Peptide nucleic acids

Abstract

A novel class of compounds, known as peptide nucleic acids, bind complementary ssDNA and RNA strands more strongly than a corresponding DNA. The peptide nucleic acids generally comprise ligands such as naturally occurring DNA bases attached to a peptide backbone through a suitable linker.

What is claimed is:  
     
         1 . A compound comprising a polyamide backbone bearing a plurality of ligands that are individually bound to aza nitrogen atoms located within said backbone, at least one of said ligands being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group.  
     
     
         2 . The compound of  claim 1  wherein said aza nitrogen atoms are separated from one another in said backbone by from 4 to 6 intervening atoms.  
     
     
         3 . A compound having the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 n is at least 2,  
 each of L 1 -L n  is independently selected from the group consisting of hydrogen, hydroxy, (C 1 -C 4 )alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, heterocyclic moieties, and reporter ligands, at least one of L 1 -L n  being a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group;  
 each of A 1 -A n  is a single bond, a methylene group or a group of formula:  
                     
 where:  
 X is O, S. Se, NR 3 , CH 2  or C(CH 3 ) 2 ;  
 Y is a single bond, O, S or NR 4 ;  
 each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10;  
 each of r and 5 is zero or an integer from 1 to 5, the sum r+s being not more than 10;  
 each R 1  and R 2  is independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl which may be hydroxy- or alkoxy- or alkylthio-substituted, hydroxy, alkoxy, alkylthio, amino and halogen; and  
 each R 3  and R 4  is independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C 1 -C 4 )alkyl, hydroxy, alkoxy, alkylthio and amino;  
 each of B 1 -B n  is N or R 3 N + , where R 3  is as defined above;  
 each of C 1 -C n  is CR 6 R 7 , CHR 6 CHR 7  or CR 6 R 7 CH 2 , where R 6  is hydrogen and R 7  is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R 6  and R 7  are independently selected from the group consisting of hydrogen, (C 2 -C 6 )alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkylthio, NR 3 R 4  and SR 5 , where R 3  and R 4  are as defined above, and R 5  is hydrogen, (C 1 -C 6 )alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C 1 -C 6 )alkyl, or R 6  and R 7  taken together complete an alicyclic or heterocyclic system;  
 each of D 1 -D n  is CR 6 R 7 , CH 2 CR 6 R 7  or CHR 6 CHR 7 , where R 6  and R 7  are as defined above;  
 each of G 1 -G n−1  is —NR 3 CO—, —NR 3 CS—, —NR 3 SO— or —NR 3 SO 2 —, in either orientation, where R 3  is as defined above;  
 Q is —CO 2 H, —CONR′R″, —SO 3 H or —SO 2 NR′R″ or an activated derivative of —CO 2 H or —SO 3 H; and  
 I is —NHR′″R′″ or —NR′″C(O)R″″, where R′, R″, R′″ and R″″ are independently selected from the group consisting of hydrogen, alkyl, amino prot cting groups, r porter ligands, intercalators, chelators, peptides, proteins, carbohydrates, lipids, steroids, oligonucleotides and soluble and non-soluble polymers.  
 
     
     
         4 . The compound of  claim 3  having the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 each L is independently selected from the group consisting of hydrogen, phenyl, heterocyclic moieties, naturally occurring nucleobases, and non-naturally occurring nucleobases;  
 each R 7′  is independently selected from the group consisting of hydrogen and the side chains of naturally occurring alpha amino acids;  
 n is an integer from 1 to 60,  
 each k and m is, independently, zero or 1;  
 each 1 is zero or an integer from 1 to 5;  
 R h  is OH, NH 2  or —NHLysNH 2 ; and  
 R f  is H or COCH 3 .  
 
     
     
         5 . The compound of  claim 4  having formula:  
       
         
           
           
               
               
           
         
         ach L is independently selected from th group consisting of the nucleobases thymin, ad nine, cytosine, guanine, and uracil;  
         each R 7′  is hydrogen; and  
         n is an integer from 1 to 30.  
       
     
     
         6 . A compound having one of the following formulas:  
       
         
           
           
               
               
           
         
       
       wherein: 
 L is selected from the group consisting of hydrogen, hydroxy, (C 1 -C 4 ) alkanoyl, naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, and heterocyclic moieties, reporter ligands, wherein: 
 at least one of L 1 -L n  is a naturally occurring nucleobase, a non-naturally occurring nucleobase, a DNA intercalator, or a nucleobase-binding group; and  
 amino groups are, optionally, protected by amino protecting groups;  
 
