WO1993024507A1 - Conformationally restrained oligomers containing amide or carbamate linkages for sequence-specific binding - Google Patents

Abstract

The invention relates to novel modified nucleosides and oligomers, the construction thereof, and their use in oligonucleotide-based therapies. More specifically, the invention is to novel oligonucleotides having modified internucleoside linkages which are resistant to nucleases, having enhanced ability to penetrate cells, and which are capable of binding target oligonucleotide sequences in vitro and in vivo. The modified oligonucleotides of the invention are particularly useful in oligonucleotide-based therapies utilizing the modified oligonucleotides to interrupt protein synthesis or transcription or to otherwise inactivate messenger RNA or double stranded DNA.

Description

CONFO MATIONALLY RESTRAINED OLIGOMERS CONTAINING AMIDE OR CARBAMATE LINKAGES FOR SEQUENCE-SPECIFIC BINDING

Field of the Invention

The invention relates to novel modified oligonucleotides, the synthesis thereof, their use in oligomer-based therapies and their use as diagnostic reagents. More specifically, the invention is to oligomers having modified nucleosides which are resistant to nuclease, having enhanced ability to penetrate cells, and which are and which are capable of binding target base sequences .in vitro and in vivo. The modified oligonucleotides of the invention are particularly useful in oligonucleotide-based therapies utilizing the modified oligonucleotides to interrupt protein synthesis or transcription or to otherwise inactivate messenger RNA or double-stranded DNA.

Background of the Invention

The application of oligonucleotides and oligonucleotide analogs (oligomers) for therapeutic uses represents a relatively new development in drug design and discovery. Several different therapeutic approaches that utilize oligomers have been proposed.

One approach is based largely on interfering with gene expression through oligomer binding to a complementary RNA sequence. This application is known as "antisense" therapy because the oligomer base sequence is identical to the antisense strand of the gene that gave rise to the RNA (Uhlmann, E. , et al., Chem Reviews (1990) 9):543-584; and Stein, CA. , et al., Cancer Res (1988) 4_8:2659-2668) . Another approach, referred to herein as "triple helix" therapy utilizes oligomers that bind to duplex DΝA as detailed below. Binding to a target DΝA is sequence specific but involves different base pairing binding. Both antisense and triple helix therapies exert therapeutic effects via binding to nucleic acid sequences that are responsible for disease conditions. Such sequences are found in the genome of pathogenic organisms including bacteria, protozoa, yeasts, parasites, fungi or viruses or may be endogenous sequences (oncogenes, cytokines, etc) . By modulating the expression of a gene important for establishment, maintenance or elimination of a disease condition, the corresponding condition may be cured, prevented or ameliorated.

Another therapeutic approach that is based on the use of oligomers includes generation of "aptamerε" and is disclosed and claimed in commonly owned application nos. 745,215, 659,980 and 658,849. This approach utilizes oligomers that specifically bind to proteins thereby interfering with their function. The use of oligomers that mimic the structure of certain RΝA molecules that are bound by intracellular proteins has also been adduced as a therapeutic approach as described in international application no. PCT/US91/01822.

Antisense oligonucleotides are synthetic oligonucleotides which bind complementary nucleic acids (i.e. sense strand sequences) via hydrogen bonding, thereby inhibiting translation of these sequences. Therapeutic intervention at the nucleic acid level using antisense oligonucleotides offers a number of advantages. For example, gene expression can be inhibited using antisense or triple helix oligomers. Inhibition of gene expression is more efficient than inhibition of the protein encoded by the gene since transcription of a single DNA sequence gives rise to multiple copies of mRNA which, in turn, are translated into many protein molecules.

Antisense and triple helix therapies for diseases whose etiology is characterized by, or associated with, specific DNA or RNA sequences, are particularly useful. The oligomer employed as the therapeutic agent can be directly administered and is one that is complementary to a DNA or RNA needed for the progress of the disease. The oligomer specifically binds to this target nucleic acid sequence, thus disturbing its ordinary function.

An oligomer having a base sequence complementary to that of an mRNA which encodes a protein necessary for the initiation, maintenance or progress of the disease, is useful for interfering with synthesis of the protein. By hybridizing specifically to this mRNA, the synthesis of the protein will be interrupted. However, it is also possible to bind double-stranded DNA using an appropriate oligomer capable of effecting the formation of a specific triple helix by inserting the administered oligomer into the major groove of the double-helical DNA. The resulting triple helix structure can then interfere with transcription of the target gene (Young, S.L. et al., Proc Natl Acad Sci (1991) 88;10023- 10026). An important feature of therapeutic oligomers is the design of the backbone of the administered oligomer. Specifically, the backbone should contain internucleotide linkages or structures that are stable in vivo and should be structured such that the oligomer is resistant to endogenous nucleases, such as nucleases that attack the phosphodiester linkage. At the same time, the oligomer must also retain its ability to hybridize to the target DNA or RNA. (Agarwal, K.L. et al., Nucleic Acids Res (1979) -3009; Agarwal, S. et al., Proc Natl Acad Sci USA (1988) £5:7079.) In order to ensure these properties, a number of modified oligonucleotides have been constructed which contain alternate internucleotide linkages. Several of these oligonucleotides are described in Uhlmann, E. and Peyman, A. , Chemical Reviews (1990) 9_0:543-584. Among these are ethylphosphonates (wherein one of the phosphorous-linked oxygens has been replaced by methyl) ; phosphorothioates (wherein sulphur replaces one of these oxygens) and various amidates (wherein NH_ or an organic amine derivative, such as morpholidates or piperazidates, replace an oxygen) .

These substitutions confer enhanced stability, for the most part, but suffer from the drawback that they result in a chiral phosphorous in the linkage, thus leading to the formation of 2ⁿ diastereomers where n is the number of modified diester linkages in the oligomer. The presence of these multiple diastereomers considerably weakens the capability of the modified oligonucleotide to hybridize to target sequences. Some of these substitutions also retain the ability to support a negative charge and the presence of charged groups decreases the ability of the compounds to penetrate cell membranes. There are numerous other disadvantages associated with these modified linkages, depending on the precise nature of the linkage. It has also been suggested to use carbonate diesterε. However, these are highly unstable, and the carbonate diester link does not maintain a tetrahedral configuration exhibited by the phosphorous in the phosphodiester. Similarly, certain carbamate linkages, while achiral, confer trigonal symmetry and it has been shown that poly dT having this linkage does not hybridize strongly with poly dA (Coull, J.M., et al., Tet Lett (1987) 2^:745; Stirchak, E.P., et al., J Orσ Chem (1987) 52:4202. WO 91/15500, published October 17, 1991, teaches various oligonucleotide analogs in which one or more of the internucleotide linkages are replaced by a sulfur based linkage typically sulfamate diesters which are isosteric and isoelectric with the phosphodiester. WO 89/12060, published December 14, 1989, similarly discloses linkages containing sulfides, sulfoxides, and sulfones.

WO 86/05518, published September 25, 1986, suggests a variant of stereoregular polymeric 3',5' linkages.

U.S. Patent No. 5,079,151 to Lampson et al., discloses a sDNA molecule of branched RNA linked to a single strand DNA via a 2',5' phosphodiester linkage. 2',5' linkages that may be incorporated into oligomers are described in commonly owned, pending U.S. patent application attorney docket number 24610-20042, S. Swaminathan et al., inventors, filed June 1, 1992, the entire disclosure of which is incorporated herein by reference. Commonly owned, pending U.S. Patent Application

No. 690,786, filed April 24, 1991, the entirety of which is incorporated by notice, describes modified linkages of the formula -Y'CX^, ₂Y'- wherein Y' is independently O or S and wherein each X' is a stabilizing substituent and independently chosen.

Modifications of oligomers that enhance their affinity for target molecules will generally improve the therapeutic potential for those compounds. Previous approaches to improve binding affinity for complementary nucleic acids have centered primarily on (i) covalent linkage of intercalating agents to oligomers (Asseline, U., et al., Proc Natl Acad Sci (1984) 81-3297-3401), (ϋ) introduction of modified bases to form more stable base pairs (Inoue, H. et al., Nucl Acids Res (1985) 11:7119; commonly owned pending U.S. application serial numbers 07/799,842, 07/787,920 and 07,643,382; commonly owned pending U.S. application serial number 07/887,507 and (iii) altering the charge characteristics of oligomer internucleotide linkages (Letsinger, R.L. et al., J Am Chem Soc (1988) 110:4470; pending U.S. application serial numbers 07/806,710 and 07/990,848). Morpholino-type internucleotide linkages are described in U.S. Patent 5,034,506 and in some cases give rise to an increased affinity of the oligomer for complementary target sequences.

Commonly owned pending U.S. Patent Applications No. 07/763,130, 07/806,710 and No. 07/690,786 disclose modified oligonucleotides having modified nucleoside linkages which are capable of sequence specific hybridization to target RNA and DNA.

The modified internucleotide linkages described in the 07/806,710 and 07/990,848 disclosures were identified on the basis of computer modeling studies that defined sterically the size, bond lengths, angles and torsions that were compatible with the new linkages. Synthesis of modified linkages that were compatible with linkages predicted by the computer modeling studies was accomplished. When these linkages were introduced or incorporated into oligonucleotide analogs they were found to be binding competent.

