Classifications

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

Definitions

• This invention relates to methods for the preparation of oligomeric compounds having pendant groups conjugated thereto through a novel haloacetyl linker. This invention also relates to compounds containing such haloacetyl linkers, and to methods and intermediates useful for their preparation.
• Oligonucleotides and their analogs have been developed and used in molecular biology in a variety of procedures as probes, primers, linkers, adapters, and gene fragments. Modifications to oligonucleotides used in these procedures include labeling with nonisotopic labels, e.g. fluorescein, biotin, digoxigenin, alkaline phosphatase, or other reporter molecules. Other modifications have been made to the ribose phosphate backbone to increase the nuclease stability of the resulting analog. Examples of such modifications include incorporation of methyl phosphonate, phosphorothioate, or phosphorodithioate linkages, and 2 ' -O-methyl ribose sugar units. Further modifications include those made to modulate uptake and cellular distribution. With the success of these compounds for both diagnostic and therapeutic uses, there exists an ongoing demand for improved oligonucleotides and their analogs .
• oligonucleotides especially oligonucleotides which are complementary to a specific target messenger RNA (mRNA) sequence.
• mRNA target messenger RNA
• oligonucleotides are currently undergoing clinical trials for such use.
• Phosphorothioate oligonucleotides are presently being used as such antisense agents in human clinical trials for various disease states, including use as antiviral agents .
• oligonucleotides can serve as competitive inhibitors of transcription factors to modulate their action.
• Several recent reports describe such interactions (see Bielinska, A., et. al., Science, 1990, 250, 997-1000; and Wu, H., et . al., Gene, 1990, 89, 203-209).
• diagnostic tests can be performed using biological fluids, tissues, intact cells or isolated cellular components.
• oligonucleotides and their analogs are also widely used as research reagents. They are useful for understanding the function of many other biological molecules as well as in the preparation of other biological molecules. For example, the use of oligonucleotides and their analogs as primers in PCR reactions has given rise to an expanding commercial industry.
• PCR has become a mainstay of commercial and research laboratories, and applications of PCR have multiplied.
• PCR technology now finds use in the fields of forensics, paleontology, evolutionary studies and genetic counseling.
• kits which assist non-molecular biology-trained personnel in applying PCR.
• Oligonucleotides and their analogs are employed as primers in such PCR technology.
• Oligonucleotides and their analogs are also used in other laboratory procedures . Several of these uses are described in common laboratory manuals such as Molecular
• Oligonucleotides and their analogs can be synthesized to have customized properties that can be tailored for desired uses .
• a number of chemical modifications have been introduced into oligomeric compounds to increase their usefulness in diagnostics, as research reagents and as therapeutic entities.
• modifications include those designed to increase binding to a target strand (i.e.
• Tm melting temperatures
• nucleoside or oligonucleotide having a free hydroxyl group is reacted with a protected cyanoethyl phosphoramidite monomer in the presence of a weak acid to form a phosphite- linked structure.
• Oxidation of the phosphite linkage followed by hydrolysis of the cyanoethyl group yields the desired phosphodiester or phosphorothioate linkage.
• Synthetic oligonucleotides having 5 ' -terminal functional or reporter groups are employed in a number of bioanalytical and antisense applications.
• a number of procedures and reagents that allow their preparation have been described, including conjugation of electrophiles to oligonucleotides via tethers carrying nucleophilic groups.
• conjugation of electrophiles to oligonucleotides via tethers carrying nucleophilic groups For the opposite process of conjugating nucleophiles to electrophilic sites, the methods available to generate these sites in oligonucleotides are limited. These include periodate oxidation of a terminal ribose moiety or the use of an internal abasic site within the oligonucleotide sequence.
• Heterobifunctional reagents that bear a phosphoramidite moiety along with an electrophilic functional group that is reactive in orthogonal conditions are therefore of particular interest.
• the modified oligonucleotide could be converted into a variety of conjugates by treatment with linkers of different length and substituents that bear a primary amino group .
• haloacetyl linker that is reactive towards a variety of nucleophiles has been introduced previously into oligonucleotides by postsynthetic reaction in solution. See Goodchild, J. Bioconjugate Chem . 1990, 1, 165. Ligands carrying amino groups may be either less available or less desirable for attachment than those with other nucleophilic groups. Therefore, more universal methods for preparation of oligonucleotide conjugates with the aid of heterobifunctional phosphoramidite building blocks are greatly desirable. This invention is directed to this, as well as other, important ends.
• R x is a phosphorus protecting group
• R 2 is chlorine or a pendant group
• R 3 is -N(R 4 ) 2 , or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
• R 4 is straight or branched chain alkyl having from 1 to 10 carbons, v is 0 or 1 ; n is 1 to about 10; Q has one of the Formulas II or III:
• R 5 is a hydroxyl protecting group
• B is a nucleobase
• R 6 is F, O-R 20 , S-R 20 or N-R 20 (R 21 );
• R 20 is alkyl, or a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic or heterocyclic; and wherein any available hydrogen atom of said ring system is each replaceable with an alkoxy, alkylamino, urea or alkylurea group; or R 20 has one of the formulas :
• R 7 is H, a hydroxyl protecting group, or a linker connected to a solid support; w is 0 to about 100; to form a compound of Formula IVa :
• R 1X is 0 or S .
