AU693622B2

AU693622B2 - Amino acid nucleic acids

Info

Publication number: AU693622B2
Application number: AU42341/96A
Authority: AU
Inventors: Kandasamy Ramasamy; Wilfried Seifert; Guangyi Wang
Original assignee: ICN Pharmaceuticals Inc
Current assignee: Bausch Health Americas Inc
Priority date: 1994-11-02
Filing date: 1995-11-02
Publication date: 1998-07-02
Anticipated expiration: 2015-11-02
Also published as: PL320084A1; KR100393336B1; CN1171112A; UA48150C2; KR970707144A; MX9703188A; PL185852B1; SI9520112A; RU2154638C2; EP0789707A4; JPH10508312A; EP0789707A1; HUT77435A; SK2694U; AU4234196A; CA2202274A1; WO1996014330A1; HU218086B

Description

WO 96/14330 PCT/US95/14599 AMINO ACID NUCLEIC ACIDS Field of Invention: The invention is in the field of polynucleotide analogs 5 lacking furanose rings.

Background of the Invention: Oligonucleotides that bind sequence specifically to icomplementary nucleic acids sense strand) by hydrogen bonding so as to inhibit gene expression are commonly referred to as antisense oligonucleotides. These synthetic oligonucleotides bind to target (mRNA) and thereby inhibit translation of the messenger RNA. This antisense principle (Uhlmann, E. et al., Chem. Reviews, 1990, 90, 543-584; and Stein, C. A. et al., Cancer Res., 1988, 48, 2659-2688) is used I in nature to regulate gene expression. This antisense principle has been used in the laboratory not only to inhibit but also to .,tivate gene expression. Zamecnik and Stephenson were the first to propose, in 1978, the use of synthetic Soligonucleotides for therapeutic purposes (Stephenson, M. L.; and Zamecnik, P. Proc. Natl. Acad. Sci. USA, 1978, 75, 280 and 285). The specific inhibition of antisense polynucleotide is based on the specific Watson-Crick base pairing between the heterocyclic bases of the antisense oligonucleotide and of 1 viral nucleic acid. The process of binding of the 25 oligonucleotides to a complementary nucleic acid is called hybridization. An oligomer having a base sequence complementary to that of an mRNA which encodes protein necessary for the progress of the disease is of particular interest. By hybridizing specifically to the mRNA, the synthesis of proteins encoded by the mRNA may be disturbed.

The preparation of unmodified oligonucleotides, i.e., oligonucleotides having a DNA structure, has been the center I WO 96/14330 PCT/US95/14599 of interest for many research groups in the past decade. The synthesis via phosphoramidites according to Caruthers (McBride, and Caruthers, M. Tetrahedron Letts., 1983, 24, 245), originally introduced by Letsinger (Letsinger, and Lunsford, W. B. J. Amer. Chem. Soc., 1976, 8., 3655) as the phosphite triester method, is currently the most efficient method for the preparation phosphodiester oligonucleotides. When normal, unmodified, oligonucleotides are used as antisense oligonucleotides, the 0 problems of instability to nucleases and insufficient membrane penetration have arisen. For antisense oligonucleotides to be able to inhibit translation they must reach the interior of the cell unaltered. The properties useful for oligonucleotides to be used for antisense inhibition include: stability of the oligonucleotides towards extra- and intracellular enzymes; (ii) ability to penetrate through the cell membrane; and (iii) ability to hybridize the target DNA or RNA (Agarwal, K. L. et al., Nucleic Acids Res., 1979, 6, 3009; Agawal, S. et al., Proc Natl Acad Sci. USA. 1988, 85, 7079). Thus, it is of interest to provide polynucleotide analogs that have superior properties for use as antisense or for use as primers or hybridization, probes.

Modified polynucleotides have been synthesized in the past, these polynucleotide modifications include methylphophonates, phosphorothioates, various amidates and the sugar moieties of the nucleic acid species. These backbone substitutions confer enhanced stability to some degree but suffer from the drawback that they result in a chiral phosphorous in the linkage, thus leading to the formation of 2 n 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 olgonucleotide to hybridize to target sequences. Some 2 WO 96/14330 PCT/US95/14599 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.

Some oligonucleotide analogs containing sugar modifications have been synthesized. Previously used sugar modifications of (deoxy)ribose nucleic acids include a-DNA, homo DNA, morpholino and thio nucleosides and Peptide Nucleic Acids (PNA) to provide what has been perceived to be improved structures, especially structures which have improved cell uptake. The general synthetic scheme for arriving at such analogs has been to involve the primary hydroxyl group of a nucleoside or its nucleotide, either bound to a polymeric carrier or to a sequence-specified 3'-nucleotide with phosphorus atom in either the pentavalent or trivalent oxidation state. Specific coupling procedures have been referred to as the phosphite triester (phosphoramidite), the phosphorus diester, and the hydrogen phosphcnate procedures.

Commercially available monomers and polymeric carriers-bound monomers are available for such methods having protected bases A, C, T, U and other heterocycles) along with protected phosphorus atoms to allow storage and prevent non-specific reactions during the coupling process.

Nucleic acid species containing modified sugars, nonionic backbones or acyclic polyamides (PNA) having, to some degree, one or more of the following properties useful for gene modulation: to enhance the duplex stability (hybridization efficiency), increased target specificity, stability against nucleases, improved cellular uptake, and assistance in the important terminating events of nucleic 3 Mh_ ,-rri i WO 96/14330 PCT/US95/14599 acids RNase H activity, catalytic cleavage, hybridization arrest, and others). It has also been suggested to use carbonate diesters. However, these compounds are highly unstable, and the carbonate diester link does not maintain a tetrahedral configuration exhibited by the phosphorous in the phosphodiester. Similarly, 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., Tetrahedron Letts., 1987, 28, 745; Stirchak, E. P. et al., J. Org. Chem., 1987, 52, 4202).

More recently, reports of acyclic sugar analogs have appeared in the literature (Augustyns, K. A. et al., Nucleic Acids Res., 1991, 19, 2587-2593). Incorporation of these acyclic nucleosides into oligonucleotides caused a drop in Tm, 1depending on the number of linkers built in the oligomers.

These oligonucleotides are found to be enzymatically stable and form base pairing with the complementary sequence. Given the shortcomings of polynucleotides and known polynucleotide analogs, it is of interest to provide new polynucleotide analogs for use in antisense inhibition and other techniques employing oligomers.

Such attempts at modifying both the sugar and the backbone components have some shortcomings for use as therapeutics and in other methods. Hence, still greater improvements in these qualities is required before effective therapeutics, diagnostics and research tools become available.

Accordingly, there is a long-felt need for improved oligomer analogs of oligonucleotides as pharmaceuticals compounds.

The present invention provides novel oligonucleotides, and structural precursor thereof, which have improved resistance to nuclease digestion, and which have increased 4 WO 96/14330 PCT/US95/14599 stability under physiological conditions, and which can be neutral or positively charged that could enhance cell permeation. Furthermore, the novel oligonucleotides of the present invention improved hybridization properties with respect to nucleic acid hybridization targets.

The oligomers of the present invention are generally characterized as comprising a series of constrained linkers or monomers that is appropriate for binding of heterocyclic bases to a target nucleic acid in F -equence specific manner. The constrained linkers described herein, when incorporated into oligomers, may have a force greater than a single hydrogen bond, thereby favoring formation of the binding competent conformation.

The nucleomonomers of the present invention are generally characterized as moieties or residues that replace the furanose ring, that is found in naturally occurring nucleotides, with an amino acid or a modified amino alcohol residues. Exemplary monomers and oligomers of the invention are shown in formulae 1 through 41. Incorporation of these monomers described herein into oligonucleotides permits synthesis of compounds with improved properties, these improved properties include increased lipophilicity whc-h results from eliminating the charge associated with phosphodiester linkages (Dalge, J. M. et al., Nucleic Acids Res., 1991, 19, 1805) and (ii) resistance to degradation by enzymes such as nucleases. Oligomers containing these monomers may be quite stable for hybridization to target sequences and superior to unmodified nucleoside residues in one or more applications.

5 I i- e e -e I I us 6 Summary of the Invention The present invention provides various novel oligonucleotide analogs having one or more properties that make the subject compounds superior to conventional oligonucleotides for use in procedures employing oligonucleotides. The compounds of the invention are oligonucleotide analogs in which the furanose ring of a naturally occurring nucleic acid is replaced with an amino acid or a modified amino alcohol residue. Some embodiments of the novel compounds of the invention are particularly useful for the antisense control of gene expression. The compounds of the invention may also be used as nucleic acid hybridization probes or as primers.

Another aspect of the invention is to provide monomeric precursors of the oligonucleotide analogs of the invention. These monomeric precursors may be used to synthesize the subject polynucleotide analogs.

Another aspect of the invention is to provide formulations of the subject polynucleotide analogs that are designed for the treatment or prevention of disease 15 conditions. Yet another aspect of the invention is to provide methods for treating or i preventing diseases, particularly viral infections and cell growth disorders. The subject disease treatment methods comprise the step of administering an effective amount of the 0 0 subject polynucleotide analogs for use as antisense inhibitors.

According to a first embodiment of the invention there is provided a compound of S 20 compound 1 in any of Groups I-V: :00

W-L

o

W--L

N-Z

Y

Compound 1 Group I, where: X is CHR 2 OH where R 2 is H, lower alkyl amine or lower alkyl imidazole; Y is Base-(CH2)n-, where Base is a non-halogenated variable nucleosidic base, and n is 1 to 7; A is carbonyl; Z is H or OR 3 where R 3 is H, lower alkyl, lower alkyl amine or lower alkyl imidazole; W is CHR 4 0H, where R 4 is H or lower alkyl amine or lower alkyl imidazole; N is N(nitrogen); and

RAI

4 L is not present; [N:\libff 942:MCN r 6a Group II, where: X is Base-(CH 2 where Base is a non-halogenated variable nucleosidic base, and n is 1 to 7; Y is CHR20H where R 2 is H, lower alkyl amine or lower alkyl imidazole: A is carbonyl or CH 2 Z is H or OR 3 where R 3 is H, lower alkyl, lower alkyl amine or lower alkyl imidazole; W is CHR40H, where R 4 is H or lower alkyl amine or lower alkyl imidazole; N is N(nitrogen); and L is not present; Group III, where: X is CHR 2 OH where R 2 is H, lower alkyl amine or lower alkyl imidazole; Y is Base-(CH 2 where Base is a non-halogenated variable nucleosidlc base, and n is 1 to 7; S 15 A is CH 2 0 Z is OH or OR 3 and where R 3 is H, lower alkyl, lower alkyl amine or lower alkyl imidazole; ,W is CHR40H, where R 4 is H or lower alkyli amine or lower alkyl imidazole; N is N(nitrogen); and L is not present; Group IV, where: SX is Base-(CH 2 where Base is a non-halogenated variable nucleosidic base, and Sn is 1 to 7; i Y is COOH or CHR 2 OH where R 2 is H, lower alkyl amine or lower alkyl imidazole; A is carbonyl or CH 2 Z is CH 2 W is CH 2 N is N(nitrogen); and L is CHNHR 5 where R 5 is H, OH or OR 3 and R 3 is H, lower alkyl, lower alkyl amine or lower alkyl imidazole; Group V, where: X is CH 2 0H, CH 2

NH

2

CONH

2 or COOH; Y is not present; Z is CH 2 or CHO-L 1

-B;

W is O, S or CH 2 N is CH; and L and A are independently COOH, CHCOOH, CHCH 2 COOH, NH 2

CHNH

2

L

1

NH-L

2 -B or CH-L 1

-NH-L

2 where B is H or a nucleoside base, and L 1 and L 2 are 72 o independently (CH2)n, where n= 1-3 or (CH2)nCO, where n 0-2.

[N:\libff]00942:MCN

M

I

6b Brief Description of the Figures Figures 1 through 30 are depictions of chemical reaction sequences usable for synthesizing monomers and oligonucleotides of the present invention. More specifically, at #4 o Co.

a,.

B

WO 96/14330 PCTflUS95/14599 WO 96/14330 PCT/US95/14599 Figure 1 shows the synthesis of L-serinol coupled thymine monomer phosphoramidite with a -CH 2 -CO- linkage between thymine and serinol.

Figure 2 shows the synthesis of L-se.nol coupled thymine monomer phosphoramidite with a -CH 2

-CH

2 linkage between thymine and serinol.

Figure 3 and 4 depicts the synthesis of substituted Lserinol coupled thymine monomer phophoramidites with a -CH,- CO- :Lnkage between thymine and serinol.

Figure 5 shows the synthesis of T-T dimer with 5 atom long inter nucleotide linkage having hydroxylamine in the middle of the internucleotidc linkage with a -CH 2 -CO- linkage between thymine and serinol.

Figure 6 depicts the synthesis of thymine monomer phosphoramidite in wh' thymine is connected to an Nethylhydroxylamine through a -CH,-CO- linkage.

Figure 7 shows the synthesis of L-serinol coupled thymine monomer phosphoramidite in which the NH 2 group of L-serine is connected to 2-hydroxyacetyl group and the hydroxy function is blocked with DMT group. This building block is used for connection. This figure also depicts the synthesis of thymine monomer in which the NH 2 group of L-serine is connected to a 2'-hydroxyethyl function.

Figure 8 shows the synthesis T-T dimer having a hydroxamate backbone with linkage. In this dimer one building block is made from L-aspartic acid and thymine and the other is from L-serine and thymine. This dimer has two additional amide bond in the backbone.

7 ~F I WO 96/14330 PCT/US95/14599 Figure 9 depicts the synthesis T-T dimer having hydroxamate backbone with linkage. In this dimer one building block is made from L-aspartic acid and thymine and the other is from L-serine and thymine. This dimer lacks amide Sbond between in the backbone.

Figure 10 shows the synthesis of L-serinol-b-alanine coupled thymine monomer phosphoramidite in which 3-alanine links thymine and serinol.

*t Figure 11 shows the synthesis of L-serinol-akylamine coupled thymine monomer phosphoramidite with alkyamine links thvmine and serinol.

Ei:ure 12 depicts the synthesis T-T dimer having hydroxamate backbone with linkage. In this, the dimer is made from two L-aspartic acid units and two thymine units with an acetyl linker between thymine and aspartic acid.

Figure 13 depicts the synthesis T-T dimer having hydroxamate backbone with linkage. In this, tne dimer is made from two L-aspartic acid units and two thymine units with an ethyl linker between thymine and aspartic acid.

Figure 14 shows the synthesis of N-hydroxyamino acid coupled thymine building block.

Figure 15 shows the synthesis of L-aspartic acid coupled thymine building block with an N-hydroxylamine linker between thymine and aspartic acid.

Figure 16 depicts the synthesis T-T dimer having a hydroxamate backbone with linkage. In this, the 8 I carboxylic acid group is coupled to thymine building block through an N-hydroxylamine linker.

Figure 17 depicts the synthesis thymidineacetic acid substituted N-hydroxyamino acid building block 150 and its analogue 149. These monomer building blocks are useful to create nucleic acid with hydroxamate backbones.

SFigure 18 shows the synthesis of thymidineacetic acid substituted hydroxylamine containing amino acid building blocks 157 and 158. These monomers are useful to design nucleic acid having amide backbone with hydroxylamine functionality.

o Figure 19 shows the synthesis of L-serinol coupled i 4n :1 n 15 thymidine building block 166 having a hydroxylamine moiety between thymine and serinol. This building block is useful to devise nucleic acid of linkages.

Figure 20 depicts the synthesis of glutamic acid-glycine o. coupled Thymidine monomer 174. This monomer building block is S 20 useful to generate nucleic acid with amide backbones and linkages.

Figure 21 shows the synthesis of glycinol-glycine coupled thymidine building block 181 and 182 having a hydroxylamine moiety between thymine and glycinol. These building blocks are useful to prepare nucleic acid of linkages.

Figures 22 through 24 indicate the synthesis of ribose amino acid coupled thymidine building blocks 191, 199 and 2U..

These building blocks are useful to prepare oligonucleotides having ribose-amide backbone.

9 i ;I

NH-L

2 -B or CH-L 1

-NH-L

2 where B is H or a nucleoside base, and L 1 and L 2 are independently (CH2)n, where n= 1-3 or (CH2)nCO, where n 0-2.

/3 Figure 25 depicts the solid phase synthesis of oligonucleotide 211 having ribose-amide backbone.

Figure 26 shows the synthesis of l-O-(4,4'-Dimethoxy- Strityl)-2-[amino(thyminylacetyl)]-L-propan-3-O-(N,N-diisopropy 1)-2-cyanoethylphosphoramidite.

Figure 27 shows the synthesis of 1-0-(4,4'-Dimethoxytrityl)-2-[amino(thyminylacetyl)]-D-propan-3-O-(N,N-diisopropyl)-2-cyanoethylphosphoramidite.

Figure 28 shows the synthesis of 2-[(2-(4,4'-Dimethoxytrityl)-0-acetyl)amino]-3-thyminyl-L-propan-1-0-(N,N-diisoprop a. fyl)-2-cyanoethylphosphoramidite.

ft Figure 29 shows the synthesis of N-(Thyminylacetyl)-Nfill Figure 30 shows the synthesis of (2R,4S)-l-(tertft. Butyloxycarbonyl)-2-[N 3 -benzoyl(thymin-l-yl)] methyl-4-phthalim ido-pyrrolidine.

Detailed Description of the Specific Embodiments: I A. Definitions and Abbreviations: In describing the present invention, the following terms will be employed, and are intended to be defined as indicated below.

As used herein, "antisense" therapy refers to administration or in situ generation of DNA or RNA oligomers, or their analogs thereof, which bind specifically to a complementary target nucleic acid sequence. The binding may be by conventional base pair complementarily, or the binding may through other mechanisms, for example, in the case of binding 10 WO 96/14330 PCT/US95/14599 to DNA duplexes, through specific interactions in the major groove of the double helix. In general, "antisense" refers to a range of techniques generally employed under this description in the art, and includes any therapy which relies on specific binding to oligonucleotide sequences. The techniques of antisense gene regulation are well known to the person of ordinary skill in the art of molecular biology descriptions of antisense gene regulation can be found, for example, in U.S. Patent 5,107,065, U.S. Patent 5,166,195, U.S.

Patent 5,087,617, and Crooke, Annual Review Pharmacology Toxicology 1992 32; 329-376.

The terms "Oligomer" or "Oligonucleotide" are used interchangeably and include naturally occurring compounds such as RNA and DNA, as well as synthetic analogs thereof, including compounds of the invention. Unless indicated otherwise, the terms "oligomer" and "oligonucleotide" refer to both DNA/RNA and to synthetic analogs thereof. The term "oligomer" refers to compounds comprising two or more nucleomonomers covalently attached to each other by a phosphodiester linkage or any other substitute linkages.

Unless indicated otherwise, a lengthy limitation should not be read into the term "oligomer". Thus, an oligomer can have as few as two covalently linked nucleomonomers a dimer )or may be significantly longer. Oligomers can be binding competent and, thus, can base pair with single-stranded or doublestranded nucleic acid sequences. Oligomers dimers hexamers) are also useful as synthons for longer oligomers as described herein. Oligomers may contain abasic sites and pseudonucleosides.

The Oligomers includes oligonuclectides, oligonucleosides, polydeoxyribo-nucleotides (containing 2'deoxy-D-ribose or modified forms thereof), DNA, 11 WO 96/14330 PCT/US95/14599 polyribonucleotides containing D-ribose or modified forms thereof), RNA, and any other type of polynucleotide which is an N-glycoside or C-glycoside of purine or pyrimidine base, or modified purine or pyrimidine base. Oligomer as used Sherein is also intended to include compounds where adjacent nucleomonomers are linked via hydroxamate linkages. Elements ordinarily found in oligomers, such as the furanose ring and/or the phosphodiester linkage can be replaced with any suitable functionally equivalent element. The term "Oligomer" is intended to include any structure that serves as a chassis or support for the bases wherein the chassis permits binding to target nucleic acids in a sequence-dependent manner.

Oligomers that are currently known can be defined into four groups that can be characterized as having phosphodiester or phosphodiester analog (phosphorothiaoate, methylphosphonate, etc) linkages, (ii) substitute linkages that contain a non-phosphorous isostere (riboacetal, formacetal, carbamate, etc), (iii) morpholino residues, carbocyclic residues or other furanose sugars, such as arabinose, or a hexose in place of ribose or deoxyribose and (iv) nucleomonomers linked via amide bonds or acyclic nucleomonomers linked via any suitable substitute linkage.

The term "Nucleoronomer" as used herein refers to a moiety comprising a base covalently linked to a second moiety. Nucleomonomers include nucleosides, nucleotides or bases connected t an amino alcohol. Nucleomonomers can be linked to form oligomers that bind to target or complementary base sequences of nucleic acids in a sequence specific manner.

A "second moiety" as used herein refers to a compound linked to a Nucleomonomer, and includes an amino acid/amino alcohol moiety, usually serinol, aspartic acid, glutamic acid, glycine, and those species which contain modifications of the 12 I- c WO 96/14330 PCT/US95/14599 amino acid moiety, for example, wherei- one or more of the hydrogen is replaced with other functionality (see formulae 24-41), or one carboxylic acid is functionalized to an alcohol, amines, thiols, hydroxylamines, and the like.

Nucleomonomers as defined herein are also intended to include a base linked to an amino acid or amino alcohol and/or amino acid/alcohol analog having a free carboxyl/hydroxyl group and/or a free amino group and/or protected forms thereof.

The term "Nucleoside" as used herein refers to an amino acid and amino alcohol derivative thereof, as described further below, carrying a purine, pyrimidine, or analogous forms thereof, as defined below, but lacking a linking moiety such as a phosphodiester analog or a modified internucleoside linkage. By nucleoside is meant the nucleoside which provides the 5' carbon coupling point to the linker. The end of the linker couples to the 5' nucleoside. The end of the linker joins to the 3' position on the next nucleoside.

If a modified nucleoside is present which does not precisely include a 3' and/or a 5' carbon, it is understood by the person skilled in the art that this and terminology to describe strand polarity used by analogy to DNA and RNA.

The term "Nucleoside" as used herein refers to a base covalently attached to an amino alcohol/ amino acid analog and which contain a linker between base and the amino acid/amino alcohol. The term nucleoside normally includes ribonucleosides, deoxyribonucleosides, or any other nucleoside which is an N-glycoside or C-glycoside of a base.

The term "Nucleotide" as used herein refers to a nucleoside having a phosphate group or a phosphate analog (groups with phosphorous in the same oxidation state as in the phosphate group e.g. thiophosphate, amidate).

13 WO 96/14330 PCT/US95/14599 The term "Base" as used herein refers to a wide variety of nucleoside base, including purine and pyrimidine heterocycles and heterocyclic analogs and tautomers thereof.

Purines include adenine, guanine and xanthine and exemplary purine analogs include 8-oxo-N6-methyladenine and 7deazaxanthine. Pyrimidines include uracil and cytosine and their analogs such as 5-methylcytosine, 5-(1-propynyluracil), 5-methyluracil and 4,4-ethanocytosine.

"Bases", when joined to a suitable molecular framework, e.g. a phophodiester backbone, are capable of entering into a base pairing relationship that occur in double-stranded DNA or other double-stranded nucleic acids of similar structure.