Computer modeling studies based on the amide linkage (Nielsen, P.E. et al., Science (1991) 254:1497 1500) and the riboacetal type linkages that are described in application 07/806,710 and 07/990,848 revealed a set of oligomers, described herein, that fall within the structural domain of compounds that can be incorporated into oligomers which are binding competent. The compounds have a heterocyclic base covalently attached, but lack a furanose moiety and do not contain a phosphorus atom in the linkage between monomers. When incorporated into oligomers, the compounds give rise to oligomers that are more ordered toward a binding competent conformation as shown in Figures 8 - 13 in free solution than the amide linked oligomers previously described (Nielsen, P.E. et al., Science (1991) 254:1497- 1500) . A binding competent conformation, as used herein, refers to the spatial orientation of heterocyclic bases in an oligomer required for binding to duplex or single stranded DNA or RNA in a sequence-specific manner. Conformationally restricted oligomers containing binding competent riboacetal linkages with high affinity for binding to single strand as well as double strand target nucleic acid sequences were described in 07/806,710 and 07/990,848. The invention linkages were arrived at using modeling studies and were compared with the riboacetal linkage as shown in Figure 1. The riboacetal linkage studies verified computer model predictions that the conformational restriction could improve affinity in some cases. Nielsen et al., (Science (1991) 254:1497-1500) showed that an oligomer conjugate composed of amide linked nucleomonomers and a lysine residue had high affinity binding to single stranded oligonucleotides. Comparison of the riboacetal type linkage (Figure 1) with the amide-based linkage (Figure 1) , indicated that by substituting carbonyl (C3- 022) in the place of 03, N4 in the place of atom 4, NH(N7-H21) in the place of atom 7, carbonyl (C8-023) in the place of 08, N10 in the place of atom 10 and carbonyl (C10-024) in the place of atom 011 and by eliminating atoms 16,17,18 and 19, the polyamide-based oligomer in Figure 1 can be produced. It is to be noted that the elimination of atoms 16, 17, 18 and 19 removes the conformational restriction in the riboacetal linkage, but a new conformational restriction is introduced with the formation of the internal hydrogen bond between H21 and 022. In the modeling studies when the substitution of atoms and deletions were executed to convert the riboacetal linkage to the amide-based linkage, there was very little distortion from the riboacetal linkage structure and the hydrogen bond between H21 and 022 was naturally formed as shown in Figure l. Figures 2a shows a computer-generated structure for the riboacetal linkage. Figure 2b shows the structure obtained after minimization of atoms that were substituted in the riboacetal linkage to generate the amide linkage, while

Figure 2c shows a composite of both structures. As shown in Figure 2c, there is a nearly identical spatial overlap between the linkages when the structures are superimposed. The structural correspondence between the riboacetal linkage and the polyamide-based linkage is the most persuasive evidence supporting the underlying assumption regarding the structure of polyamide-based oligomers when bound to target nucleic acid sequences. The oligomers disclosed herein fall within the same spatial group of binding competent species as the amide and riboacetal structures. The novel insight into the postulated structure of the amide-linked oligomer as shown in Figure 1 coupled with the structural correspondence of the new class of amide and carbamate linkages as shown in Figure 2c with both amide and riboacetal linkages leads to the validation of the new oligomers described herein as binding competent species. Oligomers containing amide- or carbamate-linked monomers, "amide-linked oligomers", that are in a binding competent conformation will have improved target nucleic acid binding properties. The improved properties include an enhanced rate for binding which results in an increased affinity of the oligomer for its target nucleic acid sequence. Recognition of the conformation assumed by oligomers containing amide linkages when bound to a target nucleic acid in a sequence specific manner is the key discovery that underpins the rationally designed compounds described herein. The discovery of the binding competent conformation for amide-linked oligomers as described herein led to the discovery of a common structural motif that is shared by these new classes of^" high affinity oligomers, including new types of amide- linked oligomers, that can bind tc target nucleic acid sequences in a sequence specific manner. The oligomers of the present invention are generally characterized as being comprised of a series of constrained linkers or nucleomononers which correspond in space to εeven-membered rings or to seven-membered ring equivalents so as to attain a spatial conformation that is appropriate for binding of the heterocyclic base to a target nucleic acid in a sequence specific manner. As used herein, a seven-membered ring equivalent includes seven-sided structures or the equivalent of a seven-sided structure which may have one or more sides comprised of, for example, a hydrogen bond or a covalent structural equivalent that conforms to a seven-sided ring. As used herein, a seven-membered ring equivalent is any constrained arrangement of atoms which has a spatial conformation substantially identical to the formula:

The seven-membered ring and its equivalent, designated herein as X^a and referred to herein as the seven-membered ring equivalent, is a common structural motif in the oligomers of the present invention that can be arrived at through several different structural "modes". Such structural modes include structures, which when incorporated into oligomers, form a covalently linked structure that is a seven-membered ring or its - covalently linked equivalent. Such structures are exemplified by the oligomers shown in Figure 10 which represents a covalently closed seven-membered ring or Figure 11 which represents a covalently closed seven- membered ring equivalent. In addition, a conformation favoring the seven-membered ring or its equivalent can be attained in oligomers by structural modes that include (i) torsional constraints imposed by a covalent structure, such as a covalent ring, within the monomers that comprise an oligomer, as exemplified by the oligomers shown in Figures 9 and 13, and (ii) torsional constraints imposed by noncovalent forces such as hydrogen bonds, hydrophobic interactions, ionic interactions or steric constraints. In the case of a torsional constraint imposed by a noncovalent force such as a hydrogen bond, as exemplified by the oligomer shown in Figure 12, the hydrogen bond will favor a conformation in the oligomer that comprises a seven-membered ring equivalent that itself has as one of its sides, a hydrogen bond.

The previously described amide linkages (Nielsen et al., IScience (1991) 254.:1497-1500) can be characterized as having a single hydrogen bond that favors formation of the seven-membered ring or its equivalent that is required for a bonding competent conformation. The constrained linkers described herein, when incorporated into oligomers, can be characterized as having a force greater than a single hydrogen bond that favors formation of the binding competent conformation. In the case of a pyrollidone-linked oligomer, the forces favoring binding competence are a hydrogen bond and the cyclic connection between C3 and N4 which locks in the cis 022-C3-N4-C5 conformation in preference over the trans 022-C3-N4-C5 conformation about the C3 and N4 bond as defined in space in Figure 1. Hydroxymethyl-linked. oligomers have two hydrogen bondε that favor formation of a seven membered ring, wherein one of the hydrogen bonds is one εide of a complimentary seven-membered ring that drives formation of the seven-membered ring to which the heterocycle is attached. In the case of an azepine- or norbornyl-linked oligomer, a covalent bond fixes 100% of the heterocycleε in an oligomer into a binding competent conformation due to the preεence of the covalent seven- membered ring (azepine) or its covalent equivalent (norbornyl) .

The nucleomonomerε of the present invention are generally characterized as moieties or residues that replace both the furanose ring and the phosphorus atom that is normally found in nucleotides. The discovery of these nucleomonomers and their characteristicε is based on modeling studies that both (1) predicted such analogs are compatible with a binding competent oligomer and (2) defined the range of molecular characteristics that such nucleomonomers could assume without the loss of binding competence, when incorporated into oligomers. Binding competence, as used herein, refers either to Watεon-Crick baεe pairing with single-stranded DNA or single-stranded RNA or to Hoogsteen pairing (Beal, P.A. et al., Science (1991), 251:1360-1363 with duplex nucleic acids including duplex DNA or duplex RNA. Exemplary monomers and oligomers (and methods of their synthesis) of this invention are shown in Figures 3 through 13 and are conformationally more restricted relative to the phosphodiester linkages found in unmodified DNA or RNA. Thiε conformational reεtriction is believed to underline, at least in part, their capacity for enhanced binding to complementary nucleic acid target sequences. Incorporation of the nucleomonomers described herein into oligomers permits synthesis of improved compounds with respect to properties such as (i) increased lipophilicity which results from eliminating the charge asεociated with phosphodieεter linkageε (Dalge, J.M. et al., Nucleic Acids Res (1991) .19.:1805-1810) and (ii) resistance to degradation by enzymes such as nucleases. Consequently, oligomers containing theεe nucleoside analogs are quite suitable for hybridization to target sequences or molecules, and in some aspectε are superior to unmodified phosphodiester-linked nucleoside residueε when incorporated into oligonucleotideε.

The therapeutic potential of oligomers is generally enhanced by modifications that increase oligomer uptake by cells or reduce the rate of metabolism by cellε or serum. Such modifications include (i) reduced oligomer charge, (ii) increaεed stability toward nuclease activity, and (iii) increaεed lipophilicity of the oligomer. Oligomers as described in the invention exhibit sequence-specific binding to complementary single stranded and duplex target sequences. The present invention provides a serieε of nucleomonomers that can be incorporated into binding competent oligomers. The invention oligomers are resistant to nuclease digestion, are stable under physiological conditions and are neutral so as to enhance cell permeation. Both nuclease stability and enhanced cellular permeation are important considerations for the development of oligomers that are intended to be used as therapeutic agents that function by binding to specific DNA or RNA (mRNA, hnRNA, etc.) sequences. Such specific target sequence binding underlies their therapeutic efficacy by interfering with the normal biological function of nucleic acid sequences asεociated with pathological conditionε.

Summary of the Invention

The present invention is baεed on the identification of the rationally deεigned oligomerε disclosed herein and of novel nucleomonomers and methods for their incorporation into oligomers containing the nucleomonomers.