• v is 0. In further preferred embodiments, v is 1. In still further preferred embodiments, R 2 is Cl . In yet further preferred embodiments, R 2 is Cl and R 3 is -N(R 4 ) 2 , wherein R 4 is isopropyl.
• R 2 is Cl
• R 3 is -N(R 4 ) 2
• R 4 is isopropyl
• R ⁇ is (list protecting groups) , with ⁇ - cyanoethyl being preferred.
• n is 2 to 8, with 4 to 6 being preferred, and 6 being more preferred.
• R 7 is a linker connected to a solid support.
• the pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid.
• v is 0,
• R 2 is Cl
• R 3 is -N(R 4 ) 2 , wherein R 4 is isopropyl
• R x is ⁇ -cyanoethyl
• n is 6
• R 7 is a linker connected to a solid support, and said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid.
• v is 1, R 2 is Cl, R 3 is -N(R 4 ) 2 , wherein R 4 is isopropyl, R 2 is ⁇ -cyanoethyl, n is 6, R 7 is a linker connected to a solid support, and said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid, and Q has the Formula II.
• v is 1, R 2 is Cl, R 3 is -N(R 4 ) 2 , wherein R 4 is isopropyl, R-, is ⁇ - cyanoethyl, n is 6, R 7 is a linker connected to a solid support, and said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid, and Q has the Formula III.
• Also provided in accordnce with the present invention are synthetic methods comprising: providing a compound of Formula H 2 N- (CH 2 ) n -0H wherein n is 1 to about 10; reacting said compound with a compound of Formula VIII:
• R 2 is a phosphorus protecting group
• R 3 is -N(R 4 ) 2 , or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
• R 4 is straight or branched chain alkyl having from 1 to 10 carbons. n is 1 to about 10; for a time and under conditions sufficient to form an activated phosphate compound of Formula X:
• R 3 is diisopropylamino; and R 2 is ⁇ -cyanoethyl .
• Further preferred embodiments of the methods of the inventikon further comprise reacting said activated phosphate compound with a free hydroxyl group of a nucleoside, a nucleotide, an oligonucleotide, or an oligonucleotide connected to a solid support to form a compound of Formula XI:
• R 9 is a nucleoside, a nucleotide, an oligonucleotide, or an oligonucleotide connected to a solid support .
• Still further preferred embodiments further comprise coupling a pendant group to the compound of Formula XII, preferably wherein said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid.
• a pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid.
• R 2 is halogen or a pendant group
• R 10 is a nucleobase, a nucleoside, a nucleotide, an activated nucleotide, an oligonucleotide, an oligonucleotide connected to a solid support, or a moiety of Formula VI :
• R ⁇ is H or a phosphorus protecting group
• R 3 is -N(R 4 ) 2 , or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
• R 4 is straight or branched chain alkyl having from 1 to 10 carbons; and n is from 1 to about 10.
• R 10 is a nucleoside, a nucleotide, an activated nucleotide, an oligonucleotide, or an oligonucleotide connected to a solid support. In further preferred embodiments R 10 has the Formula IV.
• compounds of the invention have the Formula VII:
• R 5 is a hydroxyl protecting group
• B is a nucleobase
• R 6 is F, O-R 20 , S-R 20 or N-R 20 (R 21 );
• R 20 is alkyl, or a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic or heterocyclic; and wherein any available hydrogen atom of said ring system is each replaceable with an alkoxy, alkylammo, urea or alkylurea group; or R 20 has one of the formulas :
• Q is 0, S or NR 2 ; m is from 1 to 10; y is from 0 to 10;
• v is 0 and q is 1. In further preferred embodiments, v is 1. In further preferred embodiments, v is 1 and q is 1.
• R 2 is Cl . In further preferred embodiments, R 2 is Cl and R 3 is -N(R 4 ) 2 , wherein R 4 is isopropyl. In still further preferred embodiments, R 2 is Cl, R 3 is -N(R 4 ) 2/ wherein R 4 is isopropyl, and R ⁇ is (list protecting groups) , with ⁇ -cyanoethyl being preferred.
• n is 2 to 8, with 4 to 6 being more preferred, and 6 being especially preferred.
• v and q are each 1; and Q has the Formula III wherein the moiety -O-P (R 3 ) -0-R a is attached to B at the N 2 position.
• v and q are each 1; and Q has the Formula II wherein the moiety -O-P (R 3 ) -0-R ⁇ is attached at the 2 '-position.
• said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid.
• v is 0; q is 1; R 2 is Cl, R 3 is -N(R 4 ) 2 , wherein R 4 is isopropyl, R x is ⁇ -cyanoethyl, n is 6, and said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid.