Bases may also be capable of entering into a base pairing relationship in a triple helix nucleic acid.

The term "Sugar Modification" as used herei; frs to any amino acid or amino alcohol moiety other than 2'deoxyribose.

I The term "Amino Acids/Alcohol" as used herein refers to any natural amino acids and alcohols of both and "S" l isomers.

The term "Nucleoside Linkages" as used herein refers to the linkage that exists within the monomer.

The term "Linkage" as used herein refers to the moiety that is used to connect the base with amino acid/amino alcohol and derivatives thereof.

The term "Internucleotide Linkages" as used herein refers to a phophodiester moiety or any other functionally equivalent moiety that covalently connects adjacent nucleomonomers.

14 WO 96/14330 PCT/US95/14599 The term "Substitute Linkages" as used herein refers to any analog of the native group or any suitable moiety that covalently couples adjacent nucleomonomers. Substitute linkages include phosphodiester analogs, e.g. such as phosphorothioate and methylphosphonate, and nonphosphorus containing linkages, e.g. such as amides, hydroxamates, hydroxylamine. Substitute linkages include the nonphosphorous containing linkages linkages, linkages and linkages) of the invention.

The term "Crosslinking moiety" as used herein refers to a group or moiety in an oligomer that forms a covalent bond with a target nucleic acid. Crosslinking moieties include covalent binding species that covalently link an oligomer to target nucleic acids either spontaneously N 4 N-ethanocytosine) or via photoactivation psoralen) and the like.

The term "Blocking Groups" as used herein refers to a substituent other than H that is covalently coupled to oligomers or nucleomonomers, either as a protecting group, a coupling group for synthesis, OPO_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 groups such as a Hphosphonate or a phosphoramidite.

The term "protecting group" as used herein refers to any group capable of protecting the O-atom, S-atom or N-atom to which it is attached from participating in a reaction or bonding. Such protecting groups for N-atoms on a base moiety in a Nucleomonomer and their introduction are conventionally known in the art. Non-limiting examples of suitable protecting 15 4 WO 96/14330 .r WO 96/14330 PCT/US95/14599 groups include: diisobutylformamidine, benzoyl, silyl and the like. Suitable protecting groups for O-atoms and S-atoms are, for example, DMT, MMT, FMOC or esters. "Protecting groups" as used herein includes any group capable of preventing the 0atom, S-atom, or N-atom to which it is attached from participating in a reaction or binding. Such protecting groups for and N-atoms in nucleomonomers are described and the methods 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.

The term "Coupling group" as used herein refers to any group suitable for generating a linkages or substitute linkage between nucleomonomers such as a hydrogen phosphonate and a phosphoramidite.

The term "Conjugate" or "conjugate moiety" as used herein refers to any group attached to the oligomer at a terminal end or within the oligomer itself. Conjugates include solid supports, such as silica gel, controlled pore glass and polystyrene; labels, such as fluorescent, chemiluminescent, radioactive atoms or molecules, enzymatic moieties and reporter groups; oligomer transport agents, such as polycations, serum proteins and glycoproteins and polymers and the like. Other conjugate moities include O-cholesterol, polyethylene glycol (PEG), amino acids, intercalators, polynucleotide clearing moieties, crosslinking functionalities, lipids, hydroxamates, alkylating agents and the like.

The term "Synthon" as used herein refers to a structural unit within an oligonucleotide analog of the invention.

16 5 WO 96/14330 PCT/US95/1499 The term "Transfection" as used herein refers to any method that is suitable for enhanced delivery of oligomers into cells.

I 5 The term "Subject" as used herein refers t, a plant or Sanimal, including mammal, particularly a human.

The term "Derivatives" and monomeric constituents thereof i oligomers include those conventionally recognized in the art.

For instance, the oligonucleotides may be covalently linked to various moieties such as intercalators, substances which interact specially 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 modified backbone linkage as part of the linkage itself. For example, intercalators, such as acridine can be linked through an R-CH 2 attached through any available -OH or SH, at the terminal 5' position of RNA or DNA, the 2' position of RNA, or an OH or SH engineered into the 5 position of pyrimidines, instead of the 5 methyl of cytosine, a derivatized form which contains -CH 2

CHCH

2 OH or -CHCH,CH 2 SH in the 5 position. A wide variety of substituents can be attached, including those bound through conventional linkages.

Accordingly the indicated OH moieties in the oligomer of formula may be replaced by phosphonate rroups, protected by standard protecting groups, or activated to prepare additional linkages to other nucleotides, or may be bound to the conjugated substituent. The 5' terminal OH is conventionally phosphorylated; the 2'-OH or OH substituents at the 3' terminus may also be phosphorylated. The hydroxyls may also be derivatized to standard protecting groups.

17 w is L-nr4un, wnere K4 is 1i or lower alkyl amine or lower alkyl imidazole; N is N(nitrogen); and B4 L is not present; L1 [N:\libfflQ0942:MC WO 96/14330 PCT/US95/14599 The term phosphodiester analog" as used herein refers to an analog of the conventional phosphodiester linkage as well as alternative linking groups. These alternative linking groups include, but are not limited to embodiments wherein the O-P(O) is replaced with P(O)S, P(O)NR 2 P(O)R, P(O)OR', where R is H or alkyl (1-7C) and R' is alkyl Not all phosphodiester analogs in the same oligomer need to identical, the only requirement being that at least one of these linkages is a modified internucleotide linkage as described herein.

"Aniialogous" forms of purines and pyrimidines are those generally known in the art, many of which are used as chemotherapeutic agents. An exemplary but not exhaustive list includes 4-acetylcytosine, 8-hydroxy-N6-methyladenine, az4ridinylcytosine, pseudoisocytosine, (carboxyhydroxymethyl) uracil, 5-fluorouracil, 5-carboxymethylaminomethyl-2-thiouracil, carboxymethylaminomethyluracil, dihydrouracil, inosine, N 6 isopentenyladenine, 1-methyladenine, 1-methylpseudouracil, 1methylguanine, 1-methylinosine, 2,2-dimethylguanine, 2methyladenine, 2-methylguanine, 3-methylcytosine, methylcytosine, N,-methyladenine, 7-methyladenine, 7methylguanine, 5-methylaminomethyluracil, methoxyaminomethyl-2-thiouracil, beta-D-mannosylguanosine, methoxycarbonylmethyluracil, 5-methoxyuracil, 2-methylthio-N6isopentenyladenine, uracil-5-oxyacetic acid methylester, acid, oxybutoxosine, pseudouracil, gueosine, 2-thiouracil, 4-thiouracil, 5-methyluracil, Nacid methylester, uracil-5-oxyacetic acid, pseudouracil, queosine, 2-thiocytosine, and 2,6-diaminopurine.

A particularly preferred analog is (abbreviated herein as "Cme") I WO 96/14330 PCT/US95/14599 The term "Isosteric" as used herein refers to the spatial and orientation properties of an internucleoside linkage and the fact that these properties are so similar to those of the native phosphodiester linkage that the modified oligonuceotide I 5 containing an is.Jsteric bond will replace, substitute for, mimic and/ or hybridize with a native oligonuclotide.

SThe term "Ribose-amide" as used herein refers to the internucleotide linkage that exists between two nucleobases.

The ribose-amide internucleotide linkage has combination of ribose/(2'-deoxy) and amino acid functionalities.

Various abbreviations are used in this application to i refer to functional groups and compounds. These abbreviations i are readily understood by the person skilled in the art of organic chemistry, for example, "Ph" refers to phenyl, "Me" Srefers to methyl, indicates that a given chain i contains anywhere from 1 to 7 carbons, etc.

Description of the Invention: The present invention provides novel oligonucleotide analogs containing modified amino acid/amino alcohol linkages between the bases and the backbones (phosphociester, iphosphorothioates and others as shown in table 1) also referred to as modified nucleotide linkages. The modifications or functional equivalent thereof, replacing the sugar moiety that lies between the backbone and the bases with an amino acid derivatives, for example as shown in formulae 24. The present invention is also provides novel nucleomonomers and methods for their incorporation into oligomers containing the nucleomonomers.

The invention provides various nucleomonomer compounds having the structures of formulae 1-23.

19 -a WO 96114330 PCTIUS95/14599 Base R,

N-R

Ho 0 Base

R

3 yR R,

N-R

Ho

O

2

R

Base

N

x Base Z% O Ho ORl 4 Bast

N

Base 0

R

31 R

N-R

6

_A

R

2 Base

R

3 R,

N-R

B

A

7

R

Base I I

R,

B

A

8R 2 Base

IR

3 /y R, N-OH HO

R

9 Base R N-OH HO

R

20 1 .72 1 WO 96/14330 WO 9614330PCTIUS95/1 4599 Base z y R,

OH

HO

R

2 12 Base

N

IOH

HOl 13 Base

R

N

Bk 14 Base B

R

2 Base

R

3

OH

R, N-R HO 0 18 Base 16 Base

R

R3)\ OH R N-R HO0

R

2 19 Base Z\ ,R

N

A

Br

R

1 R 2 17 Base

HO

Base

N

R, x HO

R

2 21 Base Z% .A

N

I~

21- SUBSh1UT SHEET (RULE2) I WO 96/14330 The oligomers of the one or more of the subjec provide a structural anal K the invention comprise tw comprises virtually any n oligomers of 200 or less to synthesize. Compounds one another through 4'-5' linkages, as can be seen j PCT/US95/14599 invention are polymers comprising t monomei compounds joined so as to og of DNA or RNA. The oligomers of o or more nucleomonomers and may umber of nucleomonomers, although nucleomonomers are generally easier of formulae 1-23, may be joined to linkages, linkages, and in formulae 24-41.

22 SUBSTITUTE SHEET (RULE 26) -i I WO 96/14330 PCT/US95/14599 I 00 O O SBase Base Base H 0 0 H O=P-O I Base 54BM Bas' 0 24A 24B The nucleotide linkages in the compounds of the invention are made from amino acids serine and glycine or derivatives thereof. The oligonucleotides of the invention are stable in vivo, resistant to endogenous nucleases and are able to hybridize to target nucleotide sequences. Exemplary compounds of this invention are shown in formulae 24 through 41 and are conformationally more restricted relative to the phosphodiester linkages found in unmodified DNA or RNA. This conformational restriction may, in part, contribute to the enhanced binding properties of the subject compounds to complementary polynucleotide target sequences; however, the use of the invention is not dependent upon this theory for enhanced binding properties.

In another embodiment, the present invention is directed to a modified oligonucleotide or derivatives thereof, wherein the furanose moiety of a natural oligonucleotide, DNA or RNA is replaced with amino acid/amino alcohol moiety and other modifications that comprises substitution at the amino acid positions are shown in the formulae 25 to 41. The internucleotide linkages between adjacent nucleomonomers is a 23 SUBSTITUTE SHEET (RULE 26) WO 96114330 WO 9614330PCTfUS95/14599 linkage between the 4' and 5' position of adjacent nucleomonomers. In other words, the phosphodiester internucleotide linkage, or functional equivalent thereof, originates from 5'-position of one nucleomonomer and connects the 41-position of adjacent monomer as exemplified by the compounds of formulae 24 -33: 00 4 Base N4 Base 0 R 0 N 0=P-U 0 R 1500=P0 4 B a se0 N37'2' Base o R 0 N-- 0 R, o R,

R

2 N Base 0OH 2

N

R, O=- 300

H

26A0 A I 26B 24- SUBSTITUTE SHEET (RULE 26) ~AO961433OPCT/US95/1 4599

R

3 N Base

R

3 Bs 0 N~ O P 01 H R 4 SBase

Z

N Bs o 0

N

1027A 0 I' R 10 271B

R,

R2 BaseR HN R2A Base Base N R 2 BaB' Rl R 0

N

2028A R R, R3R RR Bae

R

1~'25 R, N "kI Bac 1;V R 0 R. N-4 4

NF

as

R

3 R2N -R 2 NI Bs R9 02R9B P. R 4 29 9329C SUBSTITUTE SHEET (RULE 26) glycine, and those species which contain modifications of the 12 WO 96/14330 PCTIUS95/14599 XI a o X-N

R

Z-Base o X-Ni

R

0 X Base %I

R

R v\ Z- Base X -Base 30B R0 R 0

R

2 C 4i 0 R o=P-o 0I

R~

0 Baxe

R

2 N 31B

R,

0 -BWs 6 R,

R

2 z rr% o R 31A

R

0 Bawe r

R

32A 32B 26 phosphate groUg e.g. thiohosphate, aiddate).

13 mvmmq WO 96/14330 0 Base 0 -Bae

R

V R N -Ba PCT/US95/14599 +4 a- Base

X-N,

R

33C in- 33B 33A Wherein each is independently H, OH, SH, CN, OH 3 00H 3

SCH

3

ONE

2

ONH(CH

3 Ph, where is 1-7 carbon and I" is NH- 2 SE, OH, OOH, OCH 3

SOE

3 SPh, NOE, NOH(CH3), SNH 2 1 S(O)NH,, S(O) (O)NH 2

CE

3 Ph.

Wherein each "Base" is independently a nucleoside base.

Wherein each is independently H, OH, SE, ON, CE 3

OCH

3

SCH

3 ONH,, -ONH(CH 3 Ph, where is 1-7 carbon and is NH 2 SH, OH, COOR, OCH3, SCH 3 SPh, NOH,

NOH(CH

3

SNH

2

S(O)NH

2 S(0) OH 3 Ph.

Wherein each "R 2 is independently H, OH, SE-, CN, CE 3

OCH

3 1 SCE 3 1 UNH 2 1 ONE(CH 3 Ph, -(CH 2 where is 1-7 carbon and is NH 2 SE, OH, COQE, OCE 3

SCE

3 SPh, NOH,

NOE(CH

3

SNH

2 S (O)NH 2 1 S(0) CH 3 Ph.

Wherein each "R 3 is independently H, OH, SE, ON, OH 3 00E 3

SOE

3 1 ONE 2 ONH(0E 3 Ph, -(CH 2 where is 1-7 carbon and is NE 2 SE, OH, COO, 00E 3

SOE

3 SPh, NOE, 30NOH(0E 3 SNE,, S(O)NH 2 S(O) OH 3 Ph.

27- WO 96/14330 PCTIUS95/14599 4 i independentlyH, OH, SH, ON, CH3 Wherein each is3dpnety(C1,CO,5 S(0 NH NON, PCh, NRCH andFe where "x" 1 is 1- 7 Kcarbon.ad""i H1SOCOH C3 C3 ~,NH HO C3 N2 0 H2 0 0 H1C3 h Wherein each is independently (OH2j,, 00, CS, S, S S5(0) NH, NON, NCH3, NR 5 and Se, where

T

is 1- 7 carbon.

Wherein each is independently (CH 2 C0, OS, 0, S S NH, NON, NOH 3 and R, where is 1- 7 Wherein each is independently C0, OS, 0,S, S S NH, NON, NCNJ anrd NR 5 where is 1- 7 carbon.

R

5 is a N, ON, O~e, ON, NH, NON, ONCE 3

ONH

2 ,ehl propyl, lower alkyl (1-7C) Me, heteroalky! (1-70) aryl (6-

(CH

2 where is 1-7C, and is independently N, ON, SN, 00H 3 ON, SON 3

ONH

2 1 ONH (CH 3

SNH

2 S (0)NH 2

SO)(ONH

2 ,CNH Ph.

Wherein each is independently a phosphodiester analog, phosphorothioates, methylphosphonates, phosphorodithioates, boronphosphonates, selenophosphonates, phosphorainidates, acetamidate, oxyformanido, oxyacetamido, diisopropylsilyl, carbamate, dimethylene sulfide, dixnethylene sulfoxide, dimethylene sulfone and! or two to four atom long internucleoside linkage is selected from carbon, nitrogen, 28 WO 96/14330 PCT/US95/14599 oxygen, sulfur and selenium. The length of the oligomer may vary from a dimer to a 200mer, or longer. Preferred modified internucleotide linkages include the structures for are shown in Table I.

Additionally, the compounds of formulae may be conjugated to one or more conjugate moiety. Suitably, conjugate moieties include O-cholesterol, polyethylene glycol, amino acids, intercalators, cleaving moieties imdazole), crosslinking functionalities psoralen), lipids, peptides, alkylating agents, hydroxamaes, and fluorescent labels. The conjugate moiety may independently replace one or more of R, Ri, R 2

R

3

R

4 and R s In yet other embodiment, the subject invention provides oligomer structures as indicated in formulae 34-36 and derivatives thereof: PN yBase

I

0 R R 3 -Base 0 0 34A Base o R, 0=P-0 0. R, 0

O=P-O

O R, R2- ,Z-Base

N-Y

0 34C 34C

R

4 34B Formula 34 29 a I -I ~I~L WO 96/14330 R2-Y Base S 0 R2-.(Y Base 0 N v PCT/US95/14599

R

3 N- i LBase \N-Y II:Bs 3511 In the compounds of formulae 34-36, the linkages between adjacent nucleomonomers are 3' to 5' linkages.

Wherein each is independently H, OH, SH, CN, CM 3 0CM 3

SCM

3 ONH,, ONH(CH 3 Ph, -(CH 2 where is 1-7 carbon and is NM 2 SM, OH, COOH, OCH 3

SCH

3 SPh, NOH,

NOM(CH

3 SNH,, S(O)NM 2 S(O) (O)NH 2

CM

3 Ph.

Wherein each "Base" is independently a nucleoside base.

Wherein each is in~dependently H, OH, SH, CN, CA,

OCH

3 SCH3, ONH 2

ONH(CH

3 Ph, -(CH 2 where is 1-7 carbon and is NM 2 SM, OM, COOM, 0'CM 3 SCMJ, SPh, NOM,

NOH(CM

3

SNM

2

S(O)NH

2 S(O) (O)NH 2

CM

3 Ph.

wherein each "R 2 is independently H, OH, SM, CN, CM 3 OH1SCM 3 ONh 2 ONH(CH3j, Ph, -(CH 2 where is 1-7 carbon and is NM 2 SM, OH, COOM, 0CM 3

SCM

3 SPh, NOM, NOM (CM 3

SNM

2 1 S N 2 S5(0) NH 2

CM

3 Ph.

30 17 SWO 96/14330 PCTIUS95/14599 Wherein each "R 3 is independently H, OH, SH, CN, CH 3

OCH

3

SCH

3

ONH

2

ONH(CH

3 Ph, -(CH 2 where is 1-7 carbon and is NH 2 SH, OH, COOH, OCH 3 SCH3, SPh, NOH,

NOH(CH

3

SNH

2 S (O)NH 2 S (O)NH 2

CH

3 Ph.

Wherein each "R 4 is independently H, OH, SH, CN, CH 3

OCH

3 SCH3, ONH,, ONH(CH 3 Ph, where is 1-7 carbon and is NH 2 SH, OH, COOH, OCH 3 SCH3, SPh, NOH,

NOH(CH

3

SNH

2 S(O)NH, S(O) CH 3 Ph.

Wherein each is independently CO, CS, S, S(0) NH, NOH, NCH 3

NR

5 and Se. Where is 1- 7.

Wherein each is independently CO, CS, S, S(O) NH, NOH, NCH 3 NPR, and Se. Where is 1- 7.

Wherein each is independently CO, CS, O, S, S(0) NH, NOH, NCH 3 and NR 5 Where is 1- 7.

Wherein each is independently CO, CS, O, S, S(O) NH, NOH, NCH 3 and NR 5 Where is 1- 7.

Wherein each is independently CO, CS, S, S S NH, NOH, NCH 3 and NR;. Where is 1- 7.

R

5 is a H, OH, OMe, CN, NH, NOH, ONCH 3

ONH

2 ethyl, propyl, lower alkyl Me, heteroalkyl aryl(6- 7C), where is 1-7C, and is independently H, OH, SH, OCH 3 CN, SCH 3 ONH, ONH(CH 3 SNH, S(0)NH 2 S(0) CH 3 Ph.

30 Wherein each is independently a phosphodiester analog, phosphorothioates, methylphosphonates, phosphorodithioates, boronphosphonates, selenophosphonates, 31 WO 96/14330 phosphorc linkage i i selenium.

i a 200mer, linkages i I PCT/US95/14599 umidates and/ or two to four atom long internucleoside .s selected from carbon, nitrogen, oxygen, sulfur and The length of the oligomers may vary from a dimer to or longer. Preferred modified internucleotide include the structures for are shown in Table I.

In another embodiment of the invention, the subject invention provides oligomers having formulae 37 to 41, or variants thereof, oligomers comprising novel internucleotide linkages that are linkages. These oligonucleotides are stable in vivo, have improved resistance to endogenous nucleases, and are able to hybridize to target oligonucleotide sequences.

37A 37B

R

4 abe 32 38A 38B 38A 38BL PCTIUS95/14599 WO 96/14330 X- A 0 0 R -Bs

X-N

39 A/ Z- Bas c

X-N,

A

39B -XN Base R v/A

R

3 B Base

N

R

Z- ZBam

A

V

B

IZ-Bac X-r4 41B 41A 33 WO 96/14330 PCTJUS95/14599 Wherein each is independently H, OH, SH, ON, OH 3

CB

3

SCH

3

ONH

2 ONH(0H 3 Ph, where is 1-7 carbon and 'IFY is NH,, SH, OH, COOH, OCH 3

SOH

3 SPh, NOH, NOH (CH 3

SNH

2 S NH 2 S NH 2

OH

3 Ph.

Wherein each "Base" is independently a nucleoside base.

Wherein each is independently H, OH, SH, ON, OH 3

OCH

3

SOB

3 ONH,, ONH(0H 3 Ph, -(OB 9 where is 1-7 carbon and 'IF" is NB 2 SH, OH, COOB, OCBj, SOH 3 SPh, NOB, NOH(0H 3

SNH

2

S(O)NH

2 S(O) (O)NH 9

OH

3 Ph.

Wherein each is independently H, OH, SB, ON, OH 3 00H 3

SOB

3

ONH

2 ONH(0H 3 Ph, -(0H 2 where is 1-7 1carbon and 'IF" is NH 2 SH, OH, OOOH, OOH 3 1 SOB 3 SPh, NOH, NOB(0H 3

SNTH

2 1 S(O)NB 2 S(O) (O)NH 9

OH

3 Ph.

Wherein each is independently H, OH, SH, ON, OH 3 00H 3

SOB

3 oNB,, ONB(0H 3 Ph, -(0H 9 where' is 1-7 carbon and 'IF" is NH,, SH, OH, OOOH, 00H, SOH 3 SPh, NOH, NOH (0B 3

SNH

9 S (0)NH 2 S5(0) NH,, OH 3 Ph.

Wherein each I'R 4 is independently H, OH, SH, ON, OH 3

CH

3 1 SOBH 3 ON~H, ONH (0H 3 I Ph, (CH F; where is 1-7 and 'IF" is NH 2 SH, OH, 00011, 00H 3

SOB

3 SPh, NOB, NOH(0H 3

SNH

2

S(O)N

2

OH

3 Ph.

Wherein each is independently 00, OS, S, S(0) S5(0) NH, NOB, NCH 3

NR

5 and Se; where is 1- 7 carbon.