In one embodiment, the present invention iε directed to an oligomer which compriεeε the presence of one or more invention nucleomonomers which are discloεed herein and are exemplified by formulaε I - VIII:

35 where B is a purine or pyrimidine base or an analogous form thereof;

X¹ is S, O, SO, S0₂, CH₂, CHF, CF₂ NR or CH- lower alkyl including CH-methyl, CH-ethyl, CH-propyl and CH-butyl, provided that adjacent X¹ are not both 0; and each R is independently H, F, OH, OMe, CH₃ or lower alkyl including ethyl, propyl and butyl provided that both R attached to the same carbon atom are not both OH or are not OH and OMe together. In preferred embodiments, all of the nucleomonomers of formulas I -. VIII in a given oligomer or domain of an oligomer are chirally pure at poεitionε where an X¹ or R group iε located.

The oligomerε of the invention contain at leaεt one domain compriεing invention nucleomonomers that contain a seven-membered ring or a seven-membered ring equivalent that conforms in space to structures that are compatible with the binding competent oligomers described herein. A domain of an oligomer, as used herein, is defined to mean a part or region of an oligomer that contains at least three linked heterocyclic baseε or three linked monomer or linker residues (one or more of which residues may in some cases be abasic, i.e. not containing a heterocyclic base) . An "amide linked domain", as used herein, referε to amide linked nucleomonomerε while a "rigid domain", as used herein, referε to a domain that containε other types of linked nucleomonomers or may have a mixture of nucleomonomer types and may include one or more amide linked nucleomonomerε. An oligomer may contain one or more amide linked domainε that are comprised of a single type of nucleomonomer unit or may contain a mixture of nucleomonomerε.

In general, the invention oligomers will be either (1) comprised solely of invention nucleomonomers or (2) comprised of a domain comprised solely of invention nucleomonomers coupled to a domain comprised of nucleomonomers coupled via linkages or via noninvention substitute linkages (e.g. thioate, ethylphosphonate, riboacetal, 3',5' for acetal, 2 ' , 5 ' formacetal and the like) . The invention nucleomonomerε do not contain a furanoεe moiety and can be coupled to each other via methodε similar to thoεe uεed in peptide synthesis and which are disclosed herein. Oligomers are conveniently produced from dimers or tri ers as synthonε for solid phase or solution phase syntheεis using standard methods known in the art. However, oligomers of any length may be prepared including 10-merε (10 nucleomonomerε) , 20-merε, 50-merε, 100-mers, 200-mers, 500-mers or oligomerε of greater length. Oligomerε containing 2 to 30 nucleomonomerε are preferred. In general, the invention oligomerε will be εyntheεized by solid phase methods which sequentially add nucleomonomers to an oligomer bound to a support. Additional preferred embodiments include oligomers as exemplified in Figureε 9 through 13 wherein Y and Y¹ are independently an oligomer, a blocking group such as FMOC or tBOC, hydrogen, an activated ester coupling group εuitable for εolid phase peptide synthesis a label (radioisotope, enzyme or chro ophore) or a solid support (polystyrene and the like) . These are exampleε of valence satisfying ligands for the terminuε of the molecule.

Preferred εeven-membered ring or seven-membered ring equivalent linkers or nucleomonomers have the base attached to the linker or nucleomonomer through a moiety which consists of two covalent bonds. Preferred moieties contain at least one carbon atom linked to at least one of the two covalent bonds. Some of the oligomerε of the preεent invention may be represented as structural formulas XVII, XVIII and XIX:

wherein Y, Y¹ and B have the same definitions as above; and X^s is a seven-membered ring or a seven- membered ring equivalent with B covalently connected to the backbone through a moiety which consiεts of two covalent bonds. In preferred embodiments, the seven- membered rings or their equivalents are coupled to each other through three bonds internal to the seven-membered ring or equivalent and three covalent bonds external to the seven-membered ring or equivalent. In yet other embodiments, the invention is directed to methods for treating diseaεeε mediated by the preεence of a nucleotide sequence which comprise adminiεtering to a subject in need of such treatment an amount of an invention oligomer capable of specifically binding the nucleotide sequence effective so to inactivate (or modulate) the nucleotide sequence.

The analogs may be utilized in oligomers that contain additional modifications of other nucleomonomers that comprise the oligomer. An exemplary liεt of such modifications include oligomers where (i) one or more furanose-containing nucleomonomers iε modified at the 2' poεition, (ii) one or more croεslinking moieties have been incorporated, (iii) switchback linkers have been incorporated as described in copending U.S. application serial no. 07/559,958, incorporated herein by reference, (iv) other substitute internucleotide linkages have been included and (v) baεe analogs that facilitate duplex or triplex formation, such as 8-oxo-N⁶-methyladenine, 5-propynyluracil, 5-propynylcytosine, or 7-deazaxanthine have been included. One or more of εuch modifications may advantageously be incorporated into a given oligomer depending upon target nucleic acid sequences.

These and other embodiments of the present invention will readily occur to those of ordinary skill in the art in view of the diεclosure herein. Brief Deεcription of the Ficrureε

Figure 1 shows a comparison of the riboacetal and amide linkages.

Figures 2a, 2b and 2c show a to-scale representation generated by computer of the spatial correspondence between riboacetal and amide linkages.

Figure 3 depicts the synthesis of a norborynl monomer that can be incorporated into oligomerε.

Figure 4 (scheme 1) depicts the synthesis of an azepine monomer that can be incorporated into oligomerε; (scheme 2) depicts an alternative synthetic pathway.

Figure 5 shows the synthesis of a hydroxymethyl monomer.

Figure 6 depicts the synthesiε of an oligomer uεing hydroxymethyl monomers.

Figure 7 (scheme 1) shows the syntheεiε of a pyrolidone monomer; (scheme 2) shows an alternative synthetic pathway.

Figure 8 showε an oligomer containing an amide linkage with a ciε-fused cyclopentyl monomer.

Figure 9 shows a spatial comparison of an amide linked oligomer with an amide linked oligomer composed of linked pyrollidone monomers or linkers.

Figure 10 shows a spatial comparison of an amide linked oligomer with an amide linked oligomer compoεed of linked azepine monomers or linkers.

Figure 11 shows a spatial comparison of an amide linked oligomer with an amide linked oligomer composed of linked norbornyl monomers or linkers. Figure 12 showε a εpatial comparison of an amide linked oligomer with an amide linked oligomer compoεed of linked hydroxymethyl monomerε.

Figure 13 εhows a spatial comparison of an amide linked oligomer with an amide linked oligomer composed of linked cyclopentyl monomers. Description of the Invention

The practice of the present invention will employ, unless otherwise indicated, conventional techniques of chemistry, molecular biology, biochemistry, protein chemistry, and recombinant DNA technology, which are within the skill of the art. Such techniques are explained fully in the literature. See, e.σ.. Oligonucleotide Synthesis (M.J. Gait ed. 1984) ; Nucleic Acid Hybridization (B.D. Hames & S.J. Higgins eds. 1984) ; Sambrook, Fritsch & Maniatis, Molecular Cloning: A

Laboratory Manual. Second Edition (1989) ; and the series Methods in Enzymology (S. Colowick and N. Kaplan eds., Academic Press, Inc.).

Structural Formulas

Structural formulas described herein are designated aε roman numeralε (I, II, etc) and chemical compoundε are designated by an underlined numeral (1 , 2_, etc) .

Detailed Description of the Invention Definitions

Nucleomonomer. As used herein, the term "nucleomonomer" means a moiety comprising (1) a base covalently linked to (2) a εecond moiety. Nucleomonomerε of the invention comprise a base linked to a "second moiety" that is an amino acid, amino acid analog or related compound having a free carboxyl and a free amine group or protected forms thereof. Invention nucleomonomers are exemplified by compounds of formulas I - VIII as disclosed herein. The invention nucleomonomers lack a sugar or furansoe moiety εuch aε ribose or deoxyribose. Nucleomonomers also include nucleosides and nucleotides. Nucleomonomers can be linked to form oligomerε that bind to target or complementary base sequenceε in nucleic acids in a sequence specific manner. A "second moiety" aε used herein alεo includeε a sugar moiety, usually a pentoεe, and those species which contain modifications of the sugar moiety, for example, wherein one or more of the hydroxyl groupε are replaced with a halogen, a heteroatom, an aliphatic groupε, or are functionalized aε ethers, amines, thiols, and the like. The pentose moiety can be replaced by a hexoεe or an alternate εtructure such aε a cyclopentane ring, a 6-member morpholino ring and the like.