• v and q are each 1;
• R 2 is Cl,
• R 3 is -N(R 4 ) 2 , wherein R 4 is isopropyl, R ⁇ is ⁇ -cyanoethyl, n is 6, said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid, and
• Q has the Formula II wherein the moiety -O-P (R 3 ) -0-R ⁇ is attached at the 2 '-position.
• R 2 is Cl
• R 3 is -N(R 4 ) 2/ wherein R 4 is isopropyl
• R 2 is ⁇ - cyanoethyl
• n 6
• said pendant group is an amine, a polyamine, a thiol, a protein, a peptide, or an amino acid
• Q has the Formula III wherein the moiety -0-P (R 3 ) -O-R ! is attached at the N 2 -position.
• the present invention provides methods for the preparation of oligomeric compounds having at least one pendant group conjugated thereto.
• the phosphite linkage is then oxidized or sulfurized by standard techniques to form a phosphotriester or phosphorothiotriester linkage, as represented by Formula IVb:
• R is 0 or S.
• the compound of Formula IVb is then contacted with a pendant group having, in preferred embodiments, a primary amino or thio group, for a time and under conditions sufficient to form the conjugate.
• reacting means placing the indicated moieties together under conditions that will cause the moieties to perform the chemical reaction indicated.
• the term contacting means placing the indicated moieties together in a container under conditions that will cause the indicated reaction to occur.
• the term "pendant group” means a functional or reporting group as is used in bioanalytical and/or antisense applications.
• Representative pendant groups include amines such as long and short straight, branched chain or cyclic alkylamines, polyamines such as spermine and spemidiine, polyalkylamines, thiols including aliphatic and aromatic mercaptans such as n-C 18 H 37 SH, thiocresol, benzylmercaptan, and thiocholesterol, proteins, peptides, and amino acids.
• pendant groups are amenable to the present invention. It is only required that the pendant group have a nucleophilic moiety such as an amino group, a sulfur atom, or an oxygen atom which is capable of displacing the halogen atom of the electrophilic linker and thus effecting the linkage of to the pendant group.
• a nucleophilic moiety such as an amino group, a sulfur atom, or an oxygen atom which is capable of displacing the halogen atom of the electrophilic linker and thus effecting the linkage of to the pendant group.
• the haloacetyl linker is incorporated into an activated nucleoside phosphoramidite synthon (i.e., embodiments wherein variable "v" is 1 in the Formulas herein) .
• the electrophilic haloacetyl linker can be attached to an activated nucleoside phosphoramidite synthon at the sugar moiety thereof, preferably through the 2'- hydroxyl (e.g., embodiments wherein variable "v” is 1 and Q has Formula II) .
• the electrophilic haloacetyl linker also can be attached to an activated nucleoside phosphoramidite synthon at the base moiety thereof, preferably through the N-2 nitrogen (e.g., embodiments wherein variable "v” is 1 and Q has Formula III) .
• the electrophilic haloacetyl linker also can be attached to the intersugar linkage of an oligonucleotide. Acordingly, in some preferred embodiments, the linker can be prepared as a phosphoramidite (e.g., embodiments wherein variable "v” is 0 in the Formulas herein) of Formula X:
• haloacetyl linker can be conveniently introduced into an oligonucleotide during standard oligonucleotide synthesis at the 3 '-terminus, the 5 '-terminus, or at any position in the oligonucleotide .
• the methods of the invention provide the additional feature that a pendant group of interest can be attached postsynthetically to the fully protected support- bound oligonucleotide via either thiol or primary amino group.
• the postsynthetic coupling of pendant group may be carried our both in aqueous and organic solvent .
• the concentration of the compound to be attached is from about 0.05M to about 1.0M. It is preferred that the coupling of pendant groups through thiol groups be performed in the presence of a tertiary amine as a catalyst, preferably from about 0.5M to about 1.0M. Suitable preferred catalysts include triethylamine, ethyldiisopropylamine, and 1,8- diazabicyclo [4.5.0] undec-7-ene (DBU) .
• the resulting oligonucleotide having the electrophilic haloacetyl linker attached thereto is typically coupled to a pendant group through the linker, and the completed oligomer is then released from the solid support and simultaneously deprotected by contacting with aqueous ammonia.
• Typical isolated yields of modified oligonucleotides is 20 to 70% depending on the length of the oligonucleotide, and the reactivity of particular pendant group.
• the methods of the invention can be used to prepare oligomers having a variety of internucleoside linkages, represented by Z in the formulas herein.
• oligonucleotides prepared by the methods of the invention can have a wide variety of substitutents at their 2 ' -positions, in addition to the haloacetyl linker described herein. These include those described for substituent R 6 of the formulas described herein.
• the methods of the invention provide for oligonucleotides having one, two or a plurality of pendant groups attached through novel linkers described herein.