-34 ~s~~ll__l WO 96/14330 PCT/US95/14599 Wherein each is independently (CH 2 CO, CS, S, S(O) NH, NOH, NCH 3

NR

5 and Se; where is 1- 7 carbon.

Wherein each is independently (CH 2 CO, CS, O, S, S(0) NH, NOH, NCH 3 and NR 5 where is 1- 7 carbon.

Wherein each is independently (CH2z), CO, CS, S, carbon.

R, is a H, OH, OMe, CN, NH, NOH, ONCH 3

ONH

2 ethyl, propyl, lower alkyl Me, heteroalkyl aryl(6- 7C), where is 1-7C, and is independently H, 1OH, SH, OCH 3 CN, SCH 3 ONHj O SNH 2

S(O)NH

2 S(0) CH3, Ph.

Wherein each is independently a phosphodiester analog, phosphorothioates, methylphospho.ates, phosphorodithioates, boronphosphonates, selenophoshonates, phosphoramidates and/ or two to four atom long internucleoside linkage is selected from carbon, nitrogen, oxygen, sulfur and selenium. The length of the oligomer varies from dimer to 200mer. Preferred modified internucleotide linkages include the structures for are shown in Table I.

In other embodiments of the invention, the subject invention is directed to an oligomer of the following fornulae (formula 42) and monomeric constituents thereof (formulae 85-90).

35 '14330 PCT/US95/14599 -B N -B Y R N-X

Z

R Y X-B Z X-B R Y R B Z 4 N-X 42A 42B 42C Y Z NH Z z> R R :y Y R N X-B H-N X-B N-H Z H Z 0

R

20

R

42D^' 42Eq N -H 42F2wherein, X is selected from the group consisting of (CH 2 where n=l- 3, CO(CH 2 where n= 0-2, and (CH 2 )nS0 2 where n=1-2, Y is selected from the group consisting of CH,, CO, COOH, CS, and SO,, Y'is selected from the group consisting of CH,, CO, COOH, CS, and SO, Z is selected from the group consisting of O, S, NH, and CH, 36 I T- i- q ~11 I s I WO096/14.330 PCTJUS95/14599 R is selected from the group consisting ofCHOH, CH,NE,,

CH

2 NHCEO, CONE 2 and COOE, B is a nucleoside base.

COOH X -B Z X-Z z N'

BZ

H

HR

86 87 Y /H y X-BY -BfHl-N1 I z z

ZH,

R I N-H y H Hi 88 89 wherein, is selected from the group consisting of (CH9~ where n=1- 3, CO(CH 2 where n= 0-2, and (CH 2 where n=1-2, Y is selected from the group consisting of CH2, 00, COOH-, CS, and SO 2 Ylis selected from the group consisting of CE 2 CO, COOH, CS, and SO 2 Z is selected from the group consisting of 0, S, NH, and CH,, R is selected from the group consisting ofCH,OH, CE-,NH7, 2 NH-CHO, CONH,, and COON, B is a nucleoside base.

37 WO 96/14330 PCT/US95/14599 In other embodiments, the invention provides methods for treating diseases mediated by the presence of a nucleotide sequence which comprise administering to a subject in need of such treatment an amount of the above modified oligonucleotides capable of specifically binding the nucleotide sequence effective to inactivate the nucleotide sequence.

In the oligonucleotides of the invention, at least one of the phosphodiester groups included within the "Vs" of Formulae 24-41 is substituted by the modified internucleoside linkages described herein. Desirably, multiple phosphodiester linkages in the unmodified oligonucleotide are substituted by the modified internucleoside linkage may be used repeatedly in this structure, or, if desired, a variety of modified internucleotide linkages may be used in an individual oligonucleotide. In a preferred embodiment of the subject oligonucleotides these substituent linkages are non-chiral so as to enhance the ability of the oligonucleotide to hybridize to a desired target; however, useful compounds of the invention include those embodiments in which chiral forms are used.

-38 L i WO 96/14330 PCT/US95/14599 Perferred modified internucleotide linkages include the structures f or "V' t are shown in the Table 1.

Table 2: -0- -Se- -Si-

-NH-

-NOH-

-0-CH,- -CH,-0- -0-0W2-CR 2

-CH

2 -CH-0-

-O-CH

2 -0

-S-CHW-

-CH

2

-S-

-S-CRC,

-CH,-CH,-S-

-CH

2 -S-C H,-

-S-CW,-S-

0CS

-S-OW--

-s -0H 2 -S -S -OR,-0H -CH -CH -S -CH -S -CRH 25-S(0)-

CH

2 -S 2 -S -CH,-0-

-OH

2 -0H 2 -S -S

-CH,-CH

2 -S

CH

2 -S

-OH,-

-CW,-S -0-CH 2 -S -S -CH,-0- (0- 39 WO 96/14330 PCTJUS95/14599 -S -S -Se-CR,-

-CR

2 -Se- -Se-CH-,-CH,-

-CH,-CH

2 Se-

-CR

2 -Se-CH 2 _S e-CHW-Se- -Se-CH 2 -0- -Se (0)-CH 2 -CR,-Se -Se -CH,-CH,- -CH -CH -Se -OW,-Se -CIJ!L- -Se -OW-Se -O-CH,-Se -Se -CH,-O- -Se -CR,- -CH,-Se -Se -CH,-CH,-Se -OW2-Se -CR,- -Se -CH 2 -Se -Se-Se- -Se -Se -Se -Se -0-CR 2 -Se -Se -0R 2

-O-

-S-CR

2 -Se- -Se-OW,-S -S -CH,-Se -Se -CH2-S -CR,-Se -Se -CR,-S -S -Se-Se- -Se -Se -Se -Se -N (R 5

-OH

2 -0H 2 -N (R 5 -N (R 5

-CH

2

-CH

2

-CH,-CR

2 -N (R 5

(R

5

-OH,-

-N (R 5 -0- -0-N (R 5 -N (R 5 -0-OH 2

-CH

2 -0-N (R 5 -CH,-N (R 5 -0- -0-N (Rs) -OH7- 27 WO 96114330 V 5 A 10 PCTIUS95/14599

-C-CH

2 -N (R 5 -N (R 5

-CH

2

-C-

-N (R 5

-S-

-S-N (R 5 -N (R 5

-CH

2 -S-N (R 5

CR

5 -S-N (R 5

-OH

2

-S-CH

2 -N (R 5 -N (R 5

-CH

2

-S-

-N (R 5 -S -S -N (R 5 -N (R 5 -S -N (R 5

-CH

2 -N (R 5 -S -S -N CR 5

-CH,-

-CH2-N CR 5

-N(R

5 -CH,-S

-NC(R

5 -S C0) -SC(0) C0) -NCRS)

-NC(R

5 -S CC) C0) -CH,- -CIL-S C) -NC(R 5 -0H 2 -N (R 5 -S CC) C) -SCO) C) -NC(R3)-CH 2 -S CC) CC) -CH,-N CR 5

-NC(R

5

-CH

2 -S CC) CC) -0-N CR 5 -S-N CR 5

-C-

-C-N CR 5 -S CC) -S CC) -N CR 5

-C-

-C-NCR

5 -S CC) C) -S (0)C0) -NC(R 5

-C-

-C-S-C-

-C-SCC) -C- -C-SCC) C)-C-

-NC(R

5 -S-N CR 5 -NC(RS) SC0) -NC(RO

-NC(R

5 )-SCC0) C)-NC(R 5

-CH

2 -S -C-

-CH

2 -S CC) -C- -CH-SCC0) C) -C- -CH,-C CC) -C-

-CH

2 -C CS) -C-

-CH

2 -N CR 5 -CCC0) -C-

-CH

2 -N CR 5 -C CS) -C- -N (R 5 -CCC0) -NC(R) -C CS) -0CH,- -C-C CC) -N CR 5

-C-

-C-C CS) -N CR 5

-C-

-C-C CC) -N CR 5

-OH

2 -C-C CS) -N CR 5

-CH,-

41 WO 96/14330 PCT/US95/14599 -0-C -CH2-N CR5) -0-C(S -CH 2 N -0-C -CH -0-N -0-C (R 5 )-0--CH 2 -0-C -N (R 5

-O-CH

2 -0-N (R 5 -C -0-CH- -0-N (R -C -O-CH-- -CH,-O-C -N (R 5 -0-

-CH

9 -0-C -N (R 5 -0-

-CH

2 -0-C -N (R 5

-CH,-

-CH

2 -0-C -N (R 5

-CR,-

-CH -0-C -CH -N CR) -CH,-0-C -CH 2 -N (R 5 -CH,-0-C -N (R -N (R 5 -CH -0-C -NC(R5) -0- -CH,-0-C -N (R 5 -0-

-CH

2 -0-N CR 5 -C -0-

-CH

2 -0-N CR 5 -C CS) -0- -CH,-N CR 5 -C -CH,-N (R 5 -C

-NC(R

5 -C -S-CH,- -N CR 5 -C -S-CH- -S-C -NC(R 5 -0- -0-C -N CR 5 -S-C -N (R 5

-CR

2 -S-C -N CR 5

-CH,-

-S-C CO) -CH,-N (R 5 -S-C -CH,-N CR 5 -S-C -CH 2 -0-N CR 5 -0-C -CH 2 -S-N CR 5 -0-C -N CR 5

-S-CHW-

-S-C -N(R 5 -0-CH 2 -S-N CR 5 -C -0-CHW- -0-N CR 5 -C -S-CR 2

-CR

2 -S-C -N CR 5 0-

-CH

2 -0-C -N CR 5

-CR

2 -S-C -N CR 5

-CH-,

-CH

2 -S-C -N CR 5

-CR,-

-CR,-S-C (R 5

-CH

2 -S-C -CR,-N CR 5

-CH

2 -S-C -NC(R 5

-CH

2 -S-C (5S) -N CR 5

-CR

2 -S-C N CR 5 -0-

-CR

2 -S-C -N CR 5 -0-

-CR

2 -S-N CR 5 -C -0-

-CR

2 -S-N CR 5 -C -0-

-CH

2

-NC(R

5

-S-

-CR

2 -0-C -N CR 5

-CR

2 -0-N (R 5 -C 42 WO 96/14330 PCTJUS95/14599

-CH

2 -0-N (R 5 -C -S- -N (R 5 -N (R5) -N (R 5 -N CR 5

-CR

2 -CH,-N (K 5 -N CR 5 -N=C (NH 2 -N (R 5 -N (R5) -N=C (NH 2 -S -CH-0- -0-CH 2 -S -S-CH CR 5 -0- -0-CR (R 5

-S-

-0-CH 2

-CH=CH-

S-CHW-CH=CH-

-S-CH

2

-C=C-

-N CR 5

-CH

2 -N (R 5 -N (R 5 -C -N (R 5 -N (R 5 -C -N (R 5 -N (R 5 -C -S- -N CR 5 -C -N CR 5 -C -0-

-NC(R

5 -C -0- -0-C -NCR 5 -0-C -N CR 5 -S-C -NCR 5 -S-C -N CR 5

R

5 is a H, OH, OMe, CN, NH, NOH, ONCH 3

ONH

2 ethyl, propyl, lower alkyl Me, heteroalkyl arylCG- 7C), where is 1-7C, and is independently H, OH, SH, OCH3, CN, SCH3, ONH 2 ONH (CH 3

SNH

2 S NH 2 S(0) (0)NH 2

CR

3 Ph. Additionally, conjugate one or more moieties may be joined to the linkage so as to produce an oligomer conjugate. Suitable conjugate moieties include, 0cholesterol, polyethylene glycol, amino acids, intercaiulators, cleaving moieties imdazole), crosslinking functionalities psoralen), lipids, peptides, alkylating agents, hydroxamates, and fluorescent labels.

Particularly preferred linkages include phosphodiester, phosphorothiates, metylphosphonates, carboxamide, thiocarboxamide, hydroxanate, sulfonamide, 43 WO 96/14330 PCT/US95/14599 hydroxviamine and carbamate. The same modifications are preferred for and linkages as well.

The oligomers of the invention are not limited to oligomers of homogeneous linkage type, and that alternating or randomly distributed substitute linkages including the linkages are included. Since the oligomers of the invention can be synthesized one nucleomonomer residue at a time, each individual linkage, and/or substitute linkage, and the nature of each individual "Base" substituent may be selected independently so as to produce oligonucleotides having a desired seauence.

The oligomers of the invention may contain any desired number of the substitute linkages. These substitute linkages may be iaentical to each other or different by virtue of the embodiments chosen for including other noninvention substitute linkages. Since the oligomers are prepared sequentially, any pattern of linkage or substitute linkage types, bases and sugar modifications may be used.

In preferred embodiments of the invention, the substitute linkages of the invention alternate in a regular pattern. For example, one substitute linkage is followed by two phosphodiester linkages followed by one invention substitute linkage, etc. Additional embodiments include, for example, alternating linkages such as a substitute linkage followed by a phosphodiester analog thioate, etc.), followed a substitute linkage of the invention followed by a phosphodiester analog, etc., the oligomer of the invention may comprise a one-by-one alternation of the two types of substitute linkages. Oligomers of the invention comprising more than one type of linkage may have any of a number of regular patterns formed by alternations between the 44 e_ ii WO 96/14330 PCT/US95/14599 different linkage types present between the subunits of the oligomer.

Sugar modifications may be made to one or more nucleomonomer residues in oligomers of the invention; however, and nucleotide linkage between amino acid residues are preferred when such modifications are to be incorporated. Where this is the case, further abbreviation may be used to represent the base sequence of the oligonucleotide analog. For example, in standard DNA (or RNA) the sequences are generally denoted by the sequence of bases alone, such as, for example, ATG CGC TGA. In general, .t is simply stated in advance whether this represents an RNA or DNA sequence. A corresponding notation system is used herein so as to represent oligonucleotide analogs with a given base sequence.

Additional Nucleomonomer Modifications: Oligomers of the invention may also comprise of various modifications in addition to the substitute linkages of the j 20 invention. Additional modifications i .ude oligomers where one or more nucleomonomer residues are modified at the 2', and 5' positions, (ii) one or more covalent crosslinking moieties are incorporated, (iii) other noninvention substitute linkages are included, (iv) other base analogs, such as 8-oxo-N 6 -methyladenine, are included and (v) conjugates such as intercalating agents or polylysine that respectively enhance binding affinity to target nucleic acid sequences or that enhance association of the oligomer with cells are included.

The sequence-specific polynucleotide binding properties of the oligomers of the invention for single-stranded and duplex targets is compatible with further modifications to the 45 r, -ce I- I T" ~Ct I~Y- l

IBBP"

WO 96/14330 PCT/US95/14599 oligomer. These further modifications may also confer other useful properties such as stability to nuclease cleavage (e.g.

in a domain of an oligomer of the invention having phosphodiester linkages), or enhance their ability to permeate cell membranes, and the like.

The oligomers of the invention may comprise one or more substitute linkages such as sulfide or sulfone linkages (Benner, International Publication No. WO 89/12060), sulfamate linkages (International Publication No. WO 91/155001, carbamate or other substitute linkages in morpholino-linked oligomers (Stirchak, E.P. et al Nucleic.

Acids Res_ 1989, 17, 6129-6141; Summerton, et al International Publication No. 216 860) and related linkages.

I

Thus, exemplary embodiments of invention oligomers include oiigomers having at least one substitute linkage and an amino acid that is linked to an adjacent monomer and one or more non-invention substitute linkages selected from the group consisting of phosphorothioate, methylphosphonate and thionomethylphosphonate and/or one or more phosphodiester linkages and/or purine or pyrimidine analogs that enhance binding affinity for complementary target sequences. Other exemplary oligomers would include an oligomer having invention substitute linkages at the 3' and/or 5' ends and phosphorothioate linkages elsewhere in the oligomer; oligomers having invention substitute linkages and standard purine or pyrimidine bases adenine, guanine, cytosine, thymine, or uracil); oligomers having invention substitute linkages and one or more bases that enhance binding affinity or permeation competence of the oligomer 5'(1-propynyl) uracil, 5-(l-propynl) cytosine. Also included 46 Ii WO96/14330 PCT/US95/14599 are oligomers containing nucleomonomer residues linked via hydroxamates.

Synthesis of Olicomers: The oligomers of the invention may be formed using nucleomonomers of the invention alone or in combination with conventjcnal nucleomonomers and synthesized using standard solid phase kor solution phase) oligomer synthesis techniques, which are now commercially available. In general, the invention oligomers may be synthesized by a method comprising the steps of: synthesizing a nucleomonomer or oligomer synthon having a protecting group and a base and a coupling group capable of coupling to a nucleomonomer or oligomer; coupling the nucleomonomer or oligomer synthon to an acceptor nucleomonomer or an acceptor oligomer; removing the protecting group; and repeating the cycle as needed until the desired oligomer is synthesized.

The oligomers of the present invention may be of any length including those of greater than 40, 50, 100, 200 or 500 nucleomonomers. In general, preferred oligomers contain 2-30 nucleomonomers. Lengths of greater than or equal to about 8 to 20 nucleomonomers may be useful for therapeutic or diagnostic applications provided they have a suitable base sequence. Short oligomers containing 2, 3, 4 or nucleomonomers are specifically included in the present invention and may be used as synthons.

Oligomers having a randomized sequence and containing about 6, 7 or 8 nucleomonomers may be used as primers that are used in cloning or amplification protocols that use random sequence primers, provided that the oligomer contains about 1 or 2 residues at the 3' end that can serve as a primer for 47 WO 96/14330 PCT/US95/14599 polymerases or reverse transcriptases or that otherwise do not interfere with polymerase activity.

In addition to the linkages described for the first time herein, the oligomers of the invention may comprise conventional phosphodiester linkages or can contain other substitute linkages such as phosphoramidate linkages in addition to the invention substitute linkages. These substitute linkages include, but are not limited to, embodiments wherein a moiety of the formula-O-P(O) ("phosphorothioate"), -O-P -X 2

(R

1 -0- P(S) (R ("thionoalkylphosphonate") (OR')-X 2 -0- C(0) or 2 wherein R 11 is H (or a salt) or alkyl (1-12C including methyl and ethyl) and R 3 is alkyl (1=9C) and the linkage is joined to adjacent nucleomonomers through an or bonded to a carbon of the nucleomonomer and X 2 is O or S. Phosphorothioate and phosphodiester linkages are well known. Particularly preferred substitute linkages for use in the oligomers of the present invention include phosphodiester, phosphorothioate, methylohosphonate and thionomethylphosphonate substitute linkages.

Phosphorothioate and methylphosphonate substitute linkages confer added stability to the oligomer need be identical, particularly preferred oligomers of the invention contain one or more phosphorothioate or methylphosphonate substitute linkages.

Oligomers of the invention and the segments thereof may be synthesized using methods that are known to the person.-f ordianry skill in the art. The synthetic methods known in the area and described herein can be used to synthesize oligomers containing substitute linkages of the invention, as well as other linkages or substitute linkages known in the art, using appropriately protected nucleomonomers. Methods for the synthesis of oligomers having phosphorous containing linkages 48 hy sr I WO 96/14330 PCTUS95/14599 are found, for example, in Froehler, et al., Nucleic Acids Res., 1986, 14, 5399-5467; Nucleic Acids Res., 1988, 16, 4831-4839; Nucleosides Nucleotides, 1987, 6, 287-291; Froehler, Tetrahedron Letts., 1986, 5575-5578; Caruthers, M.H. in Oligodeoxynucleorides Antisense Inhibitions of Gene Expression, 1989, J.S. Cohen, editor, CRC Press, Boca Raton, p7-24; Reese, C.B. et al, Tetrahedron Letts., 1985, 26, 2245-2248. Synthesis of the methylphosphonate linked oligomers via methyl phosphonamidite chemistry has also oeen described (Agrawal, S. et al., Tetrahedron Letts., 1987, 28, 3539-3542; Klem, et al, International Publication Number WO 92/07864).

Oligomers containing linkages of the present invention are also conveniently synthesized by preparation of dimer or trimer compounds by solution phase chemistry followed by conversion of tne synthon to a derivative that is incorporated into oligomers by either solid or solution phase chemistry.

Typical synthons are J' DMT or MMT blocked 3' phosphonate or Dhosphoramidate derivatives which are prepared by standard methods (see: Gait, M.J. ed., Oligonucleotide Synthesis; A Practical Approach 1984, :RL Press, Oxford).

Synthons that are included in the scope of the present invention include dimers, trimers, tetramers, hexamers and longer oligomer made by solid or solution phase synthesis.

Trimers and longer synthons may contain more than one type of linkage. The synthons may include any base as described above or 4' and 5' groups such as OH, DMTO, MMTO, O-allyl, phosphate, a phosphonate or an amidite as described above.

Ribose-amide oligonucleotides could be synthesized by using standard solid phase peptide synthesis (Fmoc chemistry) conditions (see figure 26).

49 WO096114330 tehePCT1US95/14599 BlocingGrops or he ymtesi oftheCom-Pound o h Suitable coupling groups are, for example, H-phosphonate, methylphosphonomidite, or a phosphoramidite.

Phosphoramidites that can be used include cyanoethylphosphoramidites (preferred).

Methylphosphonamidites, alkylphosphonarnidites (including ethylphosphonarnidites and oropyiphosphonamiditLes) can also be used. Exemplary phosphoramidlites are shown in.fligures I to 21.

Suitable "coupling groucs" at the 4' or position for oligomer synthesis via phosphorarnidite triester chc- 4 nistry, referred to herein as "amidite" chemistry, include N, N-diisopropylamino- -cyanoethoxyphosphine, N- N, diisorropylamino-methoxyphosphine, N, N-diethylaminocyanoethoxyphosohiLne, and (N-morpholino) -rethoxvphosphine (Moore, M. F. et al, j Org Chemr., 1985, 2019-2025; Uznanski, et al, -Tetrahedron Lectts.,1987,28, 3401-3404; Bjergarde, et Nuci Acids Res., 1991, 19, 5843-5850; Dahl, 0. Sulfur Reports 1991, 1 167-192) Related coupling groups such as N,N-diisopropylamino-methyl-phosphine or N, N-diethvlamino-methyl-phosrhine can also be used to prepare methylphosphonates. Meth~lphosphonate oligomers can be conveniently synthesized using coupling groups such as N,Ndiisopropylamino-methylphosphoramidi-te. Synthesis of nucleomonomer amidites of the invention can be accomplished by conventional methods (for example, Gryaznov, et al, Nucl Acids Res., 1992, 2-0 1879-1882; Vinayak, et al, Nuci 3Acids Res 1992, 2L, 1265-1269; Sinha, et al, Nucl Acids Res., 1984,.12, 4539-4557; and other references cited herein) PCTIUS95/14599 WO 96/14330 2. Proteclno Grouns.

Protecting groups such as diisobutylformamidine, benzoyl, isobutyryl, FMOC, dialkylformamidine, dialkylacetamidine or other groups known in the art can be used to protect the exocyclic nitrogen of the cytosine, adenine or guanine heterocycles. Alternatively, cytidine can be directly incorporated into oligomers without a protecting group at the exocyclic nitrogen using described methods (Gryaznov, S.M. et al, J Amer Chem Soc., 1991, 111, 5876-5877; Gryaznov, S.M. et 1 al, Nucl Acids Res., 1992, 20, 1879-1882; Kung, et al, Tetrahedron Letts., 1992, 331, 5869-5872).