Base. "Base" aε uεed herein includeε thoεe moietieε which contain not only the known purine and pyrimidine heterocycles, but also heterocycle analogs and tautomers thereof. Purineε include adenine, guanine and xanthine and exemplary purine analogs include 8-oxo-N⁶- methyladenine, 7-methyl-7-deazaguanine, 7-methyl-7- deazaadenine and 7-deazaxanthine. Pyrimidines include uracil and cytosine and their analogs such as 5- methylcytosine, 5-(l-propynyluracil) , 5-(l- propynylcytoεine) , 5-methyluracil and 4,4-ethanocytoεine. Nucleoεide. Aε uεed herein, "nucleoside" means a base covalently attached to a sugar or εugar analog and which may contain a phoεphite or phoεphine. The term nucleoεide includeε ribonucleosides, deoxyribonucleosideε, or any other nucleoside which is an N-glycoside or C-glycoside of a baεe. The εtereochemistry of the sugar carbons can be other than that of D-ribose. Nucleoεides include thoεe εpecieε which contain modificationε of the εugar moiety, for example, wherein one or more of the hydroxyl groupε are replaced with a halogen (F, Cl, Br,or I), a heteroatom (including nitrogen and εulfur) , an aliphatic group (1-9C alkyl, 1- 9C alkenyl and the like) , or are functionalized as ethers, amines, thiols, and the like. The pentose moiety can be replaced by a hexose or an alternate structure such as a cyclopentane ring, a 6-member morpholino ring and the like. The stereochemistry of the sugar carbons can be other than that of D-ribose in one or more reεidueε. The pentoεe moiety can be replaced by a hexose and incorporated into oligomers as deεcribed (Augustyns, K. , et al Nucl Acidε Res (1992) 18:4711-4716). Also included are analogs where the ribose or deoxyriboεe moiety iε replaced by an alternate εtructure such as a hexoεe or εuch aε the 6-member morpholino ring described in U.S. patent number 5,034,506. Nucleosideε as defined herein also includes a purine or pyrimidine baεe linked to an amino acid or amino acid analog having a free carboxyl group and a free amino group or protected formε thereof.

Nucleotide. Aε used herein, "nucleotide" means a nucleoside having a phosphate group or phosphate analog such as a thioate, methylphoεphonate, phoεphoramidate and the like.

Sugar Modification. Aε uεed herein, "εugar modification" means any pentose or hexose moiety other than 2'-deoxyribose. Modified sugarε include D-ribose, 2'- and 3^,-0-alkyl, 2 ' - and 3'-amino, 2 ' - and 3'-halo functionalized pentoεeε, hexoεes and the like. Sugars having a stereochemistry other than that of a D-ribose are also included.

Linkage. As used herein, "linkage" meanε a phoεphodiester moiety (-O-P(O) (O)-0-) that covalently couples adjacent nucleomonomers.

Substitute Linkages. As used herein, "substitute linkage" means any analog of the native phoεphodieεter group or any suitable moiety that covalently couples adjacent nucleomonomers and generates binding competent oligomers. Substitute linkages include 21

the nonphosphorous containing linkages of the invention. The invention substitute linkageε are amideε and carbamateε that link adjacent nucleomonomers. Noninvention substitute linkages include phosphodiester analogs, e.g. such as phosphorothioate and methylphoεphonate, and nonphoεphoruε containing linkages, e.g. such as acetalε and εulfides.

Switchback. As used herein, "switchback" means an oligomer having at least one region of inverted polarity. Switchback oligomers are able to bind to opposite strands of a duplex to form a triplex on both strands of the duplex. The linker ("switchback linker") joining the regions of inverted polarity is a substitute linkage. Crosslinking moietv. "Crosεlinking moiety" includeε a group or moiety in an oligomer that formε a covalent bond with a target nucleic acid. Crosslinking moieties include covalent bonding specieε that covalently link an oligomer to target nucleic acids either spontaneously (e.g. N ,N⁴-ethanocytosine) or via photoactivation (e.g. pεoralen and the like) .

Oligomers. "Oligomerε" are defined herein as two or more nucleomonomers covalently coupled to each other by a linkage or substitute linkage moiety. Thus, an oligomer can have as few as two convalently linked nucleomonomers (a dimer) . Oligomerε can be binding competent and, thuε, can baεe pair with cognate εingle- εtranded or double-εtranded nucleic acid εequenceε. Short oligomers (e.g. dimers - hexamers) are alεo uεeful aε εynthonε, in particular for certain noninvention oligomers aε deεcribed herein. Oligomerε can alεo contain abaεic sites and pseudonucleosides. Invention oligomers are exemplified by the structures shown in Figures 8 - 13. Oligomer as used herein is also intended to include compoundε where adjacent nucleomonomerε are linked via amide linkageε aε previouεly deεcribed (Nielsen, P.E., et al, Science (1991) 254:1497-1500; WO 92/20702) . Elements ordinarily found in oligomers, such as the furanose ring and/or the phosphodiester linkage can be replaced with any suitable functionally equivalent element. "Oligomer" is thus intended to include any structure that εerves as a scaffold or support for the bases wherein the scaffold permitε binding to target nucleic acidε in a εequence-dependent manner. Oligomer also includes oligonucleotides, oligonucleosideε, polydeoxyribo-nucleotideε (containing 2^,-deoxy-D-riboεe or modified formε thereof), i.e., DNA, polyribonucleo- tides (containing D-riboεe or modified forms thereof) , i.e., RNA, and any other type of polynucleotide which is an N-glycoside or C-glycoside of a base. Oligomers that are currently known can be defined into four groups that can be characterized as having (i) phosphodiester and phosphodieεter analog (phoεphorothioate, methylphoεphonate, etc) linkages, (ii) εubstitute linkages that contain a non-phosphorous isostere (riboacetal, formacetal, hydrazino, etc) , (iii) morpholino residues, carbocyclic residues or other furanose sugars, such as arabinose, or a hexose in place of ribose or deoxyribose and (iv) acyclic nucleomonomers linked via amide linkageε or carbamate linkages or any other suitable subεtitute linkage.

Blocking Groupε. As uεed herein, "blocking group" referε to a substituent other than H that is conventionally coupled to oligomers or nucleomonomers, either as a protecting group, a coupling group for synthesis, OP0₃ ^"2, or other conventional conjugate such as a solid support, label, antibody, monoclonal antibody or fragment thereof and the like. As used herein, "blocking group" is not intended to be construed solely as a protecting group, according to slang terminology, but is meant also to include, for example, coupling groupε such as a H-phosphonate or a phosphora idite. By "protecting group" iε meant iε any group capable of protecting the O-atom, S-atom or N-atom to which it iε attached from participating in a reaction or bonding. Such protecting groups for N-atoms on a baεe moiety in a nucleomonomer and their introduction are conventionally known in the art. Non-limiting exampleε of suitable protecting groups include diisobutylformamidine, benzoyl and the like. Suitable "protecting groups" for O-atoms and S-atoms are, for example, DMT, MMT, FMOC or esterε. Protecting group. "Protecting group" aε used herein includes any group capable of preventing the O- atom, S-atom or N-atom to which it iε attached from participating in a reaction or bonding. Such protecting groupε for 0-, S- and N-atoms in nucleomonomerε are deεcribed and methodε for their introduction are conventionally known in the art. Protecting groups also include any group capable of preventing reactions and bonding at carboxylic acids, thiols and the like.

Coupling group. "Coupling group" as used herein means any group suitable for generating a linkage or substitute linkage between nucleomonomers such as a hydrogen phosphonate, a phoεphoramidite and an alkyl ether.

Conjugate. "Conjugate" aε uεed herein means any group attached to the oligomer at a terminal end or within the oligomer itself. Conjugates include solid supports, such as εilica gel, controlled pore glasε and polystyrene; labels, such aε fluoreεcent, chemilumineεcent, radioactive atomε or moleculeε, enzymatic moieties and reporter groups; oligomer tranεport agentε, such as polycations, serum proteins and glycoproteins and polymers and the like.

Synthon. "Synthon" aε uεed herein means a structural unit within a molecule that can be formed and/or asεembled by known or conceivable synthetic operations.

Tranεfection. "Transfection" as used herein refers to any suitable method that for enhanced delivery of oligomerε into cells. Subject. "Subject" as uεed herein meanε a plant or an animal, including a mammal, particularly a human.

"Sequence-specific binding" is used in its commonly accepted senεe to define the binding which occurε between, for example, an oligomer and a DNA or RNA target sequence via pairs of baseε which form hydrogen bonds according to conventional rules.

"Series" when used to define a number of modified nucleosides shall mean 3 or more, and especially from 3 to 100, modified nucleoεides appearing in sequence linked one to another.

By "phosphodiester analog" is meant an analog of the conventional phosphodiester linkage,

-O-P(O) (O)-O-, as well as phosphorus containing subεtitute linkages. These alternative substitute linkages include, but are not limited to embodiments wherein the -P(0) (0)- is replaced with P(S)S, P(0)S,

P(0)NR", P(0)R", P(0)OR'", wherein R" is H or alkyl

(1-6C) and R"' is alkyl (1-6C) . Invention oligomerε can include any εubstitute linkage such as riboacetal and formacetal substitute linkages. In general, such linkages will be confined to a domain of an invention oligomer that does not contain any invention nucleomonomerε. Suitable riboacetal and formacetal linkageε are disclosed in copending applications having Ser. No. 07/690,786; 07/763,130, and 07/806,710, all of which are incorporated herein by reference, and include formacetal linkages εuch aε: 3'-thioformacetal (-S-CH₂-0-) , formacetal (-0-CH₂-0-) ,

3'-amino (-N-CH₂-CH₂-) , 3'-thioketal (-S-C(R¹)₂-0-) , and ketal -0-C(R¹)₂-0- where R¹ is CH₂F or, when both R¹ are taken together with the atom to which . they are attached, form a 4-membered ring or a 6-membered ring where (R¹)₂ is -CH₂-X²-CH₂-, or

-CH_?-CH_?-X²-CH_?-CH_?-;

and wherein X² iε εelected from the group conεiεting of S, SO, S0₂, O, CF₂, CHF, NH, NMe, NEt, NPr.