• X are prepared by providing a compound of Formula H 2 N- (CH 2 ) n -OH wherein n is 1 to about 10; reacting the compound with a compound of Formula VIII:
• the reaction of the compound of Formula H 2 N- (CH 2 ) n -OH with the compound of Formula VIII, and the contacting of the chloroacetylamino alkanol compound of Formula IX with the reagent of Formula (R 3 ) 2 P-0-R ! is preferably performed in an organic solvent, the selection of which is within the skil of htose in the art.
• the methods of the present invention can further include reacting the activated phosphate compound of formula X with a free hydroxyl group of a nucleoside, a nucleotide, an oligonucleotide, or an oligonucleotide connected to a solid support to form a compound of Formula XI :
• R 9 is a nucleoside, a nucleotide, an oligonucleotide, or an oligonucleotide connected to a solid support.
• this is carried out in according to standard solid phase phosphoramidite synthetic protocols.
• the phosphite compound of Formula XI is then preferably oxidizing or sulfurized to form a compound of Formula XII :
• oligomeric compound is used to refer to compounds containing a plurality of nucleoside monomer subunits that are joined by internucleoside linkages, preferably phosphorus-containing linkages, such as phosphite, phospho- diester, phosphorothioate, and/or phosphorodithioate linkages.
• oligomeric compound or “oligomer” therefore includes naturally occurring oligonucleotides, their analogs, and synthetic oligonucleotides.
• Monomer or higher order synthons having the Formulas described herein include both native (i.e., naturally occurring) and synthetic (e.g., modified native or totally synthetic) nucleosides and nucleotides .
• Methods for coupling compounds of Formula I and Formula IV of the invention include both solution phase and, preferably, solid phase chemistries. Representative solution phase techniques are described in United States Patent No. 5,210,264, which is assigned to the assignee of the present invention. In preferred embodiments, the methods of the present invention are employed for use in iterative solid phase oligonucleotide synthetic regimes.
• a preferred synthetic solid phase synthesis utilizes phosphoramidites as activated phosphate compounds .
• a phosphoramidite monomer is reacted with a free hydroxyl on the growing oligomer chain to produce an intermediate phosphite compound, which is subsequently oxidized to the P v state using standard methods .
• This technique is commonly used for the synthesis of several types of linkages including phosphodiester, phosphorothioate, and phosphorodithioate linkages.
• the first step in such a process is attachment of a first monomer or higher order subunit containing a protected 5 ' -hydroxyl to a solid support, usually through a linker, using standard methods and procedures known in the art. See for example, Oligonucleotides And Analogues A Practical Approach,
• the support-bound monomer or higher order first synthon is then treated to remove the 5 ' - protecting group, typically by treatment with acid.
• the solid support bound monomer is then reacted with a nucleoside phosphoramidite under anhydrous conditions in the presence of an activating agent such as, for example, 1H- tetrazole, 5- (4-nitrophenyl) -lH-tetrazole, or diisopropylamino tetrazolide to form a phosphite triester linkage.
• the phosphite triester linkage is subsequently oxidized or sulfurized.
• Choice of oxidizing or sulfurizing agent will determine whether the linkage will be oxidized or sulfurized to a phosphotriester, thiophosphotriester, or a dithiophosphotriester linkage.
• capping step it is generally preferable to perform a capping step, either prior to or after oxidation or sulfurization of the phosphite triester, thiophosphite triester, or dithiophosphite triester.
• a capping step is generally known to be beneficial by preventing shortened oligomer chains, by blocking chains that have not reacted in the coupling cycle.
• One representative reagent used for capping is acetic anhydride.
• Other suitable capping reagents and methodologies can be found in United States Patent
• a compound of the invention having, for example, Formula I can be introduced into the oligonucleotide chain.
• the methods of the invention provide for the introduction of linked pendant groups at any position in the oligomeric chain.
• the completed oligomer is then cleaved from the solid support.
• the cleavage step which can precede or follow depr ⁇ tection of protected functional groups, will in preferred embodiments yield the completed oligomer free from the solid support, and devoid of all phosphorus protecting groups; that is, during cleavage, the linkages between monomeric subunits are converted from phosphotriester, thiophosphotriester, or dithiophosphotriester linkages to phosphodiester, phosphorothioate, or phosphorodithioate linkages.
• the internucleoside linkages of the oligomeric compounds described herein, represented by moiety Z in the compounds and methods described herein, can be any internucleoside linkage as is known in the art, including phosphorus based linking groups such as phosphite, phosphodiester, phosphorothioate, and phosphorodithioate linkages. Such linkages can be protected, i.e., they can bear, for example, phosphorus protecting groups. As used herein, the term "phosphorus protecting group" is intended to denote protecting groups that are known to be useful to protect phosphorus-containing linkages during oligonucleotide synthesis . One such preferred phosphorus protecting group is the ⁇ - cyanoethyl protecting group .
• the methods of the invention are used for the preparation of oligomeric compounds, preferably oligonucleotides.