 A is a single bond or a group of the formula:  
                     
 where:  
 X is O, S. Se, NR 3 , CH 2  or C(CH 3 ) 2 ;  
 Y is a single bond, O, S or NR 4 ;  
 each of p and q is zero or an integer from 1 to 5, the sum p+q being not more than 10;  
 each of r and s is zero or an int ger from 1 to 5, the sum r+s being not m re than 10;  
 each R 1  and R 2  is independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C 1 -C 4 )alkyl, hydroxy, alkoxy, alkylthio, amino and halogen; and  
 each R 3  and R 4  is independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C 1 -C 4 ) alkyl, hydroxy, alkoxy, alkylthio and amino;  
 B is N or R 3 N + , where R 3  is as defined above;  
 each C is CR 6 R 7 , CHR 6 CHR 7  or CR 6 R 7 CH 2 , where R 6  is hydrogen and R 7  is selected from the group consisting of the side chains of naturally occurring alpha amino acids, or R 6  and R 7  are independently selected from the group consisting of hydrogen, (C 2 -C 6 )alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C 1 -C 6 )alkoxy, (C 1 -C 6 )alkylthio, NR 3 R 4  and SR 5 , where R 3  and R 4  are as defined above, and R 5  is hydrogen or (C 1 -C 6 )alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C 1 -C 6 )alkyl, or R 6  and R 7  taken together complete an alicyclic or heterocyclic system;  
 each D is CR 6 R 7 , CH 2 CR 6 R 7  or CHR 6 CHR 7 , where R 6  and R 7  are as defined above;  
 each E is COOH, CSOH, SOOH, SO 2 OH or an activated or protected derivative thereof; and  
 each F is NHR 3  or NPgR 3 , where R 3  is as defined above, and Pg is an amino protecting group.  
 
     
     
         7 . Th compound of  claim 6  having the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 each L is independently selected from the group consisting of hydrogen, phenyl, heterocyclic moieties, naturally occurring nucleobases, and non-naturally occurring nucleobases;  
 each R 7′  is independently selected from the group consisting of hydrogen and the side chains of naturally occurring alpha amino acids; and  
 each k, l, and m is, independently, zero or an integer from 1 to 5.  
 
     
     
         8 . The compound of  claim 7  having formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 L is selected from the group consisting of the nucleobases thymine, adenine, cytosine, guanine, uracil, 5-methylcytosine, 6-thioguanine and 5-bromouracil, and protected derivatives thereof;  
 R 7′  is hydrogen;  
 E is COOH or an activated or protected derivative th re f; and  
 F is NH 2  or NHPg, where Pg is an amino protecting group.  
 
     
     
         9 . A compound having the formula:  
       
         
           
           
               
               
           
         
       
       wherein: 
 each L is independently selected from the group consisting of hydrogen, phenyl, heterocyclic moieties, naturally occurring nucleobases, and non-naturally occurring nucleobases;  
 each R 7′  is independently selected from the group consisting of hydrogen and the side chains of naturally occurring alpha amino acids;  
 n is an integer from 1 to 60, each k, l, and m is, independently, zero or an integer from 1 to 5;  
 R h  is OH, NH 2  or —NHLysNH 2 ; and  
 R f  is H or COCH 3 .  
 
     
     
         10 . A process for preparing a compound according to  claim 1 , comprising the steps of: 
 A) providing a polymer substrate, said polymer being functionalized with a chemical group capable of forming an anchoring linkage with an amino acid;    B) coupling said polymer with a first amino acid through said anchoring linkage, said first amino acid having formula (IV):                          wherein:    L is selected from the group consisting of naturally occurring nucleobases, non-naturally occurring nucleobases, aromatic moieties, DNA intercalators, nucleobase-binding groups, heterocyclic moieties, and reporter ligands, wherein amino groups are, optionally, protected by amino protecting groups;    A is a single bond or a group of the formula:                          where:    X is O, S, Se, NR 3 , CH 2  or C(CH 3 ) 2 ;    Y is a single bond, O, S or NR 4 ;    p and q are zero or integers from 1 to 5, the sum p+q being not more than 10;    r and s are zero or integers from 1 to 5, the sum r+s being not more than 10;    R 1  and R 2  are independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C 1 -C 4 ) alkyl, hydroxy, alkoxy, alkylthio, amino and halogen; and    R 3  and R 4  are independently selected from the group consisting of hydrogen, (C 1 -C 4 )alkyl, hydroxy- or alkoxy- or alkylthio-substituted (C 1 -C 4 )alkyl, hydroxy, alkoxy, alkylthio and amino;    B is N or R 3 N + , where R 3  is as defined above;    C is CR 6 R 7 , CHR 6 CHR 7  or CR 6 R 7 CH 2 , where R 6  is hydrogen and R 7  is selected from the group consisting f the side chains of naturally occurring alpha amino acids, or R 6  and R 7  are independently s lected from the group consisting of hydrogen, (C 2 -C 6 )alkyl, aryl, aralkyl, heteroaryl, hydroxy, (C 1 -C 6 ) alkoxy, (C 1 -C 6 )alkylthio, NR 3 R 4  and SR 5 , where R 3  and R 4  are as defined above, and R 5  is hydrogen or (C 1 -C 6 )alkyl, hydroxy-, alkoxy-, or alkylthio-substituted (C 1 -C 6 )alkyl, or R 6  and R 7  taken together complete an alicyclic or heterocyclic system;    D is CR 6 R 7 , CH 2 CR 6 R 7  or CHR 6 CHR 7 , where R 6  and R 7  are as defined above;    E is COOH or an activated or protected derivative thereof; and    F is NPgR 3  where R 3  is as defined above and Pg is an amino protecting group;    C) removing said amino protecting group from said coupled first amino acid to generate a free amino group; and    D) reacting said free amino group with a second amino acid having formula (IV) to form a peptide chain.    
     