Suitable riboacetal linkageε include memberε of the group:

35

where each R³ is independently H, or a suitable blocking group such as P0₃ ^"2, a dimethoxytrityl (DMT) moiety, a monomethoxytrityl (MMT) moiety, H-phosphonate (OP0₂H) , methylphosphonate (OP0₂CH₃) or phosphoramidite; R⁴ is selected from the group consisting of H, OH, F, 1-9C 0- alkyl [including OCH₃, OC₂H₅, OCH₂CHCH₂ (O-allyl, OC_JH_J) , OC₃H₇ (O-propyl) , SCH₃, SC₂H₅, SCH₂CHCH₂ (S-allyl, SC^) , SC^H_j (S-propyl)], 1-12C alkyl, 1-12C alkenyl or 1-12C alkynyl. Methylphosphoramidite and β- cyanoethylphoεphoramidite are preferred phosphoramidite groups. Each B is independently a purine or pyrimidine base or an analogous form thereof; each X³ is independently selected from the group consisting of O, S, CH₂, CF₂ and CFH; each A is independently selected from the group consisting of O, S, SO, S0₂, CH₂, CO, CF₂, CS, NH and NR⁶ wherein R⁶ is lower alkyl (including methyl, ethyl, propyl, isopropyl, butyl and isobutyl) with the proviso that adjacent A are not both O; E is selected from the group consisting of 0, S, CH₂, CO, CF₂, CS, NH and NR⁶; J is selected from the group consisting of 0, S, CH₂, CO, CF₂ and CS; G is selected from the group consisting of CH, N, CF, CCl, and CR⁵ wherein R⁵ is lower alkyl (including methyl, ethyl, propyl, isopropyl, butyl and isobutyl) or lower fluoroalkyl (1-4C, 1-5F) . Bases (B) that are preferred are adenine, thymine, guanine, cytosine, 8-oxo-N*-methyladenine, N*,N*-ethanocytosine, and 5-methylcytosine, 5-propynyluracil, 5- propynylcy osine, 7-deazaxanthine and 7-deazaguanine. "Derivatives" of oligomers include those conventionally recognized in the art. For instance, oligomers may be covalently linked to various moieties such as intercalators, substances which interact specifically with the minor groove of the DNA double helix and other arbitrarily chosen conjugates, such as labels (radioactive, fluorescent, enzyme, etc.). These additional moieties may be (but need not be) derivatized through the substitute linkage itself. For example, intercalators, such as acridine may be linked through an -R^,-CH₂-R^,~ attached through any available -OH or -SH, e.g., at the terminal 5', 2 ' or 3' position of RNA or DNA, the 2', 5', or 3' positions of RNA, or an OH or SH engineered into the 5 position of pyrimidineε, e.g., inεtead of the 5 methyl of cytosine, a derivatized form which contains -CH₂CH₂CH₂OH or -CH₂CH₂CH₂SH in the 5 poεition. A wide variety of substituents may be attached, including those bound through conventional linkages. Terminal oligomer moieties such as -NH₂ or -COOH can be blocked or derivatized using standard procedures of routine use. In addition, any -OH moieties at the terminal 3' or 5' position in oligomers with a terminal furanose-containing nucleoεide reεidue may be replaced by phoεphonate groupε, protected by εtandard protecting groupε, or activated to prepare additional linkages to other nucleomonomers, or may be bound to the conjugated subεtituent. The hydroxylε of furanose nucleoside residues may in general be derivatized to standard blocking or protecting groupε aε deεcribed in the art. In addition, εpecifically included are the 2'- or 3'-derivatized forms of the nucleomonomers diεcloεed in pending U.S. application serial no. 07/425,857, as well as the formacetal/ketal type linkages disclosed in pending U.S. Patent Application Serial Nos. 07/559,957 and 07/690,786, all of which are incorporated herein by reference in their entirety. Synthesis of DNA oligomers and nucleomonomers with 2 ' modifications has been described for 2 ' fluoro compounds (Uesugi, S. et al.. Biochemistry (1981) 2_0:3056-3062; Codington, J.F. et al., J Organic Chem (1964) 2^:564-569; Fazakerley, G.V. et al., FEBS Letters (1985) 18_2:365-369) , 2 -0-allyl compounds (OC₃H₅) (Sproat, B.S. et al.. Nucleic Acids Reε (1991) 19:733-738 and 2^,-azido compoundε (Hobbs, J. et al., Biochemiεtrv (1973) 12:5138-5145). Theεe derivativeε are alεo specifically included and the chemiεtry iε applicable to both 2 ' and 3' position. Specific modifications that are contemplated for oligomers described in the present invention include moieties that permit duplex strand switching as deεcribed in commonly owned, pending PCT patent application No. PCT/US90/06128, moietieε such as N^N^-ethanocytosine (aziridinylcytosine) that affect covalent crosεlinking- aε deεcribed in commonly owned, pending U.S. patent application Serial Nos. 07/640,654 and 07/799,824 and moieties such as the base analog 8-hydroxy-N⁶- methyladenine or 5-propynyluracil that facilitate oligomer binding to duplex target nucleic acid as described in commonly owned, pending U.S. patent application Serial No. 643,382 and 799,824. All applications cited herein are incorporated herein by reference. A "constrained linker" is an organic chemical moiety (i.e. a nucleomonomer) having two positionε for covalently chemically binding to adjacent members in a modified oligomer of this invention while having internal covalent or ionic or hydrogen bonds or the like which lock or influence the structure of the linkage into one or a limited number (2 or 3) of distinct spatial conformations. A 2,3- or 4,5-linked divalent seven- membered ring is an example of a "conεtrained linker." Numerouε other exampleε are diεclosed herein. Not all invention subεtitute linkageε in the same oligomer need be identical, the only requirement being that at least one nucleomonomer is present. The invention iε thuε directed to new oligomerε and nucleomonomerε which are uεeful in oligomer-baεed therapieε and intermediateε in their production, as well to methods to synthesize these compounds and their intermediates. In general, the invention compounds show enhanced stability with respect to nucleases by virtue of their substitute linkages, as well as enhanced ability to permeate cells.

Syntheεis of the Compounds

Synthetic schemes that can be used to obtain the compounds diεclosed herein are shown in Figures 3 - 7. Synthesis of monomers corresponding to Formulaε V - VIII and to the oligomer shown in Figure 8 would be conducted in a manner analogous to the synthetic εchemes εhown in Figures 5 and 6, with synthesis of compounds analogous to compound 2_6 starting from protected, subεtituted ethylene diamine having the ring constraints corresponding to those shown in Formulas V - VIII. Such compounds are described in the literature.

All monomerε that are used for incorporation into oligomers are protected at the amino terminuε uεing groupε commonly employed in peptide εynthesis methodε. Preferred groups are t-butylcarbamates (tBOC) and FMOC. For incorporation of nucleomonomerε correεponding to Formulaε I - II and IV - VIII into oligomerε, the carboxylate or carbamate terminus of the nucleomonomer is derivatized to an activated carboxylic acid or an active derivative thereof. Incorporation of nucleomonomerε corresponding to Formula III into oligomers utilizes a chloroformate derivatized compound ( 6) as εhown in Figure 3 or itε equivalent.

Utility and Administration

As the oligomers of the invention are capable of significant single-stranded or double-stranded target nucleic acid binding activity to form duplexes, triplexes or other forms of stable association, these oligomerε are useful in diagnosis and therapy of diseases that are associated with expression of one or more genes such aε thoεe associated with pathological conditions. Therapeutic applications can employ the oligomers to specifically inhibit the expression of genes (or inhibit tranεlation of RNA εequences encoded by those genes) that are associated with either the establishment or the maintenance of a pathological condition. Exemplary genes or RNAs encoded by thoεe genes that can be targeted include those that encode enzymeε, hormoneε, serum proteinε, tranεmembrane proteinε, adhesion molecules (LFA-1, GPII_b/III_a, ELAM-1, VACM-1, ICAM-1, E-selectin, and the like) , receptor molecules including cytokine receptors (IL-1 receptor, IL-2 receptor and the like) , cytokines (IL-1, IL-2, IL-3, IL-4, IL-6 and the like), oncogenes, growth factors, and interleukins. Target geneε or RNAs can be asεociated with any pathological condition εuch aε thoεe aεsociated with inflammatory conditions, cardiovascular disorderε, immune reactionε, cancer, viral infectionε, bacterial infectionε and the like.