• oligonuclotide means compounds that can contain both naturally occurring (i.e. "natural") and non-naturally occurring (“synthetic") moieties, for example, nucleosidic subunits containing modified sugar and/or nucleobase portions. Such oligonucleotide analogs are typically structurally distinguishable from, yet functionally interchangeable with, naturally occurring or synthetic wild type oligonucleotides.
• oligonucleotide analogs include all such structures which function effectively to mimic the structure and/or function of a desired RNA or DNA strand, for example, by hybridizing to a target.
• synthetic nucleoside for the purpose of the present invention, refers to a modified nucleoside. Representative modifications include modification of a heterocyclic base portion of a nucleoside to give a non-naturally occurring nucleobase, a sugar portion of a nucleoside, or both simultaneously.
• nucleobases useful in the compounds and methods described herein include adenine, guanine, cytosine, uridine, and thymine, as well as other non- naturally occurring and natural nucleobases such as xanthine, hypoxanthine, 2-aminoadenine, 6-methyl and other alkyl derivatives of adenine and guanine, 2-propyl and other alkyl derivatives of adenine and guanine, 5-halo uracil and cytosine, 6-azo uracil, cytosine and thymine, 5-uracil (pseudo uracil) , 4-thiouracil, 8-halo, oxa, amino, thiol, thioalkyl, hydroxyl and other 8 -substituted adenines and guanines, 5-trifluoromethyl and other 5-substituted uracils and cytosines, 7-methylguanine .
• nucleobases include those disclosed in U.S. Patent No. 3,687,808 (Merigan, et al . ) , in chapter 15 by Sanghvi, in Antisense Research and Application, Ed. S. T.
• nucleosidic base' is further intended to include heterocyclic compounds that can serve as like nucleosidic bases including certain 'universal bases' that are not nucleosidic bases in the most classical sense but serve as nucleosidic bases. Especially mentioned as a universal base is 3-nitropyrrole .
• Representative 2' sugar modifications (position R x ) amenable to the present invention include fluoro, O-alkyl, O-alkylamino, O-alkylalkoxy, protected O-alkylamino, O-alkylaminoalkyl, O-alkyl imidazole, and polyethers of the formula (O-alkyl) m , where m is 1 to about 10.
• PEGs linear and cyclic polyethylene glycols
• (PEG) -containing groups such as crown ethers and those which are disclosed by Ouchi, et al . , Drug Design and Discovery 1992, 9, 93, Ravasio, et al . , J". Orgr.
• Sugars having 0-substitutions on the ribosyl ring are also amenable to the present invention.
• Representative substitutions for ring 0 include S, CH 2 , CHF, and CF 2 , see, e.g., Secrist, et al . , Abstract 21 , Program & Abstracts,
• alkyl includes but is not limited to straight chain, branch chain, and alicyclic hydrocarbon groups . Alkyl groups of the present invention may be substituted. Representative alkyl substituents are disclosed in United States Patent No. 5,212,295, at column 12, lines 41-50, hereby incorporated by reference in its entirety. As used herein, the term “lower alkyl” is intended to mean alkyl having 6 or fewer carbons .
• alkyl denotes alkyl groups which bear aryl groups, for example, benzyl groups.
• alkaryl denotes aryl groups which bear alkyl groups, for example, methylphenyl groups.
• aryl denotes aromatic cyclic groups including but not limited to phenyl, naphthyl, anthracyl, phenanthryl, and pyrenyl .
• a preferred alkanoyl group is the acetyl group.
• hetero denotes an atom other than carbon, preferably but not exclusively N, 0, or S.
• heterocycloalkyl denotes an alkyl ring system having one or more heteroatoms (i.e., non- carbon atoms) .
• Preferred heterocycloalkyl groups include, for example, morpholino groups.
• heterocycloalkenyl denotes a ring system having one or more double bonds, and one or more heteroatoms.
• Preferred heterocycloalkenyl groups include, for example, pyrrolidino groups .
• R 7 can be a linker connected to a solid support.
• Solid supports are substrates which are capable of serving as the solid phase in solid phase synthetic methodologies, such as those described in Caruthers U.S. Patents Nos . 4,415,732; 4,458,066; 4,500,707; 4,668,777; 4,973,679; and 5 , 132 , 418 ; and Koster U.S. Patents Nos. 4,725,677 and Re. 34,069.
• Linkers are known in the art as short molecules which serve to connect a solid support to functional groups (e.g., hydroxyl groups) of initial synthon molecules in solid phase synthetic techniques. Suitable linkers are disclosed in, for example, Oligonucleotides And Analogues A Practical Approach, Ekstein, F. Ed., IRL Press, N.Y, 1991, Chapter 1, pages 1-23.