     
         11 . The process of  claim 10  further comprising the steps of: 
 E) removing said amino protecting group from said second amino acid to generate a terminal free amino group on said peptide chain; and  
 F) reacting said free amino group on said peptide chain with a further amino acid having formula (IV) to lengthen said peptide chain.  
 
     
     
         12 . The process of  claim 11  wherein steps E and F are performed a plurality of times.  
     
     
         13 . The process of  claim 11  further comprising removing at least one protecting group remaining on the amino acid moieties of the peptide chain.  
     
     
         14 . The process of  claim 10  further comprising cleaving said anchoring linkage without substantially degrading said peptide chain.  
     
     
         15 . The process of  claim 10  wherein the polymer substrate contains polystyrene, polyacrylamide, silica, a composite material, cotton, or a derivative thereof.  
     
     
         16 . The process of  claim 10  wherein the chemical group capable of forming said anchoring linkage is chloro-, bromo- and iodo-substituted alkyl, amino-substituted alkyl, amino and aryl-substituted alkyl, amino- and alkylaryl-substituted alkyl, hydroxy-substituted alkyl, or a derivative thereof having a spacer group that can be cleaved substantially without degradation of said polypeptide.  
     
     
         17 . The process of  claim 16  wherein chloro-substituted alkyl is chloromethyl, amino-substituted alkyl is aminomethyl, amino- and alkyl-substituted aryl is α-aminobenzyl, amino- and alkylaryl-substituted alkyl is selected from the group consisting of α-amino-3- and α-amino-4-methylbenzyl, and hydroxy-substituted alkyl is hydroxymethyl.  
     
     
         18 . The process of  claim 16  wherein: 
 the chemical group is derived from an amino-containing moiety-selected from amino-substituted alkyl, amino- and aryl substituted alkyl, and amino- and alkylaryl-substituted alkyl; and  
 the chemical group includes a spacer group derived from the group consisting of 4-(haloalkyl)aryl-lower alkanoic acids, Boc-aminoacyl-4-(oxymethyl)aryl-lower alkanoic acids, N-Boc-p-acylbenzhydrylamines, N-Boc-4′-(lower alkyl)-p-acylbenzhydrylamines, N-Boc-4′-(lower alkoxy)-p-acylbenzhydrylamines, and 4-hydroxymethylphenoxy-lower alkanoic acids.  
 
     
     
         19 . A proc ss for sequence-sp cific r cognition of a d uble-stranded polynucleotide, comprising contacting said polynucleotide with a compound that is different from natural RNA and that binds to one strand of th polynucleotide, thereby displacing the other strand.  
     
     
         20 . The process of  claim 19  wherein said compound is an oligomer comprising a homogenous or heterogenous backbone to which are linked naturally occurring nucleobases, non-naturally occurring nucleobases or other ligands that individually bind by hydrogen bonding to at least one natural nucleobase in said polynucleotide strand.  
     
     
         21 . The process of  claim 20  wherein said compound is the compound of  claim 1 .  
     
     
         22 . The process of  claim 20  wherein said compound is the compound of  claim 4 .  
     
     
         23 . A process for modulating the expression of a gene in an organism, comprising administering to said organism a compound according to  claim 1  that specifically binds to DNA or RNA deriving from said gene.  
     
     
         24 . The process of  claim 23  wherein said compound is the compound of  claim 1 .  
     
     
         25 . The process of  claim 23  wherein said compound is the compound of  claim 4 .  
     
     
         26 . The process of  claim 23  wherein said modulation includes inhibiting transcription of said gene.  
     
     
         27 . The process of  claim 23  wherein said modulation includ s inhibiting r plication of said g n.  
     
     
         27 A. A process for modulating the activity of a double stranded polynucleotid, comprising contacting said polynucleotide with a compound that is different from natural RNA and that binds to one strand ofe polynucleotide, thereby displacing the other strand  
     
     
         28 . A process for treating conditions associat d with undesired protein production in an organism, comprising contacting said organism with an effective amount of a compound according to  claim 1  that specifically binds with DNA or RNA deriving from a gene controlling said protein production.  
     
     
         29 . The process of  claim 28  wherein said compound is the compound of  claim 1 .  
     
     
         30 . The process of  claim 28  wherein said compound is the compound of  claim 4 .  
     
     
         31 . A process for inducing degradation of DNA or RNA in cells of an organism, comprising administering to said organism a compound according to  claim 1  that specifically binds to said DNA or RNA.  
     
     
         32 . A process for killing cells or virus, comprising contacting said cells or virus with a compound according to  claim 1  that specifically binds to a portion of the genome of said cells or virus.  
     
     
         33 . A pharmaceutical composition comprising a compound according to  claim 1  and at least one pharmaceutically effective carrier, binder, thickener, diluent, buffer, preservative, or surface active agent.