Oligomers of the present invention are suitable for uεe in both in vivo and ex vivo therapeutic applicationε. Indicationε for ex vivo uεes include treatment of cells such as bone marrow or peripheral blood in conditions such as leukemia (chronic myelogenous leukemia, acute lymphocytic leukemia) or viral infection. Target genes or RNAs encoded by those genes that can serve as targets for cancer treatments include oncogenes, such as ras, k-ras, bcl-2, c-myb, bcr, c-myc, c-abl or overexpreεεed εequences such as mdm2, oncostatin M, IL-6 (Kaposi's sarcoma), HER-2 and translocationε such as bcr/abl. The oligmers may be used to inhibit proliferation of DNA or RNA viruseε εuch aε herpesviruses, papillomaviruses and the like. Viral gene sequences or RNAs encoded by those geneε such as polymerase or reverse transcriptase geneε of herpeεviruses such as CMV, HSV-1, HSV-2, retroviruses such as HTLV-1, HIV-1, HIV-2, or other DNA or RNA viruses such as HBV, HPV, VZV, influenza virus, rhinoviruε and the like are also suitable targets. Application of εpecifically binding oligomers can be used in conjunction with other therapeutic treatmentε. Other therapeutic indicationε for oligomers of the invention include (1) modulation of inflammatory responses by modulating expression of genes such as IL-1 receptor, IL-1, ICAM-1 or E-Selectin that play a role in mediating inflammation and (2) modulation of cellular proliferation in conditions εuch as arterial occlusion (restenoεiε) after angioplaεty by modulating the expreεsion of (a) growth or mitogenic factors such as non-muscle myosin, myc, fos, PCNA, PDGF or FGF or their receptors, or (b) cell proliferation factors εuch as c-myb. Other suitable proliferation factors or signal transduction factors εuch aε TGFα, TGF β, IL-6, γINF, protein kinaεe C, tyroεine kinaεeε (such as p210, pl90) , may be targeted for treatment of psoriaεiε or other conditionε. In addition, EGF receptor, TGFo or MHC alleleε may be targeted in autoimmune diseaseε. Delivery of oligomerε of the invention into cells can be enhanced by any suitable method including calcium phosphate, DMSO, glycerol or dextran transfection, electroporation or by the uεe of cationic anionic and/or neutral lipid compoεitions or liposomes by methodε deεcribed (International Publication Nos. WO 90/14074, WO 91/16024, WO 91/17424, U.S. Patent 4,897,355). The oligomers can be introduced into cells by complexation with cationic lipids such as DOTMA (which may or may not form liposomeε) which complex is then contacted with the cells. Suitable cationic lipids include but are not limited to N-(2,3-di(9-(Z)- octadecenyloxyl) )-prop-l-yl-N,N,N-trimethylammonium (DOTMA) and its salts, l-0-oleyl-2-0-oleyl-3- dimethylaminopropyl-β-hydroxyethylammonium and its salts and l,2-bis(oleyloxy)-3-(trimethylammonio) propane and its salts.

Enhanced delivery of the invention oligomers can also be mediated by the uεe of (i) viruses such as Sendai virus (Bartzatt, R. , Biotechnol Appl Biochem (1989) 11:133-135) or adenovirus (Wagner, E. , et al, Proc Natl Acad Sci (1992) £9.:6099-6013; (ii) polyamine or polycation conjugates using compoundε such as polylysine, protamine or Nl, N12-bis(ethyl)spermine (Wagner, E. , et al, Proc Natl Acad Sci (1991) £8:4255-4259; Zenke, M. , et al, Proc Natl Acad Sci (1990) 87:3655-3659; Chank, B.K. , et al, Biochem Biophvε Reε Commun (1988) 157:264-270; U.S. Patent 5,138,045); (iii) lipopolyamine complexes using compounds such as lipospermine (Behr, J.-P., et al, Proc Natl Acad Sci (1989) 8_6:6982-6986; Loeffler, J.P., et al J Neuroche (1990) 5±:1812-1815) ; (iv) anionic, neutral or pH sensitive lipids using compounds including anionic phospholipids such as phosphatidyl glycerol, cardiolipin, phosphatidic acid or phosphatidylethanolamine (Lee, K.-D., et al, Biochim Biophvs ACTA (1992) 1103:185-197: Cheddar, G., et al,

Arch Biochem Biophys (1992) 294:188-192; Yoshimura, T. , et al, Biochem Int (1990) 2JD:697-706) ; (v) conjugates with compounds such as transferrin or biotin or (vi) conjugateε with compoundε εuch as εerum proteinε (including albumin or antibodies) , glycoproteins or polymers (including polyethylene glycol) that enhance pharmacokinetic properties of oligomers in a subject. As used herein, transfection refers to any method that is suitable for delivery of oligomerε into cells. Any reagent such as a lipid or any agent such as a virus that can be used in transfection protocolε is collectively referred to herein as a "permeation enhancing agent". Delivery of the oligomers into cells can be via cotranεfection with other nucleic acidε such as (i) expreεεible DNA fragmentε encoding a protein(s) or a protein fragment or (ii) translatable RNAε that encode a protein(ε) or a protein fragment.

The oligomerε can thuε be incorporated into any εuitable formulation that enhances delivery of the oligomers into cells. Suitable pharmaceutical formulationε alεo include thoεe commonly uεed in applicationε where compoundε are delivered into cellε or tiεεueε by topical adminiεtration. Compoundε εuch aε polyethylene glyco. , propylene glycol, azone, nonoxonyl- 9, oleic acid, DMSO, polyamineε or lipopolyamineε can be uεed in topical preparationε that contain the oligomers.

The invention oligomers can be conveniently used as reagents for research or production purpoεeε where inhibition of gene expreεεion is desired. There are currently very few reagents available that efficiently and εpecifically inhibit the expreεεion of a target gene by any mechaniεm. Oligomerε that have been previouεly reported to inhibit target gene expression frequently have nonspecific effects and/or do not reduce target gene expreεεion to very low levels (less than about 40% of uninhibited levels) .

Thus, the oligomers as described herein constitute a reagent that can be used in methodε of inhibiting expreεεion of a εelected protein or proteinε in a subject or in cells wherein the proteinε are encoded by DNA εequenceε and the proteins are translated from RNA sequences, comprising the stepε cf: introducing an oligomer of the invention into the cellε; and permitting the oligomer to form a triplex with the DNA or RNA or a duplex with the DNA or RNA whereby expreεεion of the protein or proteins is inhibited. The methods and oligomers of the present invention are suitable for modulating gene expression in both procaryotic and eucaryotic cells such as bacterial, fungal parasite, yeast and mammalian cells.

Oligomers containing as few as about 8 modifie nucleosideε can be uεed to effect inhibition of target protein(ε) expression by formation of duplex or triplex structureε with target nucleic acid sequences. However, linear oligomers uεed to inhibit target protein expression via duplex or triplex formation will preferably have from about 10 to about 20 modified nucleoside reεidues.

Oligomers containing modified nucleosideε of the invention can be conveniently circularized as described (International Publication No. WO 92/19732; Kool, E.T. J Am Chem Soc (1991) 111:6265-6266; Prakash, G., et al. J Am Chem Soc (1992) 111:3523-3527). Such oligomerε are εuitable for binding to single-stranded or double-stranded nucleic acid targetε. Circular oligomer can be of variouε sizes. Such oligomers in a εize range of about 22-50 nucleomonomerε can be conveniently prepared. The circular oligomerε can have from about three to about six nucleomonomer residues in the loop region that separate binding domains of the oligomer aε deεcribed (Prakaεh, G. ibid) . Oligomerε can be enzymatically circularized through a terminal phoεphate by ligase or by chemical means via linkage through the 5'- and 3'- terminal sugars and/or bases. The oligomers can be utilized to modulate target gene expreεεion by inhibiting the interaction of nucleic acid binding proteinε reεponsible for modulating transcription (Maher, L. J. , et al. Science (1989) 245:725-730) or translation. The oligomers are thus suitable as sequence-specific agents that compete with nucleic acid binding proteins (including ribosomes, RNA poly erases, DNA poly erases, translational initiation factors, transcription factors that either increase or decrease transcription, protein-hormone transcription factors and the like) . Appropriately designed oligomers can thus be used to increase target protein synthesis through mechanisms such as binding to or near a regulatory site that transcription factors use to represε expression or by inhibiting the expression of a selected repressor protein itself.

The invention oligomers, comprising additional modifications that enhance binding affinity can be designed to contain secondary or tertiary structures, such as pseudoknots or pseudo-half-knots (Ecker, D.J., et al, Science (1992) 257:958-961.. Such structures can have a more stable secondary or tertiary structure than corresponding unmodified oligomers. The enhanced stability of such structures would rely on the increased binding affinity between regions of self complementarity in a single oligomer or regions of complementarity between two or more oligomers that form a given structure. Such structures can be used to mimic structures such as the HIV TAR structure in order to interfere with binding by the HIV Tat protein (a protein that binds to TAR) . A similar approach can be utilized with other transcription or translation factors that recognize higher nucleic acid structures such as stems, loops, hairpins, knots and the like. Alternatively, the invention oligomers can be used to (1) disrupt or (2) bind to such structures as a method to (1) interfere with or (2) enhance the binding of proteins to nucleic acid structures.

In addition to their use in antisense or triple helix therapies, the oligomers of the invention can also be applied as therapeutic or diagnostic agents that

SUBSTITUTE SHEET function by direct displacement of one strand in a duplex nucleic acid. Displacement of a strand in a natural duplex such as chromosomal DNA or duplex viral DNA, RNA or hybrid DNA/RNA is possible for oligomers with a high binding affinity for their complementary target sequences. Therapeutic applications of oligomerε by thiε method of uεe, referred to herein aε D-looping or "D-loop therapy" haε not previouεly been possible because the affinity of natural DNA or RNA for its complementary sequence is not great enough to efficiently displace a^" DNA or RNA strand in a duplex. Therapeutic efficacy of oligomers that function by D-looping would result from high affinity binding to a complementary εequence that reεultε in modulation of the normal biological function associated with the nucleic acid target. Types of target nucleic acids include but are not limited to (i) gene sequences including exons, intronε, exon/intron junctionε, promoter/enhancer regionε and 5' or 3' untranεlated regionε, (ii) regions of nucleic acids that utilize secondary structure in order to function (e.g. the HIV TAR stem-loop element or tRNAs) , (iii) nucleic acids that serve structural or other functions such as telomeres, centromereε or replication originε (viruε, bacteria and the like) and (iv) any other duplex region. It is clear that oligomers can be εyntheεized with discrete functional domains wherein one region of an oligomer binds to a target by D-looping while an adjacent region binds a target molecule by say, forming a triple helix or binding as an aptamer to a protein. Alternatively, a D-looping oligomer can bind to each εtrand in a duplex by switching the strand to which the oligomer binds (i.e. by having one region of the oligomer that binds to one strand and another region that binds to the complementary strand) . The controlling elements that dictate the mode of binding (i.e. triple helix or D-loop) are the sequence of the oligomer and the inherent affinity built into the oligomer. Base recognition rules in Watson-Crick duplex binding differ from those in Hoogsteen controlled triplex binding. Because of this, the oligomer base εequence can be uεed to dictate the type of binding ruleε an oligomer will utilize.