• Solid supports according to the invention include those generally known in the art to be suitable for use in solid phase methodologies, including, for example, controlled pore glass (CPG) , oxalyl-controlled pore glass (see, e . g. , Alul, et al . , Nucleic Acids Research 1991, 19,
• TentaGel Support an aminopolyethyleneglycol derivatized support ( see, e . g. , Wright, et al . , Tetrahedron Letters 1993, 34 , 3373, hereby incorporated by reference in its entirety) and Poros -- a copolymer of polystyrene/divinylbenzene .
• R 7 or R 5 can be a hydroxyl protecting group.
• hydroxyl protecting groups can be employed in the methods of the invention.
• the protecting group is stable under basic conditions but can be removed under acidic conditions.
• protecting groups render chemical functionalities inert to specific reaction conditions, and can be appended to and removed from such functionalities in a molecule without substantially damaging the remainder of the molecule.
• Representative hydroxyl protecting groups are disclosed by Beaucage, et al . , Tetrahedron 1992, 48, 2223-
• R 7 or R 5 Preferred protecting groups used for R 7 or R 5 include dimethoxytrityl (DMT) , monomethoxytrityl, 9-phenylxanthen-9-yl (Pixyl) and 9- (p- methoxyphenyl)xanthen-9-yl (Mox) .
• DMT dimethoxytrityl
• Mox 9-phenylxanthen-9-yl
• the R 7 or R 5 group can be removed from oligomeric compounds of the invention by techniques well known in the art to form the free hydroxyl.
• dimethoxytrityl protecting groups can be removed by protic acids such as formic acid, dichloroacetic acid, trichloroacetic acid, p-toluene sulphonic acid or with Lewis acids such as for example zinc bromide. See for example, Greene and Wuts, supra .
• amino groups are appended to alkyl or to other groups such as, for example, to 2 ' -alkoxy groups.
• Such amino groups are also commonly present in naturally occurring and non- naturally occurring nucleobases. It is generally preferred that these amino groups be in protected form during the synthesis of oligomeric compounds of the invention.
• Representative amino protecting groups suitable for these purposes are discussed in Greene and Wuts, Protective Groups in Organic Synthesis, Chapter 7, 2d ed, John Wiley & Sons,
• the term "protected” when used in connection with a molecular moiety such as “nucleobase” indicates that the molecular moiety contains one or more functionalities protected by protecting groups .
• Sulfurizing agents used during oxidation to form phosphorothioate and phosphorodithioate linkages include Beaucage reagent (see e . g. Iyer, R.P., et.al., J. Chem . Soc , 1990, 112 , 1253-1254, and Iyer, R.P., et . al . , J. Org. Chem . , 1990, 55, 4693-4699); tetraethylthiuram disulfide
• Useful oxidizing agents used to form the phosphodiester or phosphorothioate linkages include iodine/tetrahydrofuran/ water/pyridine or hydrogen peroxide/water or tert-butyl hydroperoxide or any peracid like m-chloroperbenzoic acid.
• sulfurization the reaction is performed under anhydrous conditions with the exclusion of air, in particular oxygen whereas in the case of oxidation the reaction can be performed under aqueous conditions .
• Oligonucleotides or oligonucleotide analogs according to the present invention are preferably hybridizable to a specific target preferably comprise from about 5 to about 50 monomer subunits.
• Such compounds comprise from about 10 to about 30 monomer subunits, with 15 to 25 monomer subunits being particularly preferrred.
• smaller oligomeric compounds are preferred.
• Libraries of dimeric, trimeric, or higher order compounds of general Formula II can be prepared for use as synthons in the methods of the invention. The use of small sequences synthesized via solution phase chemistries in automated synthesis of larger oligonucleotides enhances the coupling efficiency and the purity of the final oligonucloetides . See for example: Miura, K., et al., Chem . Pharm . Bull . ,
• the compounds of the invention are used to modulate RNA or DNA, which code for a protein whose formation or activity it is desired to modulate.
• the targeting portion of the composition to be employed is, thus, selected to be complementary to the preselected portion of DNA or RNA, that is to be hybridizable to that portion.
• the oligomeric compounds of the invention can be used in diagnostics, therapeutics and as research reagents and kits. They can be used in pharmaceutical compositions by including a suitable pharmaceutically acceptable diluent or carrier. They further can be used for treating organisms having a disease characterized by the undesired production of a protein. The organism should be contacted with an oligonucleotide having a sequence that is capable of specifically hybridizing with a strand of nucleic acid coding for the undesirable protein. Treatments of this type can be practiced on a variety of organisms ranging from unicellular prokaryotic and eukaryotic organisms to multicellular eukaryotic organisms.
• Any organism that utilizes DNA-RNA transcription or RNA-protein translation as a fundamental part of its hereditary, metabolic or cellular control is susceptible to therapeutic and/or prophylactic treatment in accordance with the invention. Seemingly diverse organisms such as bacteria, yeast, protozoa, algae, all plants and all higher animal forms, including warmblooded animals, can be treated. Further, each cell of multicellular eukaryotes can be treated, as they include both DNA-RNA transcription and RNA-protein translation as integral parts of their cellular activity. Furthermore, many of the organelles (e . g. , mitochondria and chloroplasts) of eukaryotic cells also include transcription and translation mechanisms. Thus, single cells, cellular populations or organelles can also be included within the definition of organisms that can be treated with therapeutic or diagnostic oligonucleotides.