Suitable prorecting groups are DMT (dimethoxy trityl), Bz (benzoyl), Bu (isobucyryl), phenoxyacetyl, MMT (monomethoxytrityl) or FMOC at the 5' terminus and/or hydrogen 1 phosphonate, methyl phosphoramidite, methyl phosphonamidite, g-cyanoethylphosphoramidite, TBS (t-butyldimethylsilyl) or TBDPS (t-butyldiphenylsilyl) at the 3'-terminus.

Preferred protecting groups are Bz (benzoyl), DMT (dimethoxytrityl), MMT (monomethoxytrityl) or FMOC at the terminus or position and/or TBS, hydrogen phosphonate, methylphosphoramidite, methyl-phosphonamidite, Pcyanoethylphosphoramidite at the 3'-terminus. However, it is intended that the position of the blocking groups can be reversed as needed a phosphoramidite at the 5' position and DMT at the 3'-position). In general, the nucleomonomers and oligomers of the invention can be derivatized to such "blocking groups" as indicated in the relevant formulas by methods known in the art.

30 Coniucates: The subject invention also provides for "conjugates" of the oligomers of the invention. "Conjugates" of conventional 51 i -I _-~ll~ll~l~s~a~Prsll~k

I

1 ;t i WO 96/14330 PCT/US95/14599 oligomers are known to the person of ordinary skill in the art. For example, the oligomers of the invention may be covalently linked to various moieties such as, for example, intercalators, and compounds which interact specifically with 5 the minor groove of the DNA double helix. Other moieties for conjugation to the subject oligomers include, labels, radioactive, fluorescent, enzyme) or moieties which facilitate cell association using cleavable linkers and the like.

Suitable radiolabels include 3 3S, 3H, "3!I and "1C; and suitable fluorescent labels include fluorescence, resorufin, rhodamine, BODIPY (Molecular Probes) and Texas red; suitable enzymes include alkaline phosphatase and horseradish peroxidase. Other compounds which can be used as covalently linked moieties include biotin, antibodies or antibody fragments, asialoglycoprotein, transferrin and the HIV Tat protein can also conveniently be linked to the oligomers of the invention.

These additional moieties can be derivatized through any convenient moiety. For example, intercalators, such as acridine or psoralen can be linked to the oligomers of the invention through any available OH or -SH, at the terminal 5'-position of the oligomer, the 2'-positions of RNA, or an OH, NH,, COOH or SH incorporated into the 5-position of pyrimidines. A derivatized form which contains, for example, S- CH,CH 2

CH

2 ,OH or -CH 2

CH

2

CH

2 SH in the 5-position of pyrimidines is convenient. Conjugates including polylysine or lysine can be synthesized as described and can further enhance the binding affinity of an oligomer to its target nucleic acid sequence (Lemaitre, M. et al., Proc Natl Acad Sci. USA,1987, 84, 648-652; Lemaitre, M. et al., Nucleosides and Nucleotides, 1987, 6, 311-315).

52 WO 96/14330 PCT/US95/14599 A wide variety of substituents can be attached, including those bound through linkages or substitute linkages. The -OH moieties in the oligomers can be replaced by phosphate groups, protected by standard protecting groups, or coupling groups to prepare additional linkages to other nucleomonomers, or can be bound to the conjugated substituent. The 5'-terminal OH can be phosphorylated; the 2'-OH or OH substituents at the 3'terminus can also be phosphorylated. The hydroxyls can also be derivatized to standard protecting groups.

Oligomers of the invention can be covalently derivatized to moieties that facilitate cell association usina cleavable linkers. Suitable conjugates also include solid supports for oligomer synthesis and to facilitate detection of nucleic acid sequences. Solid supports include, but are not limited to, silica gel, controlled pore glass, polystyrene, and magnetic glass beads.

Sugar Modifications: Derivatives can be made by substitution on the sugars.

Among the preferred derivatives of the oligomers of the invention are the 2'-O-allyl or 3'-allyl group appears to enhance permeation ability and stability to nuclease i degradation, but does not appear to diminish the affinity of the oligomer for single chain or duplex targets. In particular, in ribose-amide backbone oligonucleotides, different functionalities could be introduced at the 2', 4' and 5' positions of the ribose moiety to improve the pharmacokinetic properties of the corresponding oligonucleotides.

Substitute Linkages: The oligomers of the invention may also contain one or more "substitute linkages", in addition to the 53 L i I- s O96/14330 PCTIUS95/14599 WO 96/14330 and linkages disclosed herein, which are generally understood in the art. These "substitute linkages" include phosphorothioate, methylphosphonate, thionomethylphosphonate, phosphorodithioate, alkylphosphonates, morpholino sulfamide, boranophosphate (-0-P(OCH 3 siloxane (-0-Si(X 4

(X

4 X1 is 1 6C alkyl or phenyl) and phosphoramidate (methoxyethylamine (OCH,CH,OCH 3 and the like), and are synthesized as described in the generally available literature including the following references (Sood, et al J Am .Chem .Soc., 1990, 112, 9000-9001; WO 91/08213; WO 90/15065; WO 91/15500; Stirchak, E.P. et al Nucleic Acid Res., .1989, 7. 6129-6141; U.S. Paten: 5,034,506; U.S. Patent 5,142,047; Hewitz, J.M. er al, Nucleosides Nucleotides, 1992, 11, 1661-1666; Summerton, J. et al International Publication No. 216 860). Substitute linkages that can be used in the oligomers disclosed herein also include the sulfonamide (-0-SO 2 sulfide sulfonate carbamate dimethylhydrazino (-CH 2

-NCH

3 sulfamate -N- S(0) 3'-amine N-methylhydroxylamine

NCH

3 and linkages (such as carbamate 2' carbamate 5',2' methylcarbamate (2'-0-C(0)-N(CH 3 and thioformacetal Additional substitute linkages that are suitable include amide linkages described by Buchardt, O. et al, (International Publication No. WO 92/20702), and those described by Cook, P.D. et al, (International Publication No.

WO 92/20822), D- Mesmaeker, A. et al., (International Publication No. WO 92/20823) and as described in PCT/US92/04294.

Except where specifically indicated, the substitute linkages, such as a formacetal linkage, are linked to either the 2' carbon of a nucleomonomer on the 54 WO 96/14330 PCT/US95/14599 left side and to the 5' carbon of a nucleomonomer on the right side. The designations of a 2' or 5' carbon can be modified accordingly when a structure other than ribose, deoxyribose or arabinose is linked to an adjacent nucleomonomer. Such structures include xylose, a hexose, morpholino ring, carbocyclic ring cyclopentane) and the like.

The use of carbamate, carbonate, sulfide, sulfoxide, sulfone, N-methylhydroxylamine and dimethylhydrazino linkages in synthons or oligomers has been described (Vaseur, J-J. et al, J Amer Chem Soc., 1992, 114, 4006-4007; WO 89/12060; Musicki, B. et al, J Org Chem.. 1990, 55, 4231-4233; Reynolds, R.C. etal., J .Org .Chem., 1992, 51, 2983-2985; Mertes, M.P., et al, J Med. Chem., 1969, 12, 154-157; Mungall, et al, J. Org Chem., 1977, 42, 703-706; Stirchak, et al, J. Org. Chem., 1987, 52, 4202-4206; Wang, et al, Tetrahedron Letts., 1991, 32, 7385-7388; International Application No. PCT US91/03680). Substitute linkage(s) can be utilized in the oligomers for a number of purposes such as to further facilitate binding with complementary target nucleic acid sequences Fnd/or to increase the stability of the oligomers toward nucleases.

j Bases: Suitable bases for use as nu:leoside bases in the compounds of the invnetion include not only the naturally occurring purine and pyrimidine bases, but also analogs of these heterocyclic bases and tautomers thereof. Such analogs include alkylated purines or pyrimidines, acylated purines or pyrimidines, or other heterocycles. Such "analogous purines" and "analogous pyrimidines" or purine or pyrimidine analogs are those generally known in the art, some of which are used as chemotherapeutic agents. An exemplary, but not exhaustive, 55 -i r, t- I-

I

WO 96114330 PCTIU95/14599 list includes N 4 ethanocytosine, 7-deazaxanthosine, 7deazaguanosine, 8 -oxo-N6-methyl adenine, 4-acetylcytosine, (carboxyhydroxylmethyl) uracil, 5-fluorouracil, 5-carboxyiethylaminomethyl-2-thiouracil, carboxymethylaminornethyl uracil, inosine, N 0 -isopentenvladenine, 1-rethyladenine, 2 -methyl guanine, Norethy adenine, 7 -ruethyliguanine, 5-methylaninomethyl uracil, aminomethyl-2-thiouracil, 2-thiouracil, 4thiouracil, 5-(1-propynyl)-4-thiouracil, 5-(l-prorynyl)-2thiouracil, 5-(l-propynyl)-2--thiocytosine, 2-thiocytosine, and 2,6-diaminopurine. in addition to these base analogs, pvrimidine analogs including 6-azacytosine, 6-azathvmidine and described in Cook, et al, international Publication No. WO 92/02258 (incorporated herein by reference) can be conveniently incorporated into the invention oligomers.

Incorporation of 4-thiouridine and 2-thiothyrnidine into oligorners has been described (Nikiforov, T.T. et al, Tecrahedron Letts., 1992, .33, 2379-2382; Clivio, et a!.

Tetrahedron Letts., 1992 33:65-68; Nikiforov, T. et al, Tetrahedron Letts., 1991 32:2505-2508; Xu, et a! Tetrahedron Letts., 1991 32:2817-2820; Clivio, et al Tetrahedron Letts., 1992 33:69-72; Connolly, et al., Nucl. Acids Res., 1989 17:4957-4974) Preferred bases include adenine, guanine, thymine, uracil, cytosine, 5-(1-oropynyl) uracil, cytosine, 5-methylcytosine, propynyl) uracil, 5- (1-propynyl) cytosine, 8 rnethvladenitne, 7-deaza-7-rnethylguanine, 7-deaza-7methvladenine and 7-deazaxanthosine.

Covalent Bondingr Moiety: Included in some of the oligomers of the invention is a moietv which is capable of effecting at least one covalent -56 WO 96/14330 PCT/US95/14599 bond between the oligomer and the duplex. Multiple covalent bonds can also be formed by providing a multiplicity of such crosslinking moieties. The covalent bond is preferably to a base residue in the target strand, but can also be made with other portions of the target, including the saccharide or phosphodiester. The reaction nature of the moiety which effects crosslinking determines the nature of the target in the duplex. Preferred crosslinking moieties include acylating and alkylating agents, and, in particular, those positioned relative to the sequence specificity-conferring portion so as to permit reaction with the target location in the strand.

It is clear that the heterocycle need not be a purine or pyrimidine; indeed the pseudo-base to which the reactive function is attached need not be a heterocycle at all. Any means of attaching the reactive group is satisfactory so long as the positioning is correct.

Polarity of Olicomers: In their most general form, the symbol indicates a stretch of oligomer in which the linkages are consistently formed between the 5'-hvdroxyl of the amino acid residue of the nucleomonomer to the left with the (or for oligomers having linkages, or 4' for oligomers having linkages) hydroxyl of the amino acid residue of the nucleomonomer to the right a region of uniform polarity), thus leaving the 5'-hydroxyl of the rightmost nucleomonomer amino acid residue free for additional conjugation. Analogously, indicates a stretch of oligomer in the opposite orientation wherein the linkages are formed between the 3'-hydroxyl of the amino acid residue of the left nucleomonomer and the 5'-hydroxyl of the amino acid residue of the nucleomonoier on the right, thus leaving the 3'-hydroxyl of the rightmost nucleomonomer residue free for 57 9 c lc- I- C" *r i- WO 96/14330 PCT/US95/14599 additional conjugation. The same thing is true for stretch of oligomers.

Pharmaceutically Acceptable Salts: The invention also provides for various salts of all compounds disclosed herein, including pharmaceutically acceptable salts for administration to an animal or human.

Pharmaceutically acceptable salts and such salt forming materials are well known in the art. Pharmaceutically acceptable salts are preferably metal or ammonium salts of the oligomers of the invention and include alkali or alkaline earth metal salts, the sodium. potassium, magnesium or calcium salt; or advantageously easily crystallizing ammonium salts derived from ammonia or organic amines, such as mono-, di- or tri-lower (alkyl, cycloalkyl or hydroxyalkyl)-amides, lower alkylenediamines or lower (hydroxyalkyl or arylalkyl)alkylammonium bases, e.g. methylamine, diethylamine, triethylamine, dicyclohexylamine, triethanolamine, ethylenediamine, tris-(hydroxymethyl)-aminomethane or benzyltrimethylammonium hydroxide. The oligomers of the invention may form acid addition salts, preferably of therapeutically acceptable inorganic or organic acids, such as strong mineral acids, for example hydrophilic, hydrochloric or hydrobromic acid; sulfuric, phosphoric; aliphatic or aromatic carboxylic or sulfonic acids, formic, acetic, propionic, Ssuccinic, glycollic, lactic, malic, tartaric, gluconic, citric, ascorbic, maleic, fumaric, hydroxymaleic, pyruvic, phenylacetic, benzoic, 4-aminobenzoic, anthranilic, 4hydroxynbenzoic, salicylic, 4-aminosalicylic, methanesulfonic, ethanesulfonic, hydroxyethanesulfonic, benzenesulfonic, sulfanilic or cyclohexylsulfamic acid and the like.

Utility and Administration: 58 I -i I P i I 1 I WO 96/14330 PCT/US95/14599 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, with naturally occurring polynucleotides and structural analogs thereof, the oligomers of the invention may be used in most procedures that employ conventional oligomers. Thus, the oligomers of the invention may be used as, for example, polynucleotide hybridization probes, primers for the polymerase chain reaction and similar cyclic amolification reactions, sequencing primers, and the like. The oligomers of the invention may also be used in the diagnosis and therapy of diseases. Therapeutic applications of the oligomers of the invention include the specific inhibition of the expression of genes (or inhibit translation of RNA sequences encoded by those genes) that are associated with either the establishment or the maintenance of a pathological condition through the use of antisense oligomers.

The oligomers of the invention may be used to mediate antisense inhibition of numerous genetic targets. Exemplary genes or RNAs encoded by those genes that can be targeted through antisense employing the oligomers include those that encode enzymes, hormones, serum proteins, transmembrane proteins, adhesion molecules (LFA-1, GPII.,/III, ELAM-1, VACM- 1, ICAM-1, E-selection, and the like), receptor molecules including cytokine receptors, cytokines (IL-1, IL-2, IL-3, IL- 4, IL-6 and the like), oncogenes, growth factors, and interleukins. Target genes or RNAs can be associated with any pathological condition such as those associated with inflammatory conditions, cardiovascular disorders, immune reactions, cancer, viral infections, bacterial infections, yeast infections, parasite infections and the like.

Oligomers of the present invention are suitable for use in both in vivo and ex vivo therapeutic applications.

59 I L1 I i I Ii -III-II- T-~C~L I-r II III UL~rI cyosine. Also included 46 46

A

WO 96/14330 PCT/US95/14599 Indications for ex vivo uses 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 genes that can serve as targets for cancer treatments include oncogens, such as ras, k-ras, bcl-2, c-myb, bcr, cmyc, c-abl or overexpressed sequences such as mdm2, oncostatin M, IL-6 (Kaposi's sarcoma), HER-2 and translocations such as bcr-abl. Viral gene sequences or RNAs encoded by those genes such as polymerase or reverse transcriptase genes of herpesviruses 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, adenoviruses, flaviviruses, rhinovirus and the like are also suitable targets.

Application of specifically binding oligomers can be used in conjunction with other therapeutic treatments. Other therapeutic uses 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-Selection that play a role in mediating inflammation and modulation of cellular proliferation in conditions such as arterial occlusion (restenosis) after angioplasty by modulating the expression of growth or mitogenic factors such as nonmuscle myosin, myc, fox, PCNA, PDGF or FGF or their receptors, or cell proliferation factors such as c-myb. Other Ssuitable proliferation factors or signal transduction factors such as TGFx, IL-6, gINF, protein kinase C, tyrosine kinases (such as p210, p190), may be targeted for treatment of psoriasis or other conditions. In addition, EGF receptor, TGFa or MHC alleles may be targeted in autoimmune diseases.

Delivery of oligomers of the invention into cis.s can be enhanced by any suitable method including calci. s phate, DMSO, glycerol or dextran transfection, electroper:-.ion or by 60 I L-L. 1 T V 6/43 PCTIUS95/14599 the use of cationic anionic and/or neutral lipid compositions or liposomes by methods described (international Publications Nos. WO 90/14074, WO 91/16024, WO 91/17424, U.S. Patent 4,897,355). The oligomers can be introduced into cells by complexion with cat-ionic lipids such as DOTMA (which may or 2 may not form liposomes) which complex iJs then contacted with Kthe cells. Suitable cationic ip~ids include but are not K limited to N-(2,3-di(9-(Z)-octadecenyloxyl) )-prop-1--yl-N,N,Ntrimethylammonium (DOTMA) and its salts, 1-O-oleyl-2-O-oley- 3 -dimethvlaminopropyl-g-hydroxvethylammonium and its salts and 2,2-bis (oleyloxy)-3-(trimethylammonio) propane and its salts.

Enha=nced deliveryv of the invention o2-gomers can also be mediated by the use of viruses such as Sendai virus.

(BartzattL, Biorechnol Ao~1 .Eiochezn., 1989, 11, 133-135) or 1adenovirus (Wagner, E. et Proc Nat! Acad Sci. USA, 1992, 3-9, 6099-6013) (11) polyamine or polycation conjugates using compounds such as polylysine, protamine or Na, N,,-bis (ethyl)spermine (Wagner, E. et Proc Nati Acad Sci. USA, 1991, 88, 4255-4259; Zenke, M. et Proc. Nati1. Acad. Sdi.

USA-, 1990, 87, 3655-3659; Chank, B.K. eu Biochen Bioohvs Res Commruz., 1988, i. 1, 264-270; U.S. Patent 5,138,045); (iii) lipopolyarnine complexes using compounds such as lipospermine (Behr, et al, Proc Natl Acad Sdi. USA, 1989, 86., 6982- 6986; Loeffler, et J. Neurochem.,, 1990, .54A, 1812- 1815) (iv) anionic, neutral or pH sensitive ipJids using pcompounds including anionic phospholipids such as phosphatidyl glycerol, cardiolipin, phosphatidic acid or phosphatidylethanolamine (Lee, et al, Biochem Biophys ACTA, 1992, 1103, 185-197; Cheddar, G. et al, Arch Biochem Biooh-ys, 1992, 294, 188-192; Yoshimura, et al, Biochen 1990, Z2, 697-706); conjugates with compounds such as transferrin or biotin or (vi) conjugates with proteins 61 appropriately protected nucleomonomers. Method- for the synthesis of oligomers having phosphorous containing linkages 48 WO 96/14330 PCT/US95/14599 (including albumin or antibodies), glycoproteins or polymers (including polyethylene glycol) that enhance pharmacokinetic IJ properties of oligomers in a subject. As used herein, transfection refers to any method that is suitable for delivery of oligomers into cells. Any reagent such as a lipid or any agent such as a virus that can be used in transfection protocols is collectively referred to herein as a "permeation enhancing agent". Delivery of the oligomers into cells can be via cotransfection with other nucleic acids such as (i) expressable DNA fragments encoding a protein(s) or a protein fragment or (ii) translatable RNAs that encode a protein(s) or a protein fragment.

The oligomers of the invention can thus be incorporated into any suitable formulation that enhances delivery of the oligomers into cells. Suitable pharmaceutical formulations also include those commonly used in applications where compounds are delivered into cells or tissues by topical administration. Compounds such as polyethylene glycol, propylene glycol, azone, nonoxonyl-9, oleic acid, DMSO, polyamines or lipopolyamines can be used in topical preparations that contain the oligomers.

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

62 WO 96/14330 PCT/US95/14599 Thus, the oligomers as described herein constitute a reagent that may be used in methods of inhibiting expression of a selected protein or proteins in a subject or in cells wherein the proteins are encoded by DNA sequences and the proteins are translated from RNA sequences, comprising the steps of: introducing an oligomer of the invention into the cells; and permitting the oligomer to form a triplex with the DNA or RNA or a duplex with the DNA or RNA whereo. e\pression of the protein or proteins is inhibited. The methods and compound of the oresent invention are suitable for modulating gene xpression in both procaryotic and eucaryotic cells such as bacterial, fungal parasite, yeast and mammalian cells.

RNase H "competent" or RNase H "incompetent" oligomers can be easily designed using the substitute linkages of the invention. RNase H competent oligomers can comprise one or more RNase H competent domains comprised of linked RNase H competent nucleomonomers. Oligomers having modifications such as 2'-substitutions (2'-0-allyl and the like) or certain uncharged linkages (methylphosphonate, phosphoramidate and the like) are usually incompetent as a substrate that is recognized by and/or acted on by RNase H. RNase H competence can facilitate antisense oligomer function by degrading the target RNA in an RNA-oligomer duplex (Dagle, J.M. et al, Nucl Acids Res., 1990, 18, 4751-4757; Walder, J.A. et al, International Publication Number WO 89/05358). The enzyme cleaves RNA in RNA-DNA duplexes.

In order to retain RNase H competence, an oligomer requires a RNase H competent domain of three or more competent contiguous nucleomonomers located within it (Quartin, et al, Nucl Acids Res.,1989, 17, 7253-7262). Design of oligomers resistant to nuciease digestion will have terminal linkage, sugar and/or base modifications to effect nuclease 63 ;1 1 i -i

I

WO 96/14330 PCT/US95/14599 resistance. Thus, the oligomers can be designed to have modified nucleomonomer residues at either or both the and/or ends, while having an internal RNase H competent domain. Exemplary oligomers that retain RNase H competence 5 would generally have uniform polarity and would comprise about 2 to about 12 nucleomonomers at the end and at the end which stabilize the oligomer to nuclease degradation and about three to about 26 nucleomonomers that function as a RNase H competent domain between the RNase H incomDetent 3' and ends. Variations on such an oligomer would include a shorter RNase H competent domain comprising 1 or 2 RNase H competent linkages or substitute linkages, a longer RNase H incompetent domain comprising up to 15, 20 or more substitute linkages or nucleomonomers, a longer RNase H competent domain comprising up to 30, 40 or more linkages, (4) oligomers with only a single RNase H incompetent domain at the 3' end or at the 5' end.

Oligomers containing as few as about 8 nucleomonomers may be used to effect inhibition of target protein(s) expression by formation of duplex or triplex structures with target nucleic acid sequences. However, linear oligomers used to inhibit target protein expression via duplex or triplex formation will preferably have from about 10 to about nucleomonomer residues.

Oligomers containing substitute linkages of the invention can be conveniently circularized as described (International Publication No. WO 92/19732; Kool, E.T. J Am Chem Soc.,1991, 11, 6265-6266; Prakash, G. et al, J Am Chem Soc., 1992, 114, 3523-3527). Such oligomers are suitable for binding to single-stranded or double stranded nucleic acid targets.

