3, wherein R is selected from the group consisting of 1-adamantyl-2-ethyl, 1-hexadecyl, and cholesteryl-3-carboxyamino-6-hexyl. 6. 5. The oligonucleotide according to claim 4, wherein R is selected from the group consisting of 1-adamantyl-2-ethyl, 1-hexadecyl, and cholesteryl-3-carboxyamino-6-hexyl. 7. 5. The oligonucleotide according to claim 4, having the formula 5'-TGGGGGCTTACCTTGCGAACA-3 '(SEQ ID NO2). 8. The infectious agent or the gene expression of an infectious agent in a cell comprising the oligonucleotide according to claim 1 and a pharmaceutically acceptable carrier, wherein the oligonucleotide is complementary to a region of the nucleic acid essential for replication of the infectious agent or expression of the gene. A compound for inhibiting the replication of 9. Gene expression of the infectious agent or intracellular infectious agent comprising the oligonucleotide according to claim 4 and a pharmaceutically acceptable carrier, wherein the oligonucleotide is complementary to a region of the nucleic acid essential for replication of the infectious agent or gene expression. A compound for inhibiting the replication of 10. 7. Gene expression of an infectious agent or intracellular infectious agent comprising an oligonucleotide according to claim 6 and a pharmaceutically acceptable carrier, wherein the oligonucleotide is complementary to a region of the nucleic acid essential for replication of the infectious agent or gene expression. A compound for inhibiting the replication of 11. 9. The composition according to claim 8, wherein the infectious agent is CMV. 12. A composition comprising an oligonucleotide according to claim 1 wherein the oligonucleotide is complementary to a region of the nucleic acid and a pharmaceutically acceptable carrier, the expression product of which results in a disease state or inhibits expression of a nucleic acid contained therein. 13. A composition comprising an oligonucleotide according to claim 4, wherein the oligonucleotide is complementary to a region of the nucleic acid and a pharmaceutically acceptable carrier, the expression product of which inhibits expression of the nucleic acid resulting in a disease state. 14． A composition comprising an oligonucleotide according to claim 6, wherein the oligonucleotide is complementary to a region of the nucleic acid and a pharmaceutically acceptable carrier, the expression product of which inhibits the expression of the nucleic acid resulting in a disease state. 15. An infectious agent or an intracellular infectious agent gene comprising contacting a cell with an effective amount of the oligonucleotide according to claim 1, wherein the oligonucleotide is complementary to a region of the nucleic acid that is essential for replication of the infectious agent or gene expression. A method of inhibiting expression replication. 16. The infectious agent or gene of an infectious agent in a cell comprising contacting a cell with an effective amount of an oligonucleotide according to claim 4, wherein the oligonucleotide is complementary to a region of the nucleic acid that is essential for replication of the infectious agent or gene expression. A method of inhibiting expression replication. 17． An infectious agent or intracellularly comprising contacting a cell with an effective amount of an oligonucleotide according to any one of claims 6, wherein the oligonucleotide is complementary to a region of the nucleic acid that is essential for replication of the infectious agent or gene expression. A method of inhibiting the replication of gene expression of an infectious agent. 18. A method of inhibiting expression of a nucleic acid, the expression product of which comprises contacting the nucleic acid with an effective amount of the oligonucleotide according to claim 1, wherein the oligonucleotide is complementary to a region of the nucleic acid. 19. 5. A method for inhibiting infectious agent or gene expression of an infectious agent comprising contacting a cell with an effective amount of the oligonucleotide according to claim 4, wherein the oligonucleotide is complementary to a region of the nucleic acid. 20. 7. A method for inhibiting infectious agent or gene expression of an infectious agent according to claim 6, comprising contacting the effective amount of the oligonucleotide with a cell, wherein the oligonucleotide is complementary to a region of the nucleic acid.