• organelles e g. , mitochondria and chloroplasts
• compounds of the invention comprising the haloacetyl linker include those having the Formula V:
• R 2 is halogen which is preferably chlorine, or R 2 is a pendant group
• R 10 is a nucleobase, a nucleoside, a nucleotide, an activated nucleotide, an oligonucleotide, an oligonucleotide connected to a solid support, or a moiety of Formula VI :
• R ⁇ is H or a phosphorus protecting group
• R 3 is -N(R 4 ) 2 , or a heterocycloalkyl or heterocycloalkenyl ring containing from 4 to 7 atoms, and having up to 3 heteroatoms selected from nitrogen, sulfur, and oxygen;
• R 4 is straight or branched chain alkyl having from 1 to 10 carbons; and n is from 1 to about 10.
• v and q are each 0 or 1, provided that the sum of v and q is not 0; q is 0 or 1;
• R 5 is a hydroxyl protecting group
• B is a nucleobase
• R 6 is F, 0-R 20 , S-R 20 or N-R 20 (R 21 )
• R 20 is alkyl, or a ring system having from about 4 to about 7 carbon atoms or having from about 3 to about 6 carbon atoms and 1 or 2 hetero atoms wherein said hetero atoms are selected from oxygen, nitrogen and sulfur and wherein said ring system is aliphatic, unsaturated aliphatic, aromatic or heterocyclic; and wherein any available hydrogen atom of said ring system is each replaceable with an alkoxy, alkylammo, urea or alkylurea group; or has one of the formulas :
• Oligonucleotide synthesis was performed on an ABI 380B DNA Synthesizer using phosphoramidite chemistry, standard ancillary reagents, cycles, and procedures.
• 3H-1, 2-benzodithiol-3- one 1,1-dioxide (0.05 M in MeCN) was employed as the sulfur- transfer reagent.
• Phosphoramidite 1 (0.2 M in MeCN) was attached at the 5 '-terminus on the last coupling step. Both capping subroutine and detritylation are neither required nor recommended as part of the last synthetic cycle.
• the solid support bound oligonucleotide was briefly dried on an oil pump and subjected to the attachment of a pendant group.
• Solid support bound oligonucleotides were treated with amines as shown in Figure 2, under the conditions specified in the Table 1, below.
• the reaction mixture was diluted with cone, ammonia and left at room temperature for 2 hours. The solution was collected and evaporated to dryness . The residue was dissolved in water (2 mL) and treated depending on the reagent employed for the derivatization.
• 5b, 7b, and 8b solutions were neutralized with Dowex 50wx8 (PyH + ) , filtered, and analyzed by HPLC.
• the emulsion was extracted with CH 2 C1 2 (5 x 0.5 mL) , filtered, and analyzed by HPLC. For 3a, 4a, and 4b no special treatment was required. The solutions were filtered and subjected to HPLC.
• Solid support bound oligonucleotides were treated with mercaptans as shown in Figure 3 under the conditions specified in the Table 1.
• the solid support was washed with dioxane (5 x 1 mL) , concentrated aqueous ammonia was added, and the mixture was left for 2 hours at room temperature. The solution was collected and evaporated to dryness . The residue was dissolved in water (2 mL) , filtered and analyzed by HPLC.
• Crude oligonucleotides were analyzed on a DeltaPak 15m C18 300 HPLC column (3.8 x 300 mm) eluted with linear gradients a) for 3a, 4a, and 10a from 0 to 60% B in 40 minutes (0.1 M NH 4 OAc as buffer A; 0.1 M NH 4 OAc in 50 % aq MeCN as buffer B) ; b) for 4b, 5b, 7b-10b from 0 to 25% B in
• compounds of Formula I wherein v is 1 and Q has the Formula II can be prepared by reacting a tri (2-oxyethylphthalimido)borate with a 2,2'- anhydronucleoside. This is shown for the synthesis of 2'-0- aminoethyl-5 ' -O-DMT-5-methyluridine (15) in Examples 5-8 below, and in Figure 4.
• reaction mixture is stirred at 0°C for 2 hours and tested for completion of reaction by TLC (CH 2 C1 2 :CH 3 0H 9:1) .
• TLC TLC
• the reaction is complete and the reaction mixture is applied to silica gel equilibrated with CH 2 C1 2 :CH 3 0H 9:1 and eluted with the same to yield the desired compound.
• Detritylation with aqueous 80% acetic acid and evaporation, followed by desalting in a Sephadex G-25 column give modified oligonucleotides. Oligonucleotides were analyzed by HPLC,
• Compound 20 (192mg, 0.2mmol) is dissolved in 2mL of anhydrous acetonitrile and loaded onto an Expedite Nucleic Acid Synthesis system (Millipore) to synthesize the oligonucleotides.