Circular oligomers can be of various sizes. Such oligomers in a size ranae of about 22-50 nucleomonorers can be conveniently 64 WO 96/14330 PCT/US95/14599 prepared. The circular oligomers can have from about three to about six nucleomonomer residues in the loop region that separate binding domains of the oligomer as described (Prakash, G. ibid). Oligomers can be enzymatically circularized through a terminal phosphate by ligase or by chemical means via linkage through the and terminal sugars and/or bases.

The oligomers can be utilized to modulate target gene expression by inhibiting the interaction of nucleic acid binding proteins responsible for modulating transcription (Maher, et al, Science, 1989, 245, 725-730) or translation. The oligomers are thus suitable as sequencespecific agents that compete with nucleic acid binding proteins (including ribosomes, RNA polvmerases, DNA polymerases, 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 repress 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 complementary in a single oligomer or regions of complementary between two or more oligomers that form a given structure.

65 52 WO 96/14330 Such structures can be u 3 HIV TAR structure in ord i HIV Tat protein (a prote V approach can be utilized Stranslation factors that Sstructures such as stems Alternatively, the inven Sdisrupt or bind to s interfere with or en nucleic acid structures.

PCT/US95/14599 sed to mimic structures such as the er to interfere with binding by the in that binds to TAR). A similar with other transcription or recognize higher nucleic acid ,loops, hairpins, knots and the like.

tion oligomers can be used to (1) uch structures as a method to (1) hance the binding of proteins to In addition to their use in antisense or triole helix therapies, the oligomers of the invention can also be applied as therapeutic or diagnostic agents that 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 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 sequence that results in modulation of the normal biological function associated with the nucleic acid target. Types of target nucleic acids include but are not limited to gene sequences including exons, introns, exon/intron junctions, promoter/enhancer regions and 5' or 3' untranslated regions, (ii) regions of nucleic acids that utilize secondary structure in order too function the HIV TAR stem-loop element or tRNAs), (iii) nucleic acids that serve structural or other functions such as telomeres, centromeres or replication origins (virus, bacteria and the like) and (iv) any other duplex region. It is clear that oligomers can be synthesized with discrete functional domains wherein one region of an 66 _I WO 96/14330 PCT/US95/14599 oligomer binds to a target by D-looping while an adjacent Sregion binds a target molecule by say, forming a triple helix Sor binding as an aptamer to a protein. Alternatively, a Dlooping oligomer can bind to each strand in a duplex by switching the strand to which the oligomer binds 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 DOloop) are the sequence of the oligomer and the inherent affinity built into the oligomer. Base Srecognition rules in Watson-Crick duplex binding differ from those in Hoogsteen controlled triplex binding. Because of this, the oligomer base sequence can be used to dictate the type of binding rules an oligomer will utilize. L-loop structures are formed in nature by enzyme-mediated processes 15 (Harris, L.D. et al., 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 t 20 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 S helix which facilitates a strand displacement envent leading to the D-looD.

Ribozvmes containing substitute linkages of the invention can be designed in order to design species with altered characteristics. Ribozymes that cleave single stranded RNA or DNA (Robertson, et al., Nature, 1990, 344, 467-468) have been described. Therapeutic applications for ribozymes have been postulated (Sarver, ZN. ec al., Science, 1990, 247, 1222-1225; International Publication Number WO 91/04319).

Secondary or tertiary structure necessary for ribozyme function can be affected by design of appropriate oligomer 67 WO 96/14330 PCTIUS95/14599 sequences. For example, ribozymes having nuclease stable targeting domains containing substitute linkages of the invention can have higher affinity, while maintaining base pairing specificity, for target sequences. Because of the higher affinity and/or nuclease stability of the invention substitute linkages shorter recognition domains in a ribozyme (an advantage in manufacturing) can be designed which can lead to more favorable substrate turnover (an advantage in ribozvme function).

In therapeutic applications, the oligomers of the invention may be utilized in a manner appropriate for treatment of a variety of conditions by inhibiting expression of appropriate target genes. For such therapy, the oligamers can be formulated for a variety of modes of administration, including systemic, topical or localized administration.

Techniques and formulations generally can be found in Reminaton's Pharmaceutical Sciences, Merck Publishing Co., Easton, PA, latest edition. The oligomer active ingredient is generally combined with a carrier such as a diluent or excipient which can include fillers, extenders, binders, A wetting agents, disintegrants, surface-active agents, or lubricants, depending on the nature of the mode of i administration and dosage forms. Typical dosage forms include i tablets, powders, liquid preparations including suspensions, i emulsions and solutions, granules, capsules and suppositories, Sas well as liquid preparations for injections, including liposome preparations.

For systemic administration, injection is preferred, including intramuscular, intravenous, intraperitoneal, and subcutaneous. For injection, the oligomers of the invention are formulated in liquid solutions, preferably in physiologically compatible buffers such as Hank's solution or 68 WO 96/14330 PCT/US95/14599 Ringer's solution. In addition, the oligomers can be formulated in solid form and redissolved 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 mg/Kg administered once or twice per day. However, different dosing schedules can be utilized depending on 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 associated with a given target gene, or (iii) the pharmacokinetic behavior of a given oligomer.

Systemic administration can also be by transmucosal or transdermal means, or the compounds can be administered orally. For transmucosal or transdermal administration, penetrates appropriate to the barrier to be permeated are used in the formulation. Such penetrates are generally known in the art, and include, for example, bile salts and fusidic acid derivatives for transmucosal administration. In addition, detergents can be used to facilitate permeation. Transmucosal administration can be through use of nasal sprays, for example, or suppositories. For oral administration, the oligomers are formulated into conventional oral administration forms such as capsules, tablets, and tonics.

For topical administration, the oligomers of the invention are formulated into ointments, salves, 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 compositions known in the art.

69 -ii 1- I I I II WO 9614330 PCT/US95/14599 In addition to use in therapy, the oligomers of the invention can be used as diagnostic reagents to detect the presence or absence of the target nucleic acid sequences to which they specifically bind. The enhanced binding affinity of the invention oligomers is an advantage for their use as primers and probes. Diagnostic tests can be conducted by hybridization through either double or triple helix formation which is then detected by conventional means. For example, the oligomers can be labeled using radioactive, fluorescent, or chromogenic labels and the presence of label bound to solid support detected. Alternatively, the presence of a double or triple helix can be detected by antibodies which specifically recognize these forms. Means for conducting assays using such oligomers as probes are generally known.

The use of oligomers of the invention substiutce linkages as diagnostic agents by triple helix formation is advantageous since triple helices form under mild conditions and the assays can thus be carried out without subjecting test specimens too harsh conditions. Diagnostic assays based on detection of RNA for identification of bacteria, fungi or protozoa sequences often recuired isolation of RNA from samples or organisms grown in the laboratory, which is laborious and time Sconsuming, as RNA is extremely sensitive to ubiquitous j nucleases.

SThe oligomer probes can also incorporate additional modifications such as modified sugars and/or substitute linkages that render the oligomer especially nuclease stable, and would thus be useful for assays conducted in the he presence of cell or tissue extracts which normally contain nuclease activity. Oligomers containing terminal modifications often retain their capacity to bind to complimentary sequences without loss of specificity (Uhlmann 70 WO 96/14330 PCTUS95/14599 et al., Chemical Reviews, 1990, 90, 543-584). As set forth above, the invention probes can also contain linkers that permit specific binding to alternate DNA strands by incorporating a linker that permits such binding (Froehler, B.C. et al., Biochemistry, 1992, 31, 1603-1609); Home et al., J Am Chem Soc., 1990, 112, 2435-2437).

Incorporation of base analogs of the present invention into probes that also contain covalent crosslinking agents has 1the potential to increase sensitivity and reduce background in diagnostic or detection assays. In addition, the use of crosslinking agents will permit novel assay modifications such as the use of the crosslink to increase probe discrimination, incorporation of a denaturing wash step to reduce background and carrying out hybridization and crosslinking at or near the melting temperature of the hybrid to reduce secondary structure in the target and to increase probe specificity. Modifications of hybridization conditions have been previously described (Gamper et al., Nucleic Acids Res., 1986, 14, 9943).

Oligomers of the invention are suitable for use in diagnostic assays that employ methods wherein either the oligomer or nucleic acid to be detected are covalently attached to a solid support as described Patent No.

4,775,619). The oligomers are also suitable for use in diagnostic assays that rely on poiymerase 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 orotocols.

71 Utility and Administration: 58 WO96/14330 PCT/US95/14599 WO 96/14330 The oligomers of the invention are useful as primers that are discrete sequences or as primers with a random sequence.

Random sequence primers can be generally about 6, 7, or 8 nucleomonomers in length. Such primers can be used in various nucleic acid amplification protocols (PCR, ligase chain D reaction, etc.) or in cloning protocols. The substitute linkages of the invention generally do not interfere with the capacity of the oligomer to function as a primer. Oligomers of the invention having 2'-modifications at sites other than the 3' terminal residue, other modifications that render the oligomer RNase H incompetent or otherwise nuclease stable can be advantageously used as probes or primers for RNA or DNA seauences 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 invitro diagnostic assays as described Patent Nos. 4,772,548, 4,707,440 and 4,707,352). Alternatively, oligomers of the invention can be derivatized with crosslinking agents such as 5-(3iodoacetamidoprop-l-yl) 2'-deoxyuridine or bromobutyramido) prop-l-yl)-2'-deoxyuridine and used in various assay methods or kits as described (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 invitro systems by measuring the levels of expression in subject 72 -Ab MWhOE SWO96/14330 PCT/US95/14599 cells or in recombinant systems, by an suitable method (Graessmann, M. et al, Nucleic Acids Res., 1991, 19, 53-59).

The invention having been described above, the following examples are offered to better explain the invention. The examples are offered to illustrate the invention and should not be interpreted as limiting the invention.

EXAMPLES

SOverview of the Synthesis of the Nucleomonomer Synthon and Oligomers: The oligomers of the invention can be synthesized using reactions known in the art of oligonucleotide derivative synthesis. See e.g. Flandor, J. and Yam, Tetrahedron Letts., 1990, 31, 597-600; Mattson, R.J. et al., J Org Chem., 1990, 55, 2552-2554; Chung, C.K. et al., J Org Chem., 1989, 54, 2767-2769.

As can be seen from the variety of substitute linkages specifically listed in Table 1, the substitute linkages of the invention can vary so as to contain one or more nitrogen, sulfur, and/or oxygen atoms in their structure. The positions of these atoms in the substitute linkage can vary from the end, to the "middle" to the or and end.

SIn this section, a series of representative synthesis reaction figures are set forth which provide routes to various locations and combinations of nitrogen and oxygen atoms within the substitute linkages.

The synthesis illustrated in figures 1-25 be modified as is known to those practicing in the area of oligonucleotide chemistry. For example, although protection of the bases is not always indicated in the synthesis figures, such may be desirable and can be accomplished using reagents and 73 u Delivery or oigomers or tne invention into s can De enhanced by any suitable method including calci," sphate, DMSO, glycerol or dextran transfection, electropu_.-ion or by 60 WO 96/14330 PCT/US95/14599 techniques known in the art. See, e.g. Prorective Groups in Organic Synthesis (Theodora W. Greene, John Wiley and Sons, 1981). Similarly, although the use of protective groups is shown in some cases, it is not always necessary to block the reactants in order to synthesize the exemplified invention oligomers.

Examole 1 The first five steps shown in Figure 1 relate to the preparation of isobutryl protected serinol amino acid alcohol.

The sixth and subsequent steps in Figure 1 are directed to the synthesis of the serinol substituted thymine phophoramidite building block.

In step 1 of Figure 1, the amino group of the serine amino acid is protected by reacting 1 with di-tert-butyl Sdicarbonate to yield compound 2. Other equivalent protecting groups may be used. In the next step, the (-hydroxyl group of Compound 2 is blocked with dihydropyran to give fully protected amino acid 3. The amnio acid 3 is then reacted with diborane-dimethyl sulfide complex to provide alcohol 4, which on exposure to isobutryl chloride gave 5. This reduction reaction can also be carried out using isobutyl choloroformate i and sodium borohydride (see: K. Ramasamy, R. K. Olsen and T.

Emery, Synthesis, 1982, 42). Reaction of 5 with trifluoroacetic acid for 30 minutes followed by washing with i 25 NaHCO 3 afforded 6.

Thymine acetic acid 7 was prepared as described in the literature (see: L. Kosynkina, W. Wang and T. C.

Liang,Tetrahedron Letts, 1994, 35, 5173). Coupling of 7 with 6 under mixed anhydride condition provided 8.

Dimethoxyritylation of 8 with DMTCl gave compound 9, which on hydrolysis with base afforded 10. Phophysitylation of 10 under 74 SWO 96/14330 standard condition provided the building block 11. This synthon growing oligomer using conventio synthesis chemistry such as phos chemistry can be used to link mo analogous to that set.forth abov IYI- C i.-l-il--l.r jll_ I PCT/US95/14599 serinol coupled thymine can then be added into a nal chemistry. Any DNA phoramidate or phosphonate nomers or dimers in a manner e.

Example 2 In reaction Figure 2, thymine acetaldehyde 13 was 1produced by the treatment of thymine with bromoacetaldehyde dimethylactal followed by hydrolysis of 12 with aqueous TFA.

Aldehyde 13 and amine the 6 are then coupled and the corresponding intermediate was transformed to the phosphoroamidite building block 17 in a manner analogous'to the steps used in Figure 1.

Example 3 In reaction Figure 3, the starting material is a 3substituted amino acid 18. The substituted amino acid could be transformed into the phosphoroamidite building block 27 by the procedure of the steps used in Figures 1 and 2.

Example 4 In Figure 4, the starting amino alcohol 21 is oxidized with CrO3/pyridine mixture to give an aldehyde 28. The aldehyde which on reaction with alkyl halide in the presence of a base should yield compound 29. The amino alcohol 29 could then be transformed to the building block 35 in a manner analogous to the steps used in figure 1 and 2.

Example Turning to Figure 5, the first four steps are essentially the same steps as used in Figure 1, in this aspartic acid is 75 PCT/US95/14599 WO 96/14330 used instead of serine. Aspartic acid methylester 36 gave fully protected alcohol 40, which on selective deprotection with acetic acid provided 41. Oxidation of 41 with CrO3/pyridine gave the corresponding aldehyde 42. Reductive amination of the aldehyde 42 with o-benzylhydroxyl amine in the presence of sodium triaceroxyborohydride should give 43 (see: T. Kolasa and M. J. Miller, J. Org. Chem., 1990, 1/11). The alcohol 39 is then converted to an aldehyde 46, essentially using the same reaction conditions as said above but with an allylic protecting group for the hydroxyl function of 39. Coupling of the aldehyde 46 and the hydroxylamine 43 in presence of sodium triacetoxyborohydride followed by deprotec:ion of the amino protecting groups should afford the b-.samine 48. The bisamine 48 could then be converted to a dimer 53 by following the steps used in figure 1.

Example 6 In Figure 6, coupling of alcohol 54 with 0benzylhydroxylamine 55 under Mitsunobu reaction condition (see: 0. Mitsunobu, Synthesis, 1981, 1) provides compound 56.

The intermediate 56 on hydrogenation followed by acetylation should give 57. Exposure of 57 to TEA deblocks the "TBDMSi" protecting group and gives 58. Coupling of 58 with 7 followed by dimethoxytritylation could provide 60. The final building block 62 should be accomplished from 60 by base hydrolysis followed by phosphitylation.

Example 7 In Figure 7, the serinol 4 is converted to a halide 59 and alkylated with thymine to provide 63. The protecting groups in 63 are removed, coupled with DMT-protected hydroxyacetic acid and phosphitylated to yield 66.

76

I

WO96/14330 PCT/US95/14599 Example 8 In Figure 8, the alcohol 64 is coupled with Nhydroxylaminopropanoic acid 69 to give 70. Alkylation of thymine with a halide 73 gives 74 which on deprotection, coupling with 76 followed by hydrolysis could afford 78.

Condensation of 78 with 70 followed by phosphitylation should give the hydroxamate dimer Example 9 In Figure 9, N-hydroxylamino propanoic aldehyde 81 is used to couple the alcohol 64. The dimer 88 is oreoared from 83 and 86 by following the steps used in figure 8.

Example In Figure 10, alkylation (see: T. Kolasa and M. J.

Miller, J. Org. Chem., 1990, 55, 4246) of a-bromo-Paminopropanoic acid methylester 89 with thymine would produce The intermediate 90 on hydrolysis with sodium hydroxide gives an acid 91 which is coupled with 6 to provide 92. The compound 92 is then converted into the phosphoroamidite K building block 95 using the steps described in figure 1.

Example 11 In Figure 11, thymine is alkylated with an alkylamine halide 96 (see: R. K. Olsen, K. Ramasamy and T. Emery, J. Org.

Chem., 1984, 49, 3527 and Islam et al., J. Med. Chem., 1994, 37, 293-304 for the preparation of aminoalkyl halide) to give 97. Exposure of the compound 97 to TFA followed by alkylkation would afford 100. The building block 103 is obtained from 100 by dimethoxytritylation, hydrolysis, followed by ohosphitvation.

Example 12 77 W 96/14330 PCT/US95/14599 WO 96/14330 Figure 12 is an alternative route to a hydroxamate backbone dimer 111 from N-hydroxylamine 43 and an aldehyde 107 which in turn prepared from aspartic acid.

Example 13 In Figure 13, the dimer 115 is prepared from the intermediate 108 and 13 by following the same steps of reactions described in figure 2.

0 Example 14 In Figure 14, N-hydroxylthymine is prepared (see: Kim, C.

et al., Tetrahedron Letts., 1992, 33, 25-28) and coupled with N-hydroxyphthalimide to provide 117 which on exposure to hydrazine in ethanol should give 118. Treatment of 118 with DMT-protected glycerol epoxide 119 provides 120. The intermediate 120 is then transformed to the phosphoroamidite 121 using standard procedure. In second synthesis, compound 118 is coupled with amino acid aldehyde 122 under reductive amination conditions to provide 123. Protection of the secondary amino functionality with FMOCCI followed by hydrolysis should afford 125.

Example In Figure 15, 1,2-dihydroxyprooanoc acid 126 is coupled with N-hydroxylamine thymine 118 to give 127, which is then transformed into phophoramidite synthon 129 under standard conditions. The compound 118 is also coupled with adipic acid and transformed into nucleic acid building block 133.

Example 16 In Figure 16, first the building block 136 is synthesized from 118 and 134 in a similar manner described in figure 1.

78 I ~l-eC I- WO 96/14330 PCTIUS95/14599 Coupling of 139 with 118 provided 140. Treatment of 137 with 118 should provide 138 which on condensation with 140 gives the dimer 141.

Example 17 In Figure 17, an aldehyde 142 and an glycine benzylester is coupled to give 143. Treatment of 143 with 7 should provide 145 which on exposure to acetic acid gives 148. Mitsunobu alkylation of 148 with Boc-NH-O-acetylhydroxylamine should give 147 which on hydrogenation the building block 150 could be obtained. Similarly coupling of 143 with 13 and following the same reactions as above snould vield the synthon 149.

Examole 18 In Figure 18, reductive amination of the aldehyde 142 and Boc-NH-O-benzylhydroylamine gave 151. Hydrogenation of 151 followed by alylation of 152 with glycolic acid 153 B. C.

Borer and D. C. Balogh, Tetrahedron Letts., 1991, 32, 1039) should yield 154. Treatment of 154 with TFA willl remove the Boc protecting group, which on coupling would result in 155.

The hydroxyl protecting group of 155 could selectively be removed with acetic acid to give 156. The compound 156 will then be transformed to the building block 157 using standard reaction conditions. Similarly the building block 158 will be produced by coupling of 154 with 13 and following the steps used for the preparation of 157.

Example 19 In Figure 19, alkylation of thymine-N-hydroxylamine 160 with alcohol 162 will yield 163. The compound 163 could be transformed to the phosphoroamidite building block 166 by following the steps used in figure 1.

79 16 L IL- 1 L t WO 96/14330 PCT/US95/14599 ExamDle In Figure 20, first the intermediate 169 is synthesized from glutamic acid using standard reaction conditions.

Alkylation of thymine with 169 would give 170 which on treatment with TFA should produce 171. The intermediate 171 could be coupled with Boc-glycine to provide 173 which on hydrolysis would afford the monomer synthon 174. Similarly 172 Scould be prepared by coupling of 118 and Boc-aminoacetic aldehyde followed by hydrolysis of the benzylester.

Example 21 In Figure 21, the intermediate 177 is prepared from Boc- NH-O-benzylhydroxylamine and 175 using standard reaction conditions. Hydrogenation of 177 followed by coupling with Nhydroxythymine 116 would produce 178. Removal of the THP protecting group followed by dimethoxytritylation and phosphitylation should give the building block synthon 181.

Similarly 182 could be prepared by following all the above reactions and using THP-Hydroxyacetic aldehyde instead of THP- SHydroxyacetic acid.

Example 22 In Figure 22, the building block 191 could be prepared using the known starting material 183 and following the Ireaction conditions depicted at the bottom of figure 22.

Example 23 In Figure 23, synthesis of the building i ck 199 could be accomplished utilizing the starting materiai 183 and following the reaction conditions depicted at the bottom of figure 23.

Example 24 80 a r II I In Figure 24, the starting material 200 is transformed to the building block 207 by following the reaction conditions shown at the bottom of figure 24.

Example The compounds used and generated in this example are shown in Figure 1.

Serine Thymine (37.8 g, 300 mmol) was dissolved in a solution of potassium hydroxide (64.5 g, 1150 mmol) in 200 ml of water. While this solution was warmed in a water bath, a solution of bromoacetic acid (62.5 g, 450 mmol) in 100 ml of water was added over 1 h period. The reaction was stirred of another 1 h at this temperature.

It was allowed to cool to room temperature and the pH was adjusted to 5.5 with conc.

HC1. The solution was then cooled in a refrigerator for 2 h. Any precipitate (unreacted thymine) formed was removed by filtration. The solution was then adjusted to pH 2 with conc. HCI and put in a freezer for 2 h. The white precipitate was collected by filtration and dried in a vacuum oven at 40 0 C for 6 h. The yield was 44g N-Boc-L-Serine methyl ester L-Serine methyl ester (15.6 g, 100 mmol) was 15 suspended in THF/DMF (100 ml each) mixture at room temperature. To this stirred mixture was added triethylamine (11.13 g, 110 mmol) followed by di-tert-butyl dicarbonate (24.0 g, 110 mmol) and the stirring continued at room temperature for minutes. Water (20 ml) was added and the solution was stirred at room temperature for 8 h. The solution was evaporated to dryness. The residue was suspended in ethyl acetate (250 ml) an' ,-eated with potassium hydrogen sulfate (0.25 N solution, 100ml). The product was extracted immediately with ethyl acetate solution. The organic extract was washed with water (100 ml), brine (100 ml) and dried over

C.