D-loop structures are formed in nature by enzyme-mediated processes (Harris, L.D. et al., J Biol Chem (1987) 262: 9285-9292) or are associated with regions where DNA replication occurs (Jacobs, H.T. et al., Nucl Acids Res (1989) 17:8949-8966). D-loops that arise from the binding of oligomers can result from a one or two step process. Direct displacement of a target strand will give rise to a D-loop by a single binding event. However, D-looping can also occur by forming a triple helix which facilitates a strand displacement event leading to the D-loop.

Ribozymes containing modified nucleosides of the invention can be designed in order to design species with altered characteristics. Ribozymes that cleave single stranded RNA or DNA (Robertson, D.L., et al Nature (1990) 344:467-468) have been described. Therapeutic applications for ribozymes have been postulated (Sarver, N. et al, Science (1990) 2__7:1222-1225; International Publication Number WO 91/04319) . Secondary or tertiary structure necessary for ribozyme function can be affected by design of appropriate oligomer sequences. For example, ribozymes having nuclease stable targeting domains containing modified nucleosideε of the invention can have higher affinity, while maintaining baεe pairing εpecificity, for target εequences. Because of the higher affinity and/or nuclease stability of the invention modified nucleosideε, shorter recognition domains in a ribozyme (an advantage in manufacturing) can be designed which can lead to more favorable substrate turnover (an advantage in ribozyme function) .

In therapeutic applications, the oligomers are utilized in a manner appropriate for treatment of a variety of conditions by inhibiting expresεion of appropriate target genes. For such therapy, the oligomers can be formulated for a variety of odeε of administration, including syεtemic, topical or localized adminiεtration. Techniqueε and formulationε generally can be found in Remington's Pharmaceutical Sciences. Mack Publishing Co., Easton, PA, lateεt edition. The oligomer active ingredient is generally combined with a carrier such as a diluent or excipient which can 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 formε include tabletε, powderε, liquid preparationε including εuεpensions, emulsions and solutions, granules, capsuleε and εuppoεitorieε, aε well as liquid preparations for injections, including liposome preparations.

For syεtemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneouε. For injection, the oligomerε of the invention are formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank'ε solution or Ringer's solution. In addition, the oligomerε can be formulated in εolid form and rediεεolved or suspended immediately prior to use. Lyophilized forms are also included. Dosages that can 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 can be utilized depending on (i) the potency of an individual oligomer at inhibiting the activity of its target DNA or RNA, (ii) the severity or extent of a pathological disease state aεεociated with a given target gene, or (iii) the pharmacokinetic behavior of a given oligomer. Syεtemic administration can also be by tranεmucoεal or tranεdermal means, or the compounds can be administered orally. For transmucosal or tranεdermal administration, penetrants appropriate to the barrier to be permeated are uεed in the formulation. Such penetrants are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmucoεal administration. In addition, detergents can be used to facilitate permeation. Transmucoεal adminiε¬ tration can be through uεe of naεal sprays, for example, or suppoεitorieε. For oral adminiεtration, the oligomerε are formulated into conventional oral adminiεtration formε εuch as capsules, tablets, and tonics.

For topical administration, the oligomers of the invention are formulated into ointments, salveε, gels, or creams, as is generally known in the art. Formulation of the invention oligomers for ocular indications such as viral infections would be based on standard compoεitionε known in the art.

In addition to uεe in therapy, the oligomerε of the invention can be used as diagnostic reagents to detect the preεence or abεence of the target nucleic acid εequenceε to which they εpecifically bind. The enhanced binding affinity of the invention oligomers is an advantage for their use as primers and probes. Diagnostic testε cab be conducted by hybridization throug^v either double or triple helix formation which iε then defected by conventional means. For example, the oligomers can be labeled using radioactive, fluorescent, or chromogenic labels and the preεence of label bound to εolid εupport detected. Alternatively, the preεence of a double or triple helix can be detected by antibodieε which specifically recognize these forms. Means for conducting assays using such oligomers as probes are generally known.

The use of oligomers containing the invention modified nucleoεides as diagnostic agents by triple helix formation is advantageous since triple helices form under mild conditions and the assayε can thuε be carried out without εubjecting test specimens to harsh conditions. Diagnostic assays based on detection of RNA for identification of bacteria, fungi or protozoa sequences often require isolation of RNA from samples or organisms grown in the laboratory, which is laborious and time consuming, as RNA is extremely sensitive to ubiquitous nucleases. The oligomer probeε can also incorporate additional modifications such as modified sugars and/or substitute linkages that render the oligomer especially nuclease εtable, and would thuε be uεeful for assays conducted in the presence of cell or tissue extracts which normally contain nuclease activity. Oligomers containing terminal modifications often retain their capacity to bind to complementary sequences without loss of specificity (Uhlmann et al., Chemical Reviews (1990) 9 :543-584) . As set forth above, the invention probes can also contain linkers that permit specific binding to alternate DNA strandε by incorporating a linker that permitε such binding (Froehler, B.C., et al, Biochemistry (1992) 11:1603-1609); Home et al., J Am Chem Soc (1990) 112:2435-2437) . Incorporation of base analogs of the present invention into probeε that alεo contain covalent crosslinking agents haε the potential to increaεe εensitivity and reduce background in diagnostic or detection asεays. In addition, the use of crosεlinking agentε will permit novel aεsay modifications such as (1) the use of the crosslink to increase probe discrimination, (2) incorporation of a denaturing waεh step to reduce background and (3) carrying out hybridization and crosεlinking at or near the melting temperature of the hybrid to reduce secondary structure in the target and to increase probe specificity. Modifications of hybridization conditionε have been previouεly described (Gamper et al.. Nucleic Acids Reε (1986) H:9943) . Oligomers of the invention are suitable for use in diagnoεtic assays that employ methods wherein either the oligomer or nucleic acid to be detected are covalently attached to a solid support as deεcribed (U.S. Patent No. 4,775,619). The oligomerε are alεo suitable for use in diagnoεtic aεsays that rely on polymerase chain reaction techniques to amplify target sequences according to described methods (European Patent Publication No. 0 393 744) . Oligomers of the invention containing a 3' terminus that can serve as a primer are compatible with polymerases used in polymerase chain reaction methods such as the Taq or Vent™ (New England Biolabs) polymerase. Oligomers of the invention can thus be utilized as primers in PCR protocols.

The oligomers are useful as primers that are discrete sequences or as primers with a random sequence. Random εequence primerε can be generally about 6, 7, or 8 nucleomonomerε in length. Such primers can be used in variouε nucleic acid amplification protocolε (PCR, ligaεe chain reaction, etc) or in cloning protocolε. The substitute linkages of the invention generally do not interfere with the capacity of the oligomer to function aε a primer. Oligomerε of the invention having 2'- modificationε at εiteε other than the 3' terminal residue, other modifications that render the oligomer RNase H incompetent or otherwise nuclease stable can be advantageouεly uεed aε probeε or primers for RNA or DNA sequences in cellular extracts or other solutions that contain nucleases. Thus, the oligomers can be used in protocols for amplifying nucleic acid in a sample by mixing the oligomer with a sample containing target nucleic acid, followed by hybridization of the oligomer with the target nucleic acid and amplifying the target nucleic acid by PCR, LCR or other suitable methods.

The oligomers derivatized to chelating agents such as EDTA, DTPA or analogs of 1,2-diaminocyclohexane acetic acid can be utilized in various in vitro diagnostic aεsays as described (U.S. Patent Nos. 4,772,548, 4,707,440 and 4,707,352). Alternatively, oligomers of the invention can be derivatized with crosεlinking agentε εuch as 5-(3-iodoacetamidoprop-l-yl)- 2'-deoxyuridine or 5-(3-(4-bromobutyramido)prop-l-yl)-2'- deoxyuridine and used in various asεay methods or kits aε deεcribed (International Publication No. WO 90/14353) .

In addition to the foregoing uses, the ability of the oligomers to inhibit gene expression can be verified in in vitro syεtems by measuring the levels of expresεion in εubject cells or in recombinant syεtems, by any suitable method (Graeεsmann, M. , et al.. Nucleic Acids Res (1991) 19:53-59). All references cited herein are incorporated herein by reference in their entirety.

The following example iε intended to illuεtrate, but not to limit, the invention. In general, the compounds disclosed herein are syntheεized by methodε routinely used in peptide synthesis.