• the amidite concentration is 0.1M.
• the coupling efficiencies are more than 95%.
• For the coupling of the amidite 20 coupling time is extended to 10 min. and this step is carried out twice. All other steps in the protocol supplied by Millipore are used except the extended coupling time for 20.
• the oligomers are conjugated to desired amines or thiols in the controlled pore glass (CPG) supports and deprotected under standard conditions using concentrated aqueous NH 4 OH (30%) AT 55°C. 5 ' -O-DMT-containing oligomers are then purified by reverse phase high performance liquid chromatography as in the previous case .
• the reaction mixture was stirred at 100°C temperature for 12 hours and evaporated to dryness.
• the residue was dissolved in methanol (150 mL) and cooled to 0°C.
• the precipitated solid was filtered and dried.
• the nucleoside 23 (8.32 g, 10.0 mmol) is dissolved in dry pyridine (75 mL) and allowed to stir at room temperature. To this cold stirred solution is added 0.5M tetrabutylammonium fluoride (80 mL, 40 mmol, prepared in py:THF:H 2 0; 5:4:1) at once . The reaction mixture is stirred at room temperature for 15 minutes, the pH is adjusted to 7 with H + resin. The reaction is filtered, washed with methanol (50 mL) and the filtrate evaporated to dryness.
• the residue is dissolved in CH C1 2 (150 mL) , washed wtih water (50 mL) and brine (50 mL) .
• the organic extract is dried over anhydrous MgS ⁇ 4 and evaporated to dryness.
• the residue is purified by flash chromatography over silica gel using CH 2 C1 2 --> MeOH as the eluent. The pure fractions having the pure product are collected and evaporated to give the desired product .
• the nucleoside 24 (5.90 mmol) is dissolved in dry pyridine (30 mL) and evaporated to dryness.
• the dried compound is dissolved in dry pyridine (100 mL) and treated with triethylamine (1.01 g, 10 mmol) under argon atmosphere.
• To this stirred solution is added 4, 4 ' -dimethoxytrityl chloride (2.59 g, 7.67 mmol) and the stirring is continued at room temperature for 6 hours.
• the reaction mixture is quenched with methanol (20 mL) , stirred for 10 minutes and evaporated to dryness.
• the nucleoside 25 (4.3 g, 4.70 mmol) is dissolved in dry pyridine (30 mL) and evaporated to dryness. This is repeated three times to remove last traces of water and dried over solid sodium hydroxide overnight. Then it is dissolved in dry dichloromethane (100 mL) and cooled to 0°C under argon atmosphere. To this cold stirred solution is added N,N- diisopropylethylamine (1.29 g, 10 mmol) followed by ( ⁇ - cyanoethoxy) chloro (N, N-diisopropylamino) phosphane (2.36 g, 10 mmol) dropwise over a period of 15 minutes.
• the reaction mixture is stirred at 0°C for 1 hour and at room temperature for 2 hours.
• the reaction mixture is diluted with dichloromethane (100 mL) , washed with 5% NaHC ⁇ 3 solution (50 mL) , water (50 mL) and brine (50 mL) .
• the organic extract is dried over anhydrous MgS ⁇ 4 and the solvent is removed under reduced pressure.
• the residue is purified by flash chromatography over silica gel using CH 2 C1 2 --> EtOAc containing 1% triethylamine as the eluent. The main fractions are collected and evaporated to dryness.
• the phosphoramidite compound 5 ' -Dimethoxytrityl-N 2 - (N- N-chloroacetylpropylamino-2 ' -deoxyguonosine-3 ' -0- [2- cyanoethyl-N,N-diisopropyl] phosphoramidite, is utilized in the DNA synthesizer as a 0.2M solution in anhydrous CH3CN.
• Oligonucleotide synthesis is carried out in either an ABI 380B or an ABI 394 synthesizer employing the standard synthesis cycles with an extended coupling time of 10 minutes during coupling of the modified amidite into the oligonucleotide sequence. Coupling efficiency of greater than 90% is observed.
• G* represents a nucleotide functionalized to incorporate a N2- (3-N-chloroacetyl-propylamine) functionality.
• Oligomers 1 and 2 are antisense compounds targeted against the human ICAM-1 (Inter Cellular Adhesion Molecule-1) .
• oligonucleotides are synthesized as per the previous method except during the synthesis, for oxidation of the phosphite moieties, the Beaucage reagent (i.e., 3H-1, 2-benzodithioate-3-one 1,1- dioxide, see, Iyer,R. P., et al . , J. Am . Chem . Soc . 1990, 112 , 1253) is used as a 0.24 M solution in anhydrous CH3CN solvent.
• the oligonucleotides were synthesized in the "Trityl-On" mode and purified by reverse phase HPLC.
• the chloroacetyl tether in N2- position is functionalized with amines and thiols as described for the aminohexanol tether .

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