C

[N:\libff100944:MCN WO 96/14330 PCTIUS95/14599 anhydrous sodium sulfate. Evaporation of the organic solvent provided an oily residue of 26g N-Boc-L-Serine(OTHP) methyl ester The compound 2 g, 68.49 mmol) was dissolved dry CH,Cl, (100 ml) and treated with 3,4-dihydro-2H-pyran (8.4 g, 100 mmol) and catalytic amount of p-toluene sulfonic acid (100 mg) at room temperature. The reaction mixture was allowed to stir at room temperature for 12 h and evaporated to dryness. The residue was dissolved in ethyl acetate (200 ml), washed with 5% NaHCO 3 solution (100 ml), water (50 ml) and brine (50 mi). The -organic extract was dried over anhydrous NaSO, and evaporated to dryness. The residue was pure enough for the next step and used as such. Yield 15g N-Boc-L-Serinol(OTHP) Serine(OTHP) methyl ester g, 33 mmol) was dissolved in dry THE (100 ml) and cooled to 0°C in an ice bath under argon atmosphere. To this cold stirred solution was added borane-methyl sulfide complex (2 M solution in THF, 100 ml 200 mmol) during 1 h period at 0 C temperature. After the addition of borane, the reaction mixture was warmed to room temoerature and heated at 40 0 C for 6 h. The reaction mixture was cooled to 0°C, neutralized with ;i water and acetic acid to pH 6-7 and extracted with ether (3x100 ml). The ether extract was washed with water (2x100 ml) Sand brine (100ml), dried over anhydrous NaSO, and evaporated 25 i to dryness to give a crude product as an oil. The oil on purification by flash column of silica gel using hexane acetone as the eluent gave 8g of pure product.

N-Boc-L-Serine(OTHP) OIb To a stirred solution of the compound 4 (8 g, 29.09 mmol) in dry CH2C1 2 (100 ml) at 0°C was added TEA 3.54 g, 35 mmol) followed by isobutyryl chloride (3.71 g, 35 mmol) during 30 mins period. Then, the 82 Ir -II~ WO 96/14330 PCT/US95/14599 i reaction mixture was stirred at room temperature for 4 h and Ievaporated to dryness. The residue was dissolved in EtOAc (200 ml), washed with 5% NaHCO, solution (50 ml), water (50 ml) and brine (50 ml). The organic extract was dried over anhydrous NaS 4 and evaporated to dryness to give a crude product as an oil. The oil on purification by flash column of silica gel using hexane acetone as the eluent gave 7.9g o± pure i product.

1 L-Serinol(0Ib) Compound 5 (10 g, 28.98 mmol) was dissolved in CH 2 C1, (100 ml) allowed to stir at room temoerature with TFA (50 mi) for 1 h and evaporated to dryness. The residue was dissolved in methanol (50 ml) and i evaporated again. The residue was dissolved in CHCI 2 (200 ml), washed with sat. NaHCO 3 solution (2x100 mi), water (100 ml) and brine (50 ml). The organic extract was dried over Sanhydrous Na3SO and evaporated to dryness to give 4.5g (96%) of the product as an oil.

N-(Thyminylacetyl)-L-Serinol(OIb) Thymine acetic j 20 acid 7 (7.3 g, 40 mmoi) and N-methylmorpholine (4.4 ml, i mmol) were dissolved in 100 ml of DMF. The solution was 1 allcwed to cool to -20 C under argon atmosphere. To this cold stirred solution, isobutyl chloroformate (5.2 ml, 40 mmol) was added in one portion. After 15 minutes, a solution of 6 (6.44 g 40 mmol) in 30 ml of DMF (chilled to the same temperature) was added. The reaction mixture was stirred at -20 0 C for minutes, warmed to room temperature and the stirring continued for 1 h. The reaction mixture was evaporated to dryness and the residue dissolved in CHCl, (200 ml). The organic solution was washed with 5% NaHCO 3 solution (100 ml), water (100 mi) and brine (50 ml). The organic extract was dried over anhydrous NaSO and evaporated to dryness to give a crude product as foam. The crude product was purified by flash 83 WO 96/14330 PCT/US95/14599 column of silica gel using CH C1 2 acetone as the eluent to give 12g of pure product.

4,4'-Dimethoxytrityl-N-(Thyminylacetyl)-L-Serinol(OIb) The compound 8 (10 g, 30.58 mmol) was coevaporated with dry pyridine (3x50 ml) and dissolved in dry pyridine (100 ml).

To this solution was added TEA (3.54 g, 35 mmol) followed by DMTC1 (11.83 g, 35 mmol) at room temperature under argon i atmosphere. The reaction mixture was stirred for 12 h, auenched with methanol (20 ml) and stirred for 30 minutes. The solution was evaporated to dryness and dissolved in CHC1, (200 mi). The organic extract was washed with 5% NaHCO 3 solution (100 ml), water (100 ml) and brine (50 ml). The

CH

2 Cl, layer was dried over anhydrous NaSO, and evaporated to dryness to give a crude product as foam. The crude product was purified by flash column of silica gel using CH 2 Cl 2 acetone as the eluent to give 17g of pure product.

1-0-(4,4'-Dimethoxytrityl)-2-[amino (thyminylacetyl)]-Lpropan-1,3-diol The compound 9 (10 g, 15.89 mmol) was K 20 dissolved in methanol (20 mi). To this solution was added 1N SNaOH solution (20 ml, 20 mmol) at 00C temoerature. The reaction mixture was stirred for 1 h, quenched with acetic acid to pH 7. The solution was extracted with EtOAc (2x100 ml). The organic extract was washed with 5% NaHCO 3 solution I 25 (100 ml), water (100 ml) and brine (50 ml). The EtOAc layer was dried over anhydrous Na 2 SO, and evaporated to dryness to give a crude product as foam. The crude product was purified by flash column of silica gel using CH 2 acetone as the eluent to give 8.2g 92%) of pure product.

84

I

i WO 96/14330 PCT/US95/14599 -Dimethoxytrityl)-2- amino(thyminylacetyl)-L- 1 propan- (N,N-diisopropyl) cyanoethylphophoramidite The compound 10 (8.00 g, 14.31 mmol) was coevaporated with dry pyridine (3x50 ml) and dried over solid NaOH overnight under vacuum. The dried material was dissolved in dry CH 2 Cl1 (100 ml) and cooled to 0°C under argon atmosphere.

To this cold solution was added N,N-diisopropylethylamine (5.23 g, 25 mmol) foil 'ed by 2-cyanoethyl-N,Ndiisopropylchlorophosphoramidite (4.72 g, 20.00 mmol) under Sargon atmosphere. The reaction mixture was stirred at 0°C for 1 h and at room temperature for 1 h. The reaction mixture was diluted with CH,C1, (100 mi The CH-,C1 solution was washed with 5% NaHCO 3 solution (100 mi), water (100 mi) and brine ml). The CHCl1 layer was dried over anhydrous Na.SO 4 and evaporated to dryness to give a crude product as foam. The crude product was purified by flash column of silica gel using CHCl 2 acetone containing 0.1% TEA as the eluent to give of pure product. The form was dried over solid NaOH in vacuum overnight. The form was dissolved in CH,C1, (15 ml) and dropped into stirred solution of dry hexanes (2000 ml) under argon during 1 h period. After the addition of CH,CI, solution, the precipitate that formed was stirred for addition'1. I h and filtered, washed with dry hexanes (200 ml) and dried over solid NaOH overnight. Yield: 9.5g 85 -L I Example 26 (See Figure 26) N-(tert-Butyloxycarbonyl)-O-Benzyl-L-Serine O-Benzyl-L-Serine 1 (10 g, 51.28 mmol) was suspended in THF/HO 100 ml) mixture at room temperature. To this stirred mixture was added triethylamine (6.06 g, 60 mmol) followed by di-tert-butyl dicarbonate (13.08 g, 60 mmol), and the stirring continued at room temperature overnight.

The homogenous solution was evaporated to dryness and the residue dissolved in ethyl acetate (300 ml). The organic extract was washed with 0.5N solution of potassium hydrogen sulfate (100 ml), water (100 ml) and brine (50 ml). The ethyl acetate extract was dried over anhydrous sodium sulfate and evaporated to dryness to give 14 g of an oily residue.

4 N-(tert-Butyloxycarbonyl)-O-Benzyl-L-Serinol(3): N-(tert-butyloxycarbonyl)-0benzyl-L-serine 2 (6.0 g, 20.34 mmol) was dissolved in dry THF and cooled to -20 0 C under argon atmosphere. To this cold stirred solution was added TEA (2.32 g, 23 mmol) and isobutyl chloroformate (3.13 g, 23 mmol). The stirring was continued for 30 min at -20 0

C

under argon atmosphere. The reaction mixture was filtered immediately under a blanket of argon, the precipitate was washed with dry THF (50 ml). The combined filtrate was added slowly into a cold solution of NaBH 4 (7.4 g, 200 mmol) in THF/water (80:20, 200 ml) during 10 min period. After the addition, the reaction mixture was stirred for 2 h at 0°C and the pH adjusted to 7 with acetic acid. The solution was evaporated to dryness, partitioned between ethyl acetate/water (300:150 ml) and extracted in ethyl acetate. The organic extract was washed with brine (100 ml), dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by flash column chromatography over silica gel using CH 2 C1 2 >EtOAc as the eluent. The pure product was pooled together and evaporated to dryness to give 4.7 g of the pure product as an oil. 'HNMR (CDC1 3 d 1.41 9H, Boc), 3.60 3.70 4H), 3.82 2H), 4.53 2H, OCH 2 Ph), 5.20 (bs, 1H, NH) and 7.30 7.40 5H, Ph).

86 WO 96/14330 PCT/US95/14599 column chromatography over silica gel using CH 2 C!-->EtOAc as the eluent. The pure product was pooled together and evaporated to dryness to give 4.7 g of the pure product as an oil. 'HNMR (CDC1 3 6 1.41 9H, Boc), 3.60 3.70 (m, 4H), 3.82 2H), 4.53 2H, OCH,Ph), 5.20 (bs, 1H, NH) and 7.30 7.40 Ph) N-(tert-Butyloxycarbonyl)-O-Benzyl-L-Serinol-O-Ib To a dried solution of N-(tert-butyloxycarbonyl)-0-benzyl-Lserinol 3 (4.3 g, 14.3 mmol) in dry pyridine (50 ml) was added TEA (2.02 g, 20 mmol) at room temperature. To this stirred solution was added isobutyric anhydride (3.16 g, 20 mmol) and the stirring continued overnight under argon atmosphere. The reaction mixture was evaporated to dryness, partitioned between EtOAc (100 ml) and NaHCO solution, 100 ml), and extracted in EtOAc. The organic extract was washed with water (100 ml), brine (50 ml), and dried over anhya ous Na 2 SO,. The dried solution was evaporated to dryness to give a crude residue. The residue was purified by flash chromatography over silica gel using hexane EtOAc as the eluent. The pure fractions were pooled together and evaporated to give an oily product 4.5 g IHNMR (CDC13): 6 1.04 6H, IbCH 3 1.39 9H, Boc), 2.46 1H, IbCH), 3.40 2H), 3.92 (m, 2H), 4.12 1H), 4.46 2H, OCHPh), 6.84 1H, NH) and 7.24 7.40 5H, Ph).

N-(Thyminylacetyl)-O-Benzyl-L-Serinol-O-Ib N-(tert-Butyloxycarbonyl)-0- benzyl-L-serinol-O-Ib 4 (4.3 g, 12.25 mmol) was allowed to stir at room temperature in trifluoro acetic acid (20 ml) and CHC1(20 ml) for 30 min.

The reaction mixture was evaporated to dryness, dissolved in dry CHOH (10 ml) and evaporated again to dryness. The residue was dried over solid KOH under vacuum for 12 h. The dried 87 r II -i I 14 L. I rYI WO 96/14330 PCT/US95/14599 residue was used as such for further reaction without characterization.

Thymine acetic acid 5 (2.76 g, 15 mmol) was dissolved in dry DMF (75 ml) and cooled to -20°C under argon. To this cold stirred solution was added N-methylmorpholine (1.72 g, 17 mmol) followed by isobutyl chloroformate (2.31 g, 17 mmol).

After 15 min of stirring, a solution of the above TFA salt in dry DMF (50 ml) was neutralized with N-methylmorpholine (1.72 g, 17 mmol) and added into the cold stirred solution of thymine acetic acid at once. The reaction mixture was stirred at -20 0 C for 1 h, warmed to room -emoerature and the stirring continued overnight. The solution was evaporated to dryness and the residue dissolved in CH,C1, (250 ml) and water (100 mi), and extracted in CH 2 Cl,. The organic extract was washed with 5% NahCO, solution (100 ml), water (100 ml) and brine ml). The CH,CI. extract was dried and evaporated to dryness to give crude product. The crude product was purified by flasi chromatography over silica gel using CHCl, acetone as the eluent. The necessary fractions were collected and evaporated to give 4.8 g of the pure product. The pure product was crystallized from CHC1/hexane. mp: 122-124 0 C. 'HNMR (CDC1 3 6 1.04 6H, IbCH 3 1.72 3H, CH 3 2.44 1H, IbCH), 3.42 2H), 4.06 2H), 4.18 1H), 4.30 2H), 4.46 2H, OCHPh), 7.24 7.40 6H, CH and Ph), 8.22 1H, NH) and 11.22 1H, NH).

N- (Thyminylacetyl) -L-Serinol-O-Ib N- (Thyminylacetyl)-0-Enzyl- L-Serinol-O-Ib 6 (2.08 g, 5 mmol) was dissolved in ethanol (50 ml). To this solution Pd(OH) 2 (0.6 g) and cvclohexene (5 ml) were added at room temperature. The reaction mixture was heated at 70 0 C for 12 h. The catalyst was filtered, washed with methanol (20 ml). The filtrate was evanorated to dryness to give a white solid. The white solid -88

-A-

WO 96/14330 PCTJUS95/14599 was dissolved in minimum amount of MeOH and cooled to room temperature. The product crystallized as fine powder. mp: 196-198'C. Yield: 1.48 g IHNMR :6 1.04 (di, 6H, IbCH,), 1.72 3H, 2.42 (in, 1Hi, IbCH), 3.40 (m, 2F), 3.94 (mn, 2H), 4.00' (in, 1H., 4.23 2H), 4.90 1H, OH) 7. 20 1H, C6H) 8. 12 1H, NH) and 11. 22 1H,

NH).

4, 41 -Dimethoxytrity.-N- (ThyminyJlacetyl) -L-Serinol-O-Ib N- (Thvrnivlacetv) -L-Serinol'-O-Ib 7 48 g, 4. 5 rnnol) was dissolved in dry pyridine (30 ml) under argon. To this Isirred solution was add.--d T--A (0.51 g, 5 mmocl) and N, LN-dimethvlamino pyridine (0.l10g) Af ter 10 mim, 4,4' -dimethoxvtrit'. chloride (1.69 g, 3 inmol)1 was added and the stirring continued at room tempoerature under argon for overnight. The reaction mixture was quenched with MeOH ml), stirred for 10 min and evaporaced to dryness. The residue was dissolved in EtOAc (200 ml1) washed with 5% NaHCO 3 solution (100 ml), water (100 ml1) and brine (50 ml) The crganic extract was dried over annydoos Na 2

SO

4 and evaporated r:c dryness. The crude product was purified by flash column =-romatographv over silica gelI using CHCl 2 EtOAc as the e-luent. The pure fractions were pooled and evaporated to give g of foam. 1 HNMR (C)Cl 3 5 1.04 Cd, 6H, IbCH 3 1.72 3H, CH3) 2. 40 1H, IbCH) 3. 38 Cm, 2H) 3. 72 6H, 2 .OCH3) 4.12 (in, 2H), 4.20 (mn, 1H) 4.32 6.84 (mn, 4.H, Ph) 7.20 7.40 (in, 12H, C-H and Ph) 8. 30 1H, NH) and 11 .2 8 I H, NH).

1-0- -Dimethoxytrity.) [amino (thI ;.nylacetyI)]J-L-propan- 4, 4' -DimethoxytritLyl-N- (Thyinnylacetyl) -L-Serinol-O-Ib .8 (3.4 89 WO 96/14330 PCT/U395/14599 g, 5.41 mmol) was dissolved in MeOH (30 ml) and cooled to 0°C in an ice bath. To this cold stirred solution was added 2N NaOH (10 ml, 20 mmol) and the stirring continued for 30 min at 0°C. The pH of the solution was adjusted to 7 with acetic acid and evaporated to dryness. The residue was partitioned between water (50 ml) and CH 2

CI

2 (150 ml) and extracted in CHC1 2 The Saqueous layer was extracted again with CHCl, (50 ml). The combined organic extract was washed with brine (50 mi), dried and evaporated to dryness. The residue was purified by flash S0 column chromatography over silica gel using CH,C!- acetone as the eluent. Yield: 3.0 g :HNMR (CDCl 3 1.72 3H,

CH

3 3.0 2H), 3.42 2H), 3.72 6H, 2.0CH 3 3.94 1H), 4.32 2H), 4.68 1H, OH), 6.84 4H, 7.20 7.40 12H, C6H and Ph), 8.06 1H, NH) and 11.28 (bs, 1H, NH).

1-O- -Dimethoxytrityl) [amino (thyminylacetyl) -L-pr opan-3-O- (N,N-diisopropyl) cyanoethylphosphoramidite (4,4'-Oimethoxytrityl)-2-[amino(thvminylacetyl) propan-1,3-diol 9 (3.1 g, 5.55 mmol) was dried over solid NaOH under vacuum overnight and dissolved in dry CHCI- (100 m).

The solution was cooled to 0°C under argon atmosphere. To this cold stirred solution was added N,N-diisopropylehylamine (1.29 g, 10 mmol) followed by 2-cyanoethyl-N,N-diisopropylchlorophophoramidite (1.96 g, 8.3 mmol). The reaction mixture was stirred at 0°C for 1 h and at room temperature for 2 h.

2The reaction was diluted with CHCH, (100 ml) and the organic layer was washed with 5% NaHCO 3 solution (100 ml), water (100 mi) and brine (50 ml). The CH,C1, extract was dried and evaporated to dryness to give an oily residue. The residue was purified by flash chromatography over silica gel using CH,Cl,-->EtOAc containing 0.1% TEA as the eluent. The pure fractions were pooled together and evaporated to give a foam.

The foam was dried over solid NaOH under vacuum overnight. The dried foam was dissolved in dry CHC1, (20 ml) and dropped 90 into a stirred solution of dry hexane (2000 ml) under argon during lh period. After the addition, the precipitate formed was stirred for additional 1h and filtered, washed with dry hexane (100 ml) and the solid was dried over solid NaOi under for 4 h. Yield: 3.5 g Examole 27 (See Figure 27) N-(tert-Butyloxycarbonyl)-O-Benzyl-D-Serine (12): O-Benzyl-D-Serine 11 (5 g, 25.64 mmol) was suspended in THF/HO 70 ml) mixture at room temperature. To this stirred mixture was added :riethyiamine (4.04 g, 40 mmol) followed by di-tert-butyl dicarbonate (6.54 g, 30 mmol); and aoea the stirring continued at room temperature overnight. The o.o homogenous solution was evaporated zo dryness and the residue o ao 0 a dissolved in ethyl acetate (150 mi). The organic extract was a washed with 0.5N solution of potassium hydrogen sulfate (100 ml), water (100 ml) and brine (50 ml). The ethyl acetate extract was dried over anhydrous sodium sulfate and evaporated e" to dryness to give 7.56 g (100%) of an oily residue.

*oo* N N-(tert-Butyloxycarbonyl)-O-Benzyl-D-Serinol (13) N-(tert-Butyloxycarbonyl) O-benzyl-D-serine 10 (7.56 g, 25.63 mmol) was dissolved in dry THF and cooled to -20 0 C under argon atmosphere. To this cold stirred solution was added TEA (3.03 S° 25 g, 30 mmol) and isoburyl chloroformate (4.08 g, 30 mmol). The stirring was continued for 30 min at -20°C under argon atmosphere. The reaction mixture was filtered immediately under a blanket of argon, the precipitate was washed with dry THF (50 ml). The combined filtrate was added slowly into a 4 cold solution of NaBH, (7.4 g, 200 mmol) in THF/water (80:20, 200 during 10 min period. Afterthe addition, the reaction mixture was stirred for 2 h at 0°C and the pH adjusted to 7 with acetic acid. The solution was evaporated to 91 L I -4c~r In F-gure 16, first the building block 136 is synthesized from 118 and 134 in a similar manner described in figure 1.

78 WO 96/14330 PCT/US95/14599 dryness, partitioned between ethyl acptate/water (300:150 ml) and extracted in ethyl acetate. The organic extract was washed with brine (100 ml), dried over anhydrous sodium sulfate and evaporated to dryness. The crude product was purified by flash column chromatography over silica gel using CHCl.-->EtOAc as the eluent. The pure product was pooled together and evaporated to dryness to give 6.68 g of the pure product as an oil. !HNMR (CDCl 3 6 1.41 9H, Boc), 3.60 3.70 (m, 4H), 3.82 2H), 4.53 2H, OCHPh), 5.20 (bs, 1H, NH) and 1 7.30 7.40 5H, -Ph).

N- (tert-Butyloxycarbonyl) -O-Benzyl-D-Serinol-O-Ib (14) To a dried solution of N-(Cert-Butyloxycarbonyl)-0-benzyl-D-serinol 13 (6.6 g, 23.5 mmol) in dry pyridine (50 ml) was added TEA (3.03 g, 30 mmol) at room temperature. To this stirred solution was added isobutyric anhydride (4.74 g, 30 mmol) and the stirring continued overnight under argon atmosphere. The reaction mixture was evaporated to dryness, partitioned between EtOAc (200 ml) and NaHCO 3 solution, 100 ml), and extracted in EtOAc. The organic extract was washed with water (100 ml), brine (50 ml), and dried over anhvyrous Na,SO.. The dried solution was evaporated to dryness to give a crude residue.

The residue was purified by flash chromatography over silica gel using hexane EtOAc as the eluent. The pure fractions were pooled together and evaporated to give an oily product g 'HNMR (CDC 3 6 1.04 6H, IbCH 3 1.39 (s, 9H, Boc), 2.46 1H, IbCH), 3.40 2H), 3.92 2H), 4.12 1H), 4.46 2H, OCHPh), 6.84 1H, NH) and 7.24 7.40 5H, Ph).

N- (Thyminylacetyl) -O-Benzyl-D-Serinol-O-Ib (15) N-(tert-Butyloxycarbonyl)- O-benzyl-D-serinol-O-Ib 14 (5.0 g, 92 following the steps used in figure 1.

79 4 WO 96/14330 PCT/US95/14599 14.25 mmol) was allowed to stir at room temperature in trifluoro acetic acid (20 ml) and CH,Cl, (20 ml) for 30 min.

The reaction mixture was evaporated to dryness-, dissolved in dry CH 3 OH (10 ml) and evaporated again zo dryness. The residue was dissolved in CH 2 Cl 2 (150 mi), the pH was adjusted to 7 VI 'with 5% NaHCO 3 solution and extracted in CHCl 2 The organic layer was washed with water (50 ml) and brine (50 ml). The CHCl, extract was dried and evaporated to dryness. The residue that obtained was dried over solid KOH under vacuum for 12 h.

The dried residue was used as such for further reaction without characterization.