Example 1 - Synthesis of Hydroxymethyl-nucleomonomer

To prepare compound 17_/ silyl-Z-L-serine, εhown in Figure 5, a mixture of commercially available Z-L- serine (L-serine benzyl carbamate; 12.0 g, 50 mmole) and imidazole (6.9 g, 101 mmole) in 100 mL of dry DMF was stirred followed by the addition of thexyl chloride (117.9 g, 101 mmole). The mixture was kept at room temperature for 2 hours and then evaporated to drynesε. The reεidue was extracted with CH₂C1₂. The organic phase waε waεhed with brine and water, dried over Na₂S0₄ and then concentrated. 29 g of crude product waε obtained. Thiε material waε uεed without further purification. The crude material 12 obtained above waε diεεolved in 200 mL of anhydrous tetrahydrofuran (THF). and was then added to 150 mL of diisobutyl aluminum hydride (DIBAL) (1 M solution in toluene) at -78°C. After being stirred for 2 hours, the reaction mixture was quenched with 20 ml of methanol at the same temperature and then allowed to warm to room temperature. 100 mL of ethyl acetate (EtOAc) and 20 mL of water were added and then the mixture was stirred for 1 hour. 100 g of powdered Na₂S0₄ was added and the resulting mixture waε εtirred overnight. The precipitate waε removed by filtration and then washed with EtOAc until no additional UV active material was detectable. Pure product (91%) , compound 18, waε finally obtained after purification on a flaεh column.

A solution of 11.9 g (32 mmoles) of compound 18_. in 100 mL of dry CH₂C1₂ and 8 mL of triethylamine (TEA) was treated with methanesulfonyl chloride at 0°C for one hour. The mixture waε then washed with saturated Na₂C0₃ εolution and with water. The organic layer waε dried over Na₂S0₄ and evaporated to dryneεε. Purification on a flash column using hexane/EtOAc (1:1) yielded 12.0 g (83%) of compound 19.

12.0 g (27 mmole) of compound 19. and 6 g (120 mmole) of LiN₃ were stirred in 100 mL of dry DMF at 70°C overnight. The mixture waε then evaporated to dryneεε, extracted with CH₂C1₂ and waεhed with water. Further purification on a flash column yielded 7.0 g (66%) of pure compound 20.

The azido compound 2) (7.0 g, 17.8 mmole) was disεolved in 40 mL of 1,4-dioxane. Triphenylphosphine (4.67 g, 17.8 mmole) was then added and the mixture was stirred for 3 hours. 10 L of water was then added to the solution dropwise. After stirring for 30 minutes at 50°C, the reaction mixture waε evaporated to dryneεs. The residue was partitioned between CH₂C1₂ and water. 4.7 g of compound 2_1 (72%) was obtained after purification on a flash column using methanol (0- 7%)/CH₂Cl₂.

Compound 21 (4.5 g, 12.3 mmole) was dissolved in 15 mL of dry acetonitrile and 6.9 mL (20 mmole) of TEA. To this solution, ethyl iodoacetate (1.6 mL, 14 mmole) was added. After 2 hours, the reaction mixture was evaporated to dryness and then extracted with CH₂C1₂ and water. The organic layer was then dried over Na₂S0₄ and concentrated. Purification on a flash column yielded 4.4 g (80%) of compound 22.

Thyminyl acetic acid (Nielsen et al., 1991 Science 254:1497-1500; Egholm et al., 1992 J. Am. Chem. Soc. 111:1895-1897) (2.58 g, 13.2 mmole) and hydroxybenzotriazole (HOBt) (1.80 g, 13.2 mmole) were coevaporated two times with 20 mL of dry DMF and then disεolved in 5 mL of dry DMF. Dicyclohexyl carbodiimide was added to this solution and the mixture waε stirred for 30 minutes at 20°C. The solution of compound 212. (4.0 g, 8.8 mmole) in 5 L of dry DMF waε combined with preactivated thyminyl acetate and reacted for 16 hours at 20°C. After removal of DMF under reduced pressure, the residue was extracted with CH₂C1₂ and washed with 10% sodium bicarbonate solution. Purification on a flash column using methanol/CH₂C1₂ (3:97) yielded 4.0 g (73%) of compound 23. Compound 22. (4.0 g, 6.4 mmole) was hydrolyzed under basic conditions using 100 mL of 0.1 M LiOH in 1,4- dioxane and water (1:1). After 1 hour, the mixture was acidified with 1 M citric acid to reach a pH of 2.0. The mixture was then extracted three times with CH₂C1₂.

After removal of the solvent under reduced pressure, the residue was dissolved in 20 mL of EtOH and hydrogenated with Pd on active carbon. The Pd was filtered off and the filtrate was concentrated. The residue, compound 2_5, (2.4 g, 85%) was used for the next step without further purification.

1.82 g (4 mmole) of compound 5_ was combined with 10 mL of 10% Na₂C0₃ solution and 10 mL of 1,4- dioxane. FMOC-C1 (0.92 g, 4.4 mmole) chilled to 0°C was added to the reaction mixture. The resulting mixture waε εtirred for 16 hours at 20°C and then evaporated to drynesε. The reεidue was partitioned between CH₂C1₂ and water. The water layer was acidified with 1 M citric acid and extracted three times with CH₂C1₂. The organic layers were combined and dried over Na₂S0₄. After evaporation, further purification on a flash column with methanol/CH₂C1₂ followed by CH₃CN/H₂0 (20:1) yielded 1.84 g (70%) of compound 26.

Claims

Claims

What is claimed is:

1. An oligomer comprising a multiplicity of nucleomonomers wherein at leaεt one of said nucleomonomers is of the formula:

wherein B is a base; each X¹ is independently S, 0, SO, S0₂, CH₂,

CHF, CF₂, NR, CH-CH₃, CH-C₂H₅, CH-C_jH and CH-C₄H₉, provided that adjacent X¹ are not both 0; and each R iε independently H, F, OH, OMe or lower alkyl (1-5C) provided that both R attached to the εame carbon are not both OH or OH and OMe together.

2. The oligomer of claim 1 wherein εaid nucleomonomer is of Formula I wherein X is 0 or CH₂ and R is H.

3. The oligomer of claim 1 wherein said nucleomonomer is of Formula II, each X is CR₂ and each R is H.

4. The oligomer of claim 1 wherein said nucleomonomer is of Formula III and X iε O.

5. The oligomer of claim 1 wherein εaid nucleomonomer is of Formula IV. 6. The oligomer of claim 1 wherein said nucleomonomer is of Formula V and wherein X is O.

7. The oligomer of claim 1 which is a dimer, trimer or tetramer.

8. The oligomer of claim 1 coupled to a support or a label.

9. A duplex which comprises an oligomer comprised of nucleomonomerε coupled in a εequence- εpecific manner to the oligomer of claim 1.

10. An oligomer εuitable for εequence-εpecific binding to a target nucleic acid, said oligomer comprising a εequence of nucleomonomerε wherein, at leaεt one region of the oligomer is comprised of a series of constrained nucleomonomers which correspond in εpace to εeven-membered rings (εeven-membered ring equivalents) , and wherein there is a constraint or force that is greater than one hydrogen bond that favorε formation of the seven-membered ring or its equivalent; and wherein εaid bases are attached to the backbone through said seven-membered ring equivalents so as to attain a spatial conformation appropriate for said sequence-specific binding to a target nucleic acid.

11. The oligomer of claim 10 wherein said series comprises at leaεt three sequential constrained linkers.

12. The oligomer of claim 10 wherein all of the constrained linkers in a given series are identical. 13. The oligomer of claim 10 wherein εaid region iε compriεed of εeven-membered ring equivalents wherein each said seven-membered ring equivalent iε coupled through three bondε internal to εaid seven- membered ring equivalent and three covalent bonds external to εaid εeven-membered ring equivalent to the adjacent equivalent.

14. The oligomer of claim 10 wherein the baεes are attached to εaid region of the backbone through a methylene group.

15. The oligomer of claim 14 which is of the formula:

wherein each X* is independently a seven-membered ring equivalent and each B is independently a baεe, and wherein Y and Y¹ are independently a blocking group, H, OH or are valence-satisfying ligands for the termini.

16. The oligomer of claim 14 which is of the formula:

wherein X^a, B, and Y and Y¹ are as defined in claim 15.

wherein X⁸, B, and Y and Y¹ are as defined in claim 14.

18. A method to detect the presence or absence of a target nucleic acid εequence, which method compriεeε contacting a εample εuεpected of containing said sequence with the oligomer of claim 1 under conditions wherein said target nucleic acid sequence binds specifically to εaid oligomer of claim 1 to form a complex; and detecting the preεence, abεence or amount of the complex.

19. A pharmaceutical composition, comprising: a pharmaceutically acceptable carrier; and a therapeutically effective amount of an oligomer of claim 1.

20. The oligomer of claim 1 having a covalent link between the amino terminal nucleomonomer and the carboxy terminal nucleomonomer forming a circular oligomer.

21. A method of treating a diseaεe in a εubject, which diεeaεe is characterized by a particular DNA or RNA sequence, the method comprising: administering to a subject in need of such treatment a therapeutically effective amount of an oligomer of claim 1 complementary to the DNA or RNA sequence; and allowing the oligomer to have sufficient time to bind to the DNA or RNA to form a triplex or duplex.

22. A method of increasing or decreasing the expression of at least one selected protein in a cell wherein the protein is encoded by DNA sequences and the protein is translated from RNA εequenceε, comprising the steps of: introducing an oligomer of claim 1 into the cell; and permitting the oligomer to form a triplex with the DNA or RNA or a duplex with the DNA or RNA whereby expression of the protein is increased or decreased.

23. A method of introducing an oligomer of claim 1 into cells, comprising: mixing the oligomer with a permeation enhancing agent to form a complex; and contacting the complex with the cells.

SUBSTITUTE SHEET