Thymine acetic acid 5 (2.57 g, 14 mmol) was dissolved in dry DMF (50 ml) and cooled to -20°C under argon. To this cold stirred solution was added N-methylmorpholine (1.52 g, mmol) followed by isobutyl chloroformate (2.04 g, 15 mmol).

After 15 min of stirring, a solution of the above amine in dry DMF (50 ml) was added into the cold stirred solution of thvmine acetic acid at once. The reaction mixture was stirred at -20°C for 1 h, warmed to room temperature and the stirring continued overnight. The solution was evaporated to dryness and the residue dissolved in CHC1 2 (250 ml) and water (100 mil, and extracted in CHCI'. The organic extract was washed with 5% NaHCO 3 solution (100 ml), water (100 ml) and brine ml). The CH 2 CI, extract was dried and evaporated to dryness to 25give crude product. The crude product was purified by flash chromatography over silica gel using CHCl acetone as the eluent. The necessary fractions were collected and evaporated to give 2.8 g of the pure product. 'HNMR (CDCl 3 6 1.04 6H, IbCH 3 1.72 3H, CH 3 2.44 1H, IbCH), 3.42 2H), 4.06 2H), 4.18 1H), 4.30 2H), 4.46 (s, 0 2 OCH Ph), 7.24 7.40 6H, C.H and Ph), 8.22 1H, NH) and 11.22 1H, NH).

93 *u figure 23. Example 24 80 i- WO 96/14330 PCT/US95/14599 The titled compound was also prepared by using the method described for the preparation isomer. Reagents Used: Thymine acetic acid (2.2 g, 12 mmol); Isobutyl chloroformate (1.77 g, 13 mmol); N-methylmorpholine (1.52 g, 15 mmol); TFA Ssalt (3.65 g, 10 mmol); N-methylmorpholine (1.5 g, 15 mmol) and dry DMF (100 ml). Yield: 3.5 g N- (Thyminylacetyl)-D-Serinol-O-Ib N- (Thyminylacetyl)-O-Benzyl-D-Serinol-O-Ib 15 (3.5 g, 8.39 mmol) was dissolved in ethanol (50 ml). To this solution Pd(OH) 2 (1.00 g) and cyclohexene (10 ml) were added at room temperature. The reaction mixture was heated at 70 C for 12 h. The catalyst was filtered, washed with methanol (20 mi). The filtrate was evaporated to dryness to give an white solid. Yield: 2.7 g 'HNMR (Me 2 SO-d 6 6 1.04 6H, IbCH3), 1.72 3H,

CH

3 2.42 1H, IbCH), 3.40 2H), 3.94 2H), 4.06 (m, 1H), 4.28 2H), 4.90 1H, OH), 7.20 1H, C6H), 8.12 1H, NH) and 11.22 1H, NH).

4,4'-Dimethoxytrityl-N-(Thyminylacetyl) -D-Serinol--Ib N-(Thyminylacetyl)-D-Serinol-O-Ib 16 (2.7 g, 8.26 mmol) was dissolved in dry pyridine (50 ml) under argon. To this stirred solution was added TEA (1.01 g, 10 mmol) followed by 4,4'-dimetho;'ytrityl chloride (3.38 g, 10 mmol) and the stirring continued at room temperature under argon for overnight. The reaction mixture was quenched with MeOH ml), stirred for 10 min and evaporated to dryness. The residue was dissolved in EtOAc (250 ml), washed with 5% NaHCO 3 solution (100 mi), water (100 ml) and brine (50 ml). The organic extract was dried over anhydrous Na 2 SO, and evaporated to dryness. The crude product was purified by flash column chromatography over silica gel using CHCl EtOAc as the eluent. The pure fractions were pooled and evaporated to give g of foam. 'HNMR (CDC1 3 5 1.04 6H, IbCH 3 1.72 94 WO 96/14330 PCTIUJS95/14599 3H-, OH 3 2. 40 1JH, IbCH), 3. 38 (mn, 2H), 3. 72 6H-, 2.OCH 3 4 .12 (in, 2H)U 4 .20 1H) 4 .32 (di, 2H) 6.84 (in, 4H1, Ph), 7.20 7.40 (mn, 1211, C61H and Ph), 8.30 (ci, 1H1, NH) and 11.28 1H1, NH).

-Dimeth oxytri tyJ.1) [amino (thyminyJ.acetyl)]-D-propan-1,3-dio. 4,4'-Diinethoxytrityl-N-(- Thyminvlacetyl)-D-Seriflol-O-Tb 17 (5.0 g, 7.95 minol) was dissolved in MeOH (30 ml) and cooled to 000 in an ice bath. To 1this cold stirred solution was added 2N NaOH (10 ml, 20 inmol) and the stirring continued for 30 min at 000. The pH off the solution was adjusted to 7 with acetic acid and evaporated to dryness. The residue was parz-'zioned between water (50 and

CH

2 C1, (250 ml) and extracted in CH 2 The aqueous layer was extracted again with 011,012 (50 ml) The combined organic 1extract was washed with brine (50 ml), dried and evaporated to dryness. The residue was purified by flash column chromatography over silica gel using CH.,Cl- acetone as the eluent. Yield: 4. 0 g -HNMR (CDCl 3 1. 72 3H, CH 3 (mn, 2H1), 3.42 (in, 3.72 6H, 2.OCH 3 3.94 (in, 1H), 4.32 (ci, 2H), 4.68 1H, OH), 6.84 (mn, 4H, Ph), 7.20 7.40 OW, and Ph), 8.-0 6 (di, 11Hi, NH) and 11. 28 (bs, NH).

-DimethoxytrityJ.) [amino (thyminylacetyl) -Dpropan-3-O- N-diisopropyl) -cyanoethylphosphozramidite 4' -Diiethoxytrityl) [amino (thyminylacetylj I-D-Dropan-1, 3-diol IS 79 g, 5.-0 iniol) was dried over solid NaOH under vacuum overnight and dissolved in dry 011,01, (100 ml) The solution was cooled to 000 under argon atmospher-. To this cold stirred solution was added N, N-duiisopropvlethylainine 1.29 g, 10 minol) followed by 2-cyanoethyl-W, N-duisop~ropylchlorophosphoramiditLe (1.96 g, 8.3 io) The reaction mixture was stirred at 000 for 1 h and at room temperature for 2 h. The reaction was diluted with 95 82

CH

2 C1 2 (100 ml) and the organic layer was washed with 5% NaHCO, solution (100 ml), water (100 ml) and brine (50 ml). The

CH

2 Cl extract was dried and evaporated to dryness to give'an oily residue. The residue was purified by flash chromatography over silica gel using CH,C1, >EtOAc containing 0.1% TEA as the eluent. The pure fractions were pooled together and evaporated to give a foam. The foam was dried over solid NaOH under vacuum overnight. The dried foam was dissolved in dry

CH

2 Cl, (20 ml) and dropped into a stirred solution of dry hexane (2000 ml) under argon during lh period. After the addition, the precipitate formed was stirred for additional ih and filtered, washed with dry hexane (100 ml) and the-solid was dried over solid NaOH under vacuum for 4 h. Yield: 3.3 g Example 28 (Figure 28) 1" l-O-Benzyl-2-[(tert-butyloxycarbonyl)amino]-3- [N3-benzoyl(thyminyl)-L-propanol To a stirred solution of N-benzoylthymine 20 (5.75 g, 25 mmol) in dry THE (200 ml) o 06 20 under argon was added :riphenyl phosphine (10.48 g, 40 mmol) S' and No-tert-butyloxycarbonyl- -benzyloxy-L-serinol 3 (5.3 g, 18.86 mmol) at room temperature. After 15 min, diethylazodicarboxylate (6.96 g, 40 mmol) was added slowly during 30 min o period. The reaction mixture was covered with aluminum foil S" 25 and allowed to stir at room temperature under argon for 24 h, SThe solvent was evaporated to dryness and the residue dissolved in EtOAc (300 mi). The organic extract was washed Swith 5% NaHCO, solution (100 ml), water (100 ml) and brine (100 ml), and dried over anhydrous .Na 2 SO,. The dried EtOAc extract was evaporated to dryness to give an orange oil. The crude product was purified by flash chromatography over silica gel using hexane--> EtOAc as the eluent. The fraction having the required product was pooled and evaporated to give a pale 96 _i 83 WVO 96114330 PCT/US95/14599 pink oil. Yield: 8. 0 g HNMR (CDCl3): 1. 41 9H, Boc), 1.72 3H, Cl- 3 3.56 (mn, 2H), 4.20 2H)f, 4.32 (m, 4.52 2H, OCH 2 P) 5. 20 1Hb NH) 7. 06 1.9, C6H) Al and 7.20 7.60 (mn, 101i, Ph).

2- (tert-ButyJ~oxycarbony.) amino] EN 3 -benzoyl carbonyl) amino]j-3- [N 3 -benzoyl (thyninyl) -L-propanol 21 93 g, io) was dissolved in MeOH (100 ml) and treated with Pd/C 1 g) The reaction mixture was hydrogenated at 50 psi of hydrogen for 12 h. The catalystr was filtered, washed with MeOH ml) and the filtrate was evacrated to dryness. The residue was crystallized from acetone/hexane to give 3.70 g of pure product. mc: 156-159'C. IHNMR (CDCl9:) 1. 42 (s, 9H, Boc), 1.94 Cs, 3H, CH 3 3.64 (in, 4H), 3.84 (mn, 1H), 4.14 1H) 5.22 1H, NH) 7.18 1H, C6H) 7.48 Ct, 2H-, Ph), 7.62 1H, Ph) and 7.98 2H, Ph).

1-O-Isobutyy.-2-[C(tert-butyloxycarbonyl) amino] -3-

[N

3 -benzoy. (thyminyl) -I-propano. (23) 2- (Tert-Butyloxycarbonyl) amino] [N3-benzoyl t-:hviinyl)-L-propan-l-ol 22 11.60 g, 3.97 minol) was dissolved in dry pyridine (30 and allowed to stir at rooin teinperature under argon. To this stirred solution was added TEA (0.51 g, 5 imnol) and isobutyric anhydride (0.79 g, 5 iniol) The reaction inixture was stirred at rooin temperature for 12 h and evaporated to dryness. The residue was dis-solved in EtOAc (150 ml) and washed with NaHCO 3 solution (100 ml) water (100 ml) and brine (50 ml) The organic extract was dried and evaporated to dryness. The residue was purified by -flash column chromatography over 3silica gel using CHCl 2 EtOAc as the eluent. The pure fractions were collected together and evaporated to give 1.6 g of foam. The pure product was crystallized from acetone/hexane. inp: 165-167*C. HNMR (CDCl 3 1.16 6H, 97 i*rul--- WO 96/14330 PCT/US95/14599 IbCH3), 1.42 9H, Boc), 1.94 3H, CH 3 2.52 1H), 3.64 4H), 3.84 1H), 4.14 1H), 5.22 1H, NH), 7.18 1H, CH), 7.48 2H, Ph), 7.62 1H, Ph) and 7.98 2H, Ph) l-O-Isobutyryl-2- (-hydroxyacetyl) amino]-3- [N3-benzoyl (thyminyl) -L-propanol (24) 1-O-Isobutyryl-2- (tert-butyloxycarbonyl) amino]-3-[N3-benzoyl (thyminyl) -L-propanol 23 (1.6 g, 3.38 mmol) was allowed to stir in a 1mixture of TFA (5 ml) and CH 2 Cl- (10 ml) at room temperature for 30 min and evaporated to dryness. The residue was dissolved in dry MeOH (10 ml) and evaporated again. The residue that obtained was dried over solid NaOH under vacuum overnight. The dried material was usta as such for the next reaction.

To a stirred solution of glycolic acid (0.53 g, 7 mmol) in dry DMF (50 ml) was added 1-hydroxybenzotriazole (0.57 g, mmol) and 1-ethyl-3-(3-dimethylaminopropyl)-carbodiimide hydrochloride (EDC) (1.91 g, 10 mmol). After stirring for min, TEA (1.01 g, 10 mmol) and the above TFA salt in DMF ml) were added at room temperature. The reaction mixture was stirred for 12 h and evaporated to dryness. The residue was partitioned between CH 2 C1I (150 ml) and water (100 ml), and extracted in CHCl1. The organic extract was washed with brine 2(50 ml), dried and evaporated to dryness. The residue was purified by flash chromatography over silica gel using CH 2 C1 2 >acetone as the eluent. The fractions having the required product were collected and evaporated to give 1.35 g of foam. 'HNMR (CDCl3): 1.16 6H, IbCH3), 1.94 3H, CH 3 2.52 1H), 3.20 (bs, 1H), 3.80 4.30 6H), 4.56 (m, 1H), 7.14 2H, C4H and NH), 7.50 2H, Ph), 7.64 1H, Ph) and 7.94 2H, Ph).

98 L I I WO 96/14330 PCT/US95/14599 1-O-Isobutyryl-2- -dimethoxytrityl) -0-acetyl) amino] [N3-benzoyl(thyminyl)-L-propanol (25) butyryl-2-[(P-hydroxyacetyl)amino]-3- [N3-benzoyl(thyminyl) -L-propanol 24 (1.2 g, 2.78 mmol) was dissolved in dry pyridine (50 mi) and allowed co stir at room temperature under argon atmosphere. To this stirred solution was added TEA (0.35 g, 3.5 mmol) and 4,4'-dimethoxytrityl chloride (1.18 g, mmol). The reaction mixture was stirred at room temperature for 12 h, quenched with MeOH (10 ml) and evaporated to 0 dryness. The residue was dissolved in EtOAc (150 ml), washed with 5% NaHC03 solution (100 ml), water (100 ml) and brine ml). The organic extract was dried over NaSO, and evaporated to drvness. The residue was purified by flash chromatography over silica gel using CHCL >EtOAc as the eluent. The pure fractions were pooled and evaporated to give 1.7 g of pure product. HNMR (CDC 3 1.16 6H, IbCH3), 1.94 3H,

CH

3 2.52 1H), 3.74 6H, 2.OCH 3 3.80 4.30 6H), 4.56 1H), 6.82 4H, Ph), 7.14 2H, CH and NH) and 7.26 8.00 14H, Ph).

(-(4,4'-Dimethoxytrityl) -0-acetyl) amino]- 3-thyminyl-L-propanol (26) -O-Isobutyryl-2-[ (3- (4,4'-dimethoxytrityl)-0-acetyl)aminoj-3-[N 3 -benzoyl(thyminyl) -L-propanol 25 (1.55 g, 2.05 mmol) was dissolved in MeOH ml) and cooled to 0°C in an ice bath. To this cold stirred solution was added 2N NaOH (5 ml, 10 mmol) and the stirring continued for 30 min at 0°C. The DH of the solution was adjusted to 7 with acetic acid and evaporated to dryness. The residue was partitioned between water (50 ml) and CH 2 Cl 2 (150 ml) and extracted in CHC1 2 The aqueous layer was extracted again with CHC1, (50 ml). The combined organic extract was washed with brine (50 ml), dried and evaporated to dryness.

The residue was purifie by flash column chromatography over silica gel using CHCil--> acetone as the eluent. Yield: 1.0 g 99

~I~

86 r r WO 96/14330 PCT/US95/14599 'HNMR (CDC1 3 1.94 3H, CH 3 3.74 6H, 2.OCH 3 3.80 4.30 6H), 4.56 1H), 6.82 4H, Ph), 7.14 (d, 2H, C 6 H and NH) and 7.26 8.00 14H, Ph).

-Dimethoxytrityl)-O-acetyl)amino 3-thyminyl-L-propan- 1-0-(N,N-diisopropyl)-5-cyanoethylphosphoramidite 2-[(P-(4,4'-Dimethoxytrityl)- C acetyl)amino]-3-thyminyl-L-propanol 26 (1.00 g, 2.09 mmol) was dried over solid NaOH under vacuum overnight and dissolved in dry CHCl, (50 ml). The solution was cooled to 0°C under argon atmosphere. To this cold stirred solution was added N,N-diisopropylethylamine (0.54 g, 4.2 mmol) followed by 2-cyanoethyl-N,N-diisopropylchlorophosphoramidite (0.73 g, 3.1 mmol). The reaction mixture was stirred at 0°C for 1 h and at room temperature for 2 h. The reaction was diluted with CHC1l (100 ml) and the organic layer was washed with 5% NaHCO 3 solution (100 ml), water (100 ml) and brine (50 ml). The

CH

2 C1 2 extract was dried and evaporated to dryness to give an oily residue. The residue was purified by flash chromatography over silica gel using CHC1, >EtOAc containing 0.1% TEA as the eluent. The pure fractions were pooled togecher and evaporated to give a foam. The foam was dried over solid NaOH under vacuum overnight. The dried foam was dissolved in dry CH,C1 2 (10 ml) and dropped into a stirred solution of dry hexane (800 ml) under argon during 30 min period. After the addition, the precipitate formed was stirred for min and filtered, washed with dry hexane (100 ml) and the solid was dried over solid NaOH under vacuum for 4 h. Yield: 1.3 g 100 Examroie 29 (Figure 29) t~ tezt-Butyl oxycarb~f~ On -0-ben zylhycir oxylaamife O-Benzyl hydroxylamine hydrochloride (15.9 g, 100 mmol) was suspended in THE (150 ml) and water (50 ml) mixture. To this stirred mixture was added TEAz (15.15 g, 250 mmol) followed by di-tert-butyldicarbolate (23.98 g, 110 mmol) The reaction mixture was stirred at room ternoerature for 12 h and evaporated to dryness. The residue was partitioned between EtOAc (230 ml) and water (200 ml), and extracted in EtOkc. The EtOAc extract was washed with potassium hydrogen sulfate (100 ml) and brine (100 ml), dried and evaoorated to dryness to a *give 15 g o-f clear oi4l.

15 -Chloro--2- (tetrahydro-pyranyl) oxy-ethane (Z9. I-Chloro ethanol (8.06 g, 100 mmol) was dissolved in dry CHCl, (100 and cooled to 0 0 C in an ice bath under argon. To this Stirred solution was added dihyciropyran (12.6 g, 150 mmol) a..followed by pyridinium -o-toluene -4-sulfonate (1.25 g, mmol) and the stirring continued for overnight. The reaction mixture was evaporated to dryness and dissolved in EtOAc (200 The EtOAC extract was washed with 5% NaHCO, solution (100 ml) water (100 ml) and brine (100 ml) The org'anic extract 9 ,was dried and evaporated to dryness. The crude mat _rial was a purified by flash chromatography over silica gel using h,-exane 25->0 H,C1 2 as the eluent. The pure fractiLuns collected together and evaporated to give 11 g of pure product.

N- tert-Butyl ozycarbonyl (tetrahydropyrany.) oxy) ethyl -O-ben zylhydroxylamine (30) :To a stirred solution of 3N- Cert-butyloxvcarbonyl-O-benzylhiydroxylamine 28 79 c, 25.96 mmol) in dry DMF (50 ml) was added NaH 1.2 g, mmol) slowly during 15 min period under argon atmosphere at 0CC. The reaction was allowed to stir at 00 C for 30 min and 101 and cvclohexene (5 ml) were added at room temperature. 'ine reaction mixture was heated at 700C for 12 h. The catalyst was filtered, washed with methanol (20 ml). The filtrate was evaoorated to dryness to give a white solid. The white solid 88- WO 96/14330 PCT/US95/14599 at room temperature for 1 h. l-Chloro-2-(tetrahydropyranyl)oxy- ethane 29 (4.95 g, 30 mmol) was added and the reaction mixture was heated at 800C foi 12 h. The reaction was cooled and evaporated to dryness. The residue was suspended in water (50 mi), pH of the solution adjusted to 7 and extracted in EtOAc (150 ml). The EtOAc extract was washed with water and brine, dried and evaporated to dryness. The residue was purified by flash chromatography over silica gel using hexane--> CHC1, as the eluent. The required fractions were collected and evaporated to give 6.0 g of an oily product. 'HNMR (CDC 3 6 1.48 9H, Boc), 1.49 1.84 (m, 6H, 3.4.8 3.70 4H, 3.86 2H, CH 2 4.60 IH, CH), 4.84 2H, CHPh) and 7.32 7.42 Ph).

N-tert-Butyloxycarbonyl-N- (2-hydroxy) ethyl] -0benzylhydroxylamine A stirred solution of N-tert-Butyloxycarbonyl-N- (tetrahydropyranyl)oxy]ethyl- O-benzylhydroxylamine 30 (3.51 g, 10 mmol) in THF:water: AcOH 100 ml) was heated at 70 C for 3 h. The reaction was cooled to CoC and the pH adjusted no 7 with solid NaHCO 3 The reaction mixture was extracted with EtOAc (2x75 ml). The combined organic extract was washed with water (100 ml) and brine (100 ml), dried and evaporated to dryness. The residue was purified by flash chromatography over silica gel using CHC1 2 EtOAc as the eluent. The pure fractions were pooled and evaporated to give 2.5 g of foam. 'HNMR (CDCl) 6 1.48 9H, Boc), 3.60 2H, 3.74 2H, CH 2 4.84 2H, CHPh) and 7.32 7.42 5H, Ph).

N-tert-Butyloxycarbonyl-N- (2-isobutyryl) oxy] ethyl] O-benzylhydroxylamine To a stirred solution of 102 WO 96/14330 PCT/US95/14599 mine 31 (4.2 g, 16.6 mmol) in dry pyridine (50 ml) was added TEA (2.02 g, 20 mmol) followed by isobutyric anhydride (3.16 g, 20 mmol) at room temperature under argon atmosphere. The reaction mixture was stirred at room temperature for 12 h and evaporated to dryness. the residue was dissolved in EtOAc (200 ml), washed with 5% NaHCO3 solution (100 ml), water and brine ml). The organic extract was dried and evaporated to dryness. The residue was purified by flash chromatography over silica gel using CH 2 Cl1 as the eluent. The pure fractions collected and evaporated to give 4.5 g of pure compound.

IHNMR (CDC13) 1.04(d, 6H IbCH3), 1.48 9H, Boc) 2.44 1H, IbCH), 3.60 2H, 3.74 2H, 4.84 2H, CHPh) and 7.32 7.42 5H, Ph).

benzylhydroxylamine N-tert-Butyloxycarbonyl-N- [[(2-isobutyryl)oxy]ethyl]-O-benzylhydroxylamine 32 (5.0 g, 14.84 mmol) was dissolved in CH,C1, (10 ml) and allowed to stir in TFA (12 ml) fc. 30 min. The reaction mixture was evaporated to dryness and dissolved in dry methanol (10 ml).

It was evaporated again to dryness and dried under vacuum over solid NaOH overnight. The dried material used as such for the next reaction without characterization.

Thymine acetic acid 5 (3.13 g, 17 mmol) was dissolved in dry DMF (75 ml) and cooled to -20 0 C under argon. To this cold stirred solution was added N-methylmorpholine (2.02 g, mmol) followed by isobutyl chloroformate (2.72 g, 20 mmol).

After 15 min of stirring, a solution of the above TFA salt in dry DMF (50 ml) was neutralized with N-methylmorpholine (2.02 Sg, 20 rtmol) and added immediately into the cold stirred solution of thymine acetic acid at once. The reaction mixture was stirred at -200C for 1 h, warmed to room temperature and the stirring continued overnight. The solution was evaporated 103 rractions were pooled together and evaporated to give a foam.

The foam was dried over solid NaOH under vacuum overnight. The dried foam was dissolved in dry CH 2 Cl, (20 ml) and dropped 90 to dryness and the residue dissolvc.- in CH 2 C1 2 (250 ml) and water (100 ml), and extracted in CH 2 Cl. The organic extract was washed.with 5% NaHCO 3 solution (100 ml), water (100 ml) and brine (50 ml). The CH 2 C1 2 extract was dried and evaporated to dryness to give crude product. The crude product was purified by flash chromatography over silica gel using CH 2 C1 2 acetone as the eluent. The necessary fractions were collected and evaporated to give 4.0 g of the pure product. The pure product was crystallized from CHCl 2 /hexane.

10 mp: 185-188 0 C. 'HNMR (Me 2 SO-d) 6 1.00 6H, IbCH 3 1.74 ii 10 3H, CH3), 2.44 1H, IbCH), 3.92 2H), 4.18 2H), 4.68 (bs, 2H), 4.98 2H), 7.34 1H, C 6 7.40 7.50 S(m, 5H, Ph) and 11.32 (bs, 1H, NH).

o oa o oExample 15 S 15 (Figure ao (2R,4R)-2-Carbomethoxy-4-hydroxypyrrolidine In a 250 ml round bottom flask equipped with a magnetic stir bar and a reflex condenser were placed dry methanol (40 ml) and a cooled in ice bath under argon atmosphere. To this stirred 20 solution was added acetyl chloride (4.32 g, 55 mmol) followed .4 by cis-4-hydroxy-D-proline 34 (5.00 g, 38.17 mmol) The resulting solution was heated at reflex for 7-8 h and cooled to room temperature. The solution was diluted with ether, and i the resulting white solid was collected by suction, was with i 25 ether and dried under vacuum over solid NaOH. Yield; 6.9 g(100%) 'HNMR (CDC1) 2.09 (2 dd, 1H), 2.34 1H), 3.49 3.73 3H), 3.79 3H, CHJ), 4.34 2H).

j (2R, 4R) (tert-Butyloxycarbonyl) -2-carbomethoxy- 4-hydroxypyrrolidine To a stirred solution of (2R,4)-2-Carbomethoxy-4-hydroxypyrrolidine 35 (6.9 g, 38.12 mmol) in THF/water 150 ml) was added TEA (10.1 g, 100 mmol) followed by di-tert-butyldicarbonate 0.9 g, 50 mmol) 104 L 7- SPCT/US95/14599 WO 96/14330 at room temperature. The reaction was stirred at room i temperature for 6 h and evaporated to dryness. The residue was dissolved in EtOAc (200 ml) and washed with 0.5N potassium hydrogen sulfate (50 ml), water (100 ml) and brine (50 ml).

The organic extract was dried over Na 2

SO

4 and evaporated to dryness to give 7.8 g of an oily product. The oily product on drying gave colorless solid: mp: 75-770C. IHNMR (CDC1 3 1.45 9H, Boc), 2.09 (2 dd, 1H), 2.34 1H), S3.49 3.73 3H), 3.79 3H, CH 3 4.34 (in, 2H).

(2R,4R) (tert-Butyloxycarbonyl) -2-hydroxymethyl- 4-hydroxypyrrolidine (37) (2R,4R)-1-(tert-Butyloxycarbonyl)- 2-carbomethoxy-4-hydroxypyrrolidine 36 (7.0 g, 28.6 mmol) was dissolved in dry THF (100 ml) and cooled in ice salt bath under argon atmosphere. To this cold solution was added lithium borohydride (1.88 g, 85.8 mmol) in small portions I during 15 min period. After the addition of lithium borohydride, the reaction mixture was allowed to stir at 0°C for 1 h followed by 15 h at room temperature under argon. The solution was cooled to °C and diluted with water (50 ml) and the pH was adjusted with AcOH to 6. The reaction was evaporated to dryness and dissolved in EtOAc (200 ml), washed with water (100 ml) and brine (100 ml). The EtOAc extract was dried and evaporated to dryness. The residue was purified by 11 flash column chromatography over silica gel using CH 2 C1 2 EtOAc as the eluent. The pure fractions were collected and evaporated to dryness to afford 5.00 g of clear oil. The oil on standing gave colorless solid. mp: 95-97C. 'HNMR

(CDCI

3 1.45 9H, Boc), 1.90 dd, 1H), 2.34 1H), 3.40 3.62 3H), 4.00 2H), 4.28 (bs, 1H), 4.44 1H) (2R,4R)-1-(tert-Butyloxycarbonyl)-2-(4,4' Dimethoxytrityl) oxymethyl-4-hydroxypyrrolidine (38): (2R,4R)-1-(tert- Butyloxycarbonyl)-2- hydroxymethyl- 105 L i ~i~ N- (Thyminylacetyl) -O-Benzyl-D-Serinol-O-Ib N-(tert-Butyloxycarbonyl)- O-benzyl-D-serinol-O-Ib 14 (5.0 g, 92 k WO 96/14330 PCT/US95/14599 4-hydroxypyrrolidine 37 (4.4 g, 20.28 mmol) was- dissolved in dry pyridine (50 ml) and allowed to stir under argon atmosphere. To this stirred solution was added TEA (2.53 g, mmol) followed by 4,4'-dimethoxytrityl chloride (7.45 g, 22 mmol). The reaction mixture was stirred at room temperature for 12 h and quenched with MeOH (10 ml). The solution was I evaporated to dryness and dissolved in EtOAc (200 ml). The IEtOAc layer was washed with 5% NaHCO 3 solution (100 ml), water (100 ml) and brine. The organic extract was dried over anhydrous NaSO4 and evaporated to dryness. The residue was purified by flash chromatography over silica gel using hexane EtOAc as the eluent. The required fractions were pooled together and evaporated to give 8.09 g (100%) of an orange !I foam. 'HNMR (CDCl 3 1.45 9H, Boc), 1.90 dd, 1H), 2.34 1H), 3.40 3.62 3H), 3.74 6H, 2.OC8 3 4.00 (m, i 15 2H), 4.28 (bs, 1H), 4.44 1H), 6.82 4H, Ph), and 7.26 8.00 9H, Ph).

I (2R, 4R) (tert-Butyloxycarbonyl) |i Dimethoxytrityl) oxymethyl-4- (p-toluenesulfonyl) oxy]pyrrolidin 20 e 2 R,4R)-1-(tert-Butyloxycarbonyl)-2-(4,4'-Dimethoxytrityl) oxymethyl-4-hydroxypyrrolidine 38 (8.09 g, 20.27 mmol) was dissolved in dry pyridine/ CHC1, 200 ml) an- 6 chilled in an ice bath under argon atmosphere. To this cold solution was added TEA (3.03 g, 30 mmol) followed by p-toluenesulphonyl chloride (5.7 g, 30 mmol). The reaction mixture was allowed to stir at 0 C for 3 h and below 30 0 C for 8 h. The reaction mixture was evaporated to dryness, partitioned between EtOAc (200 ml) and 5% NaHCO 3 solution (100 ml), and extracted in EtOAc. The EtOAc extract was washed with water (100 ml) and brine (100 ml), dried and evaporated to dryness. The crude product was purified by flash chromatography over silica gel using hexane EtOAc as the eluent. The pure fractions were pooled together and evapor;ted 106 -4 -1 I I I-I OCH,Ph), 7.24 40 6H, CAH and Ph) 8. 22 11H, NH) and 11.22 1H, NH",.

93 WO 96/14.330 PCTUS95/14599 to give 12 .2 g (8 of an orange oil. 'HNMR CCDCl3) 1.4.5 (s, 9H, Boc) 1. 90 C dd, 1H) 2.34 (mn, 2. 40 3H, CHJ) 3. 40 3. 62 (in, 3H) 3. 74 6H, 2.-OCH3), 4.-0 0 (mn, 2 H) 4 .2 8 (bs, 1H), 4.44 1H), 6.82 4H, Ph), and 7.26 8.00 (mn, S13H, Ph).

(2R, 4S) -1 (t-ert-Butyloxycarbonyl) -2 4' Dimethoxytrity.) oxymethyl-4-azido-pyrroJlidine (AO): (2R, 4R) (tert- Butyloxycarbonyl) 1Direthoxytrityl) oxymethyl- Cp-toluenesulfonyl) oxyypyrrolidine 39 C5.1 g, 7.58 inmol) was dissolved in dirnethylformornide (50 ml) and diluted with water (5 ml) .To this stirred solution was added sodiuin azide 0.65 g, mmiol) and heated at 80*C for 8 h. I was cooled and evaporated to dryness. The residue was partitioned between 1CH 2 1C1 2 (200 ml) and water (100 and extracted in CH 2 C1 2 The organic extract was washed with brine (50 ml), dried over Na 2

SO

4 and evaporated to dryness. The crude product was purified by flash chromatography over silica gel using hexane 4 >EtOAc as the eluent. The pure fractions were pooled together and evaporated to give 3.8 g of a clear oil.

'HNMR CCDCl 3 1.4 5 9H, Boc) 1 .90 C dd, 1H) 2. 34 Cm, 1H) 3 .40 3. 62 3H) 3.74 Cs, 6N, 2.0OCR 3 4 .00 Cm, 2H) 4.28 Cbs, 1H), 4.44 Cm, 1H), 6.82 Cd, 4H, Ph), and 7.26 -7.80 (mn, 9H, Ph).

(2R, 4S) -1-(tert-Butylorycarbonyl) -2 -hydroxymethy.- 4 -amino -pyrrolidine (41) 4S) (tert-Butyloxycarbonyl) 2-C4,4'-Dimethoxytrityl)oxymethyl-4-azido-pyrrolidine 40. (2.72 g, 5 inmol) in methanol (75 ml) was hydrogenated in the presence of 10% palladium on charcoal (0.3 g) at room temperature and 5 atm pressure. After 12 h, the catalyst was filtered, washed with methanol (20 ml) and the solvent removed under vacuum. Yield 1.0 g (93%0) IHNMR CCDC1 3 1.45 Cs, 9H, -107 30 rrr chromatography over silica gel using CHCl EtOAc as the eluent. The pure fractions were pooled and evaporated to give g of foam. 'HNMR (CDCI 3 1.04 6H, IbCH) 1.72 94 I I- WO 96/14330 PCT/US95/14599 Boc), 1.90 dd, 1H), 2.34 1H), 3.40 3.62 3H), 4.00 2H), 4.28 (bs, 1H) and 4.44 1H).

(2R, 4S) (tert-Butyloxycarbonyl) -2-hydroxymethyl- 4-phthalimido-pyrrolidine (2R,4S)-1-(tert-Butyloxycarbonyl)-2-hydroxymethyl-4-amino-pyrrolidine 41 (1.00 g, 4.63 mmol) was dissolved in dry methanol (20 ml) and treated with N-ethoxycarbonyl phthalimide (1.09 g, 5 mmol) at room temperature. The reaction mixture was stirred for 6 h and evaporated to dryness. the residue was purified by flash chromatography over silica gel using CHCl 2 EtOAc as the eluent. The pure fractions were collected and evaporated to give 1.5 g of pure compound as foam. 'HNMR (CDC13): 1.45 9H, Boc), 1.90 dd, 1H), 2.34 1H), 3.40 3.62 (m, 3H), 4.00 2H), 4.28 (bs, 1H), 4.44 1H) and 7.3 7.6 S(m, 4H, Ph).

S(2R, 4S) (tert-Butyloxycarbonyl) [N 3 -benzoyl (thymmn-l-yl)] methyl-4-phthalimido-pyrrolidine To a stirred solution of N 3 -benzoylthymine 20 (1.15 g, 5 mmol) in dry THF (70 ml) under argon was added triphenyl phosphine i. (2.62 g, 10 mmol) and (2R,4S)-1-(tert-Butyloxycarbonyl)i 2-hydroxymethyl-4-phthalimido- pyrrolidine (1.4 g, 4.05 mmol) i at room temperature. After 15 min, diethylazodicarboxylate (1.74 g, 10 mmol) was added slowly during 10 min period. The reaction mixture was covered with aluminum foil and allowed to stir at room temperature under argon for 24 h. The solvent was evaporated to dryness and the residue dissolved in EtOAc (150 ml). The organic extract was washed with 5% NaHCO 3 solution (100 ml), water (100 ml) and brine (100 ml), and dried over anhydrous Na 2

SO

4 The dried EtOAc extract was evaporated to dryness to give an orange oil. The crude product was purified by flash chromatography over silica gel using hexane--> EtOAc as the eluent. The fraction having the required product was 108

LI~

WO 96/14330 PCTIUS95/14599 pooled and evaporated to give a pale pink oil. Yield: 2.0 g 'HNMR (CDC 3 1.41 9H, Boc), 1.72 3H, CH3), 1.90 dd, 1H), 2.34 1H), 3.40 3.62 3H), 4.00 (m, 2H), 4.28 (bs, 1H), 4.44 1H), 7.06 1H, C6H) and 7.20- S 7.60 9H, Ph).

Example 31 Synthesis of Oligonucleotides: Oligonucleotides containing modified amino acid nucleic acid backbones were synthesized on an automated DNA synthesizer (Applied Biosystems model 394) using standard phosphoramidite chemistry. D-Cyanoethyl phosphoramidities, synthesis reagents and CPG polystyrene colums were purchased from Applied Biosystems (Foster City, CA). For phosphorothioate oligonucleotides, the standard oxidation bottle was replaced with tetraethylthiuram disulfide/acetonitrile, and the standard ABI phosphorothiate program was used for the stepwise thiation of the phosphate linkages. After cleavage from the controlled pore glass column, the protecting groups were removed by treating the oligonucleotides with concentrated V 20 ammonium hydroxide at 55 0 C for 8 hrs. The oligonucleotides (DMT-on) were purified by HPLC using a reverse phase semiprep C column (ABI) with a linear gradient of 5% acetonitrile in 0.1M triethylammonium acetate (buffer A) and acetonitrile (buffer The DMT protecting group was cleaved by treatment with 80% acetic acid and the product was ethanol precipitated.

The purity of the products were checked by HPLC using an analytical Cs column (Beckman). The amino acid nucleic acid monomers were incorporated at the 3'-end, 5'-end and in the middle of a DNA sequence with a coupling efficiency of 100%. A homo polymer containing 16 amino acid modified thymine was also prepared with out any problem.

109 II _I -f ;i I ~L WO 96114330 PCT/US95/14599 Example 32 Hybridization analysis: The ability of the amino acid modified oligonucleotides of the invention to hybridize to their complementary RNA and DNA sequences is determined by thermal melting analysis. The RNA complement is synthesized by Genset corporation (La Jolla, CA) and purified by denaturing urea PAGE. Natural antisense oligonucleotides or those containing functionalized at specific locations are added to either the RNA or DNA complement at stoichiometric concentrations to form hybrid duplexes. The absorbance (260 nm) hyperchromicity dependence on temperature upon duplex to random coil transition is monitored using Varian Cary 1E UV-Visible spectrophotometer. The measurements are performed in a buffer of 10 mM Na-phosphate, pH 7.4, 0.1 mM EDTA, and NaCl to yield an ionic strength of either 0.1 M or 1.0 M.

Data are analysed by a graphic representation of 1/Tm vs ln[Ct], where [Ct] is the total oligonucleotide concentration.

From this analysis the thermodynamic parameters are determined. Based on the information gained concerning the stability of the duplex or hetero-duplex formed, the placement of modified pyrimidine into oligonucleotides is assessed for its effects on helix stability. Modifications that drastically alter the stability of the hybrid exhibit reductions or enhancements in the free energy (delta G) and decisions concerning their usefulness in antisense oligonucleotides are made.

Hybridization studies were conducted with oligonucleotides containing amino acid nucleic acid backbone at 3'-end as well as at the 5'-end. Preliminary studies showed that the modified oligonucleotides form duplex with their complementary RNA and DNA sequences like unmodified oligonucleotides.

Example 33 110 111 Nuclease Resistance. Natural, phosphorothioate and modified oligonucleotides of the invention are assessed for their resistance to serum nucleases by incubation of the oligonucleotides in media containing various concentrations of fetal calf serum or adult human serum. Labeled oligonucleotides are incubated for various times, treated with protease K and then analyzed by gel electrophoresis on 20% polyacrylamide-urea denaturing gels and subsequent autoradiography or phosphor-imaging. Autoradiograms are quantitated by laser densitometry. Based upon the location of the modifications and the known length of the oligonucleotide it is possible to determine the effect of the particular modification on nuclease degradation. For the cytoplasmic nucleases, a cell line is used. A post-mitochondrial supernatant is prepared by differential centrifugation and the labeled oligonucleotides are incubated in this supernatant for various times. Following the incubation, oligonucleotides are assessed for degradation as outlined above for serum nucleolytic degradation. Autoradiography results are quantitated for comparison of the unmodified phosphorothioate and the modified oligonucleotides.

SPreliminary studies on the amino acid modified oligonucleotides showed that they are resistant to Snake Venom Phosphodiesterase.

Incorporation by Reference All patents, patents applications, and publications cited are incorporated herein by S. 20 reference.

0 o Equivalents The foregoing written specification is considered to be sufficient to enable one skilled in the art to practice the invention. Indeed, various modifications of the abovedescribed makes for carrying out the invention which are obvious to those skilled in the 25 field of molecular biology, organic chemistry, or related fields are intended to be within the scope of the following claims.

[N:\Iibff00855:MCN

Claims

1. A compound of compound 1 in any of Groups I-V: W-L X-- N--Z A Y Compound 1 Group I, where: X is CHR 2 OH where R 2 is H, lower alkyl amine or lower alkyl imidazole; Y is Base-(CH2)n-, where Base is a non-halognated variable nucleosidic base, and n is 1 to 7; o o A is carbonyl; Z is H or OR 3 where R 3 is H, lower alkyl, lower alkyl amine or lower alkyl imidazole; W is CHR40H, where R 4 is H or lower alkyl amine or lower alkyl imidazole; N is N(nitrogen); and L is not present; Group II, where: X is Base-(CH 2 where Base is a non-halogenated variable nucleosidic base, and n is 1 to 7; Y is CHR 2 OH where R2 is H, lower alkyl amine or lower alkyl imidazole; A is carbonyl or CH2; Z is H or OR3, where R3 is H, lower alkyl, lower alkyl amine lower alkyl imidazole; W is CHR40H, where R 4 is H or lower alkyl amine or lower alkyl imidazole; N is N(nitrogen); and L is not present; Group III, where: X is CHR20H where R 2 is H, lower alkyl amine or lower alkyl imidazole; Y is Base-(CH2)n-, where Base is a non-halogenated variable nucleosidic base, and n is 1 to 7; A is CH 2 Z is OH or OR 3 and where R 3 is H, lower alkyl, lower alkyl amine or lower alkyl imidazole; RA W is CHR40H, where R 4 is H or lower alkyl amine or lower alkyl imidazole; S N is N(nitrogen); and (N:\LIBA100437:MCN I 113 4 I 4 j i 4 or 4 0 e oe o i 4 4 4, 4 S6a 4 a t 4,44 4 4 a l 4444 4 4 a o t i 4 44 4 4o i a i i S 1 L is not present; Group IV, where: X is Base-(CH 2 where Base is a non-halogenated variable nucleosidic base, and n is 1 to 7; Y is COOH or CHR 2 OH where R 2 is H, lower alkyl amine or lower alkyl imidazole; A is carbonyl or CH 2 Z is CH 2 W is CH 2 N is N(nitrogen); and L is CHNHR 5 where R 5 is H, OH or OR 3 and R 3 is H, lower alkyl, lower alkyl amine or lower alkyl imidazole; Group V, where: X is CH 2 0H, CH 2 NH 2 CONH 2 or COOH; Y is not present; Z is CH 2 or CHO-L 1 -B; W is O, S or CH 2 N is CH; and L and A are independently COOH, CHCOOH, CHCH 2 COOH, NH 2 CHNH 2 L 1 NH-L 2 -B or CH-L 1 -NH-L 2 where B is H or a nucleoside base, and L 1 and L 2 are independently (CH2)n, where n= 1-3 or (CH2)nCO, where n 0-2.

2. A compound of formula 1 as defined in claim 1 and substantially as hereinbefore described with reference to any one of Examples 1 to

3. The compound according to claim 1 or 2 polymerized into an oligonucleotide characterised in that internucleotide linkages occur between W and X in Group I, W and Y in Group II, W and Z in Group III, L and Y in Group IV, and L and A in Group V. 3, characterised in that 4. The oligonucleotide internucleotide linkage comprises The oligonucleotide internucleotide linkage comprises

6. The oligonucleotide internucleotide linkage comprises

7. The oligonucleotide internucleotide linkage comprises

8. The oligonucleotide oligonucleotide into a plurality according to claim a phosphodiester. according to claim a phosphorothioate. according to claim a phosphoramidate. according to claim a hydroxamate. according to claim of monomers wherein 3, characterised 3, characterised 3, characterised in that the in that the in that the characterised in that the least one of the monomers comprises a 2'-deoxyribose nucleoside, and further comprising at least two different internucleotide linkages from the group consisting of phosphodiester, phosphorothioate, phosphoramidate and hydroxamate. [N:\UBA00437:MCN -II 100 114

9. The oligonucleotide according to claim 8, cha;acterised in that the oligonucleotide has a 2'-deoxyribose nucleoside pentafuranosyl ring wherein the ring oxygen is replaced with one of S, CH 2 or NR 6 where R6 is pentafuranosyl ring wherein the ring oxygen is replaced with one of S, CH 2 or NR 6 where R 6 is acetyl, lower alkyl, carbonyl, carbonyl lower alkyl amine, or carbonyl lower alkyl imidazole. The oligonucleotide of any one of claims 3 to 9, wherein at least one monomer satisfies Group V of claim 1, and a maximum of fifty monomers are selected from 2'- deoxyribonucleoside, ribonucleoside and 2'-methylribonucleoside.

11. A compound of formula 1 as defined in claim 1 polymerized into an oligonucleotide, substantially as hereinbefore described with reference to Example 31.

12. A pharmaceutical composition comprising the oligonucleotide of any one of claims 3 to 11, and a pharmaceutically acceptable carrier.

13. A method of inhibiting or modulating gene expression comprising administering an effective amount of the oligonucleotide of any one of claims 3 to 11, or the composition of claim 12. Ir, 14. The method of claim 13 wherein said gene expression is associated with the Sestablishment or maintenance of a pathological condition. o 15. The method of claim 14, wherein said pathological condition is selected from the group comprising inflammatory conditions, cardiovascular disorders, immune reactions, cancer, viral infections, bacterial infections, yeast infections or parasite S: infections 0 16. A method of detecting the presence or absence of a target nucleic acid Ssequence comprising a) hybridising oligonucleotides of any one of claims 3 to 11 which specifically binds to said target nucleic acid sequence; b) detecting the presence or absence of bound oligonucleotides. S17. A method of amplifying a target nucleic acid sequence comprising a) hybridisation of one or more oligonucleotides of any one of claims 3 to 11 which specifically bind to said targe sequence; b) amplifying the target sequence. Dated 19 May, 1998 ICN Pharmaceuticals Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [N:\LIBAI00437:MCN k i. i i I r I