Oligonucleotide analogues having modified intersubunit linkages and/or terminal groups

1. A process for preparing an oligomer, wherein the process comprises:

(a) deprotecting the ring nitrogen of a solid-phase-supported morpholino subunit of structure (XXXI);

wherein Formula (XXXI) is

or a salt, stereoisomer or tautomer thereof, wherein;

W is, at each occurrence, independently S or O;

X is, at each occurrence, independently —NR 8 R 9 or —OR 3 ;

Y is, at each occurrence, independently O or -NR 10 ;

R 3 is, at each occurrence, independently hydrogen or C 1 -C 6 alkyl;

R 4 is, at each occurrence, independently hydrogen, methyl, —C(═NH)NH 2 , -Z-L-NHC(═NH)NH 2 or -[C(O)CHR′NH] m H, where Z is carbonyl (C(O)) or a direct bond, R′ is a side chain of a naturally occurring amino acid or a one- or two-carbon homolog thereof, and m is 1 to 6;

R 5 is, at each occurrence, independently hydrogen, methyl or an electron pair;

R 7 is, at each occurrence, independently hydrogen C 1 -C 6 alkyl or C 1 -C 6 alkoxyalkyl;

R 8 is, at each occurrence, independently hydrogen or C 2 -C 12 alkyl;

R 9 is, at each occurrence, independently hydrogen, C 1 -C 12 alkyl, C 1 -C 12 aralkyl or aryl;

R 10 is, at each occurrence, independently hydrogen, C 1 -C 12 alkyl or -LNR 4 R 5 R 7 ;

L is an optional linker up to 18 atoms in length comprising alkyl, alkoxy or alkylamino groups, or combinations thereof;

wherein, R 8 and R 9 may join to form a 5-18 membered mono or bicyclic heterocycle or R 8 , R 9 or R 3 may join with R 10 to form a 5-7 membered heterocycle,

and wherein when X is 4-piperazino, X has the following structure (III):

wherein;

R 11 is, at each occurrence, independently C 2 -C 12 alkyl, C 1 -C 12 aminoalkyl, C 1 -C 12 alkylcarbonyl, aryl, heteroaryl or heterocyclyl;

R is, at each occurrence, independently an electron pair, hydrogen or C 1 -C 12 alkyl;

R 12 is, at each occurrence, independently, hydrogen, C 1 -C 12 alkyl, C 1 -C 12 aminoalkyl, —NH 2 , —CONH 2 , —NR 13 R 14 , —NR 13 R 14 R 15 , C 1 -C 12 alkylcarbonyl, oxo, —CN, trifluoromethyl, amidyl, amidinyl, amidinylalkyl, amidinylalkylcarbonyl guanidinyl, guanidinylalkyl, guanidinylalkylcarbonyl, cholate, deoxycholate, aryl, heteroaryl, heterocycle, —SR 13 or C 1 -C 12 alkoxy, wherein R 13 , R 14 and R 15 are, at each occurrence, independently C 1 -C 12 alkyl;

Z is a linkage to a solid support;

B is a base-pairing moiety; and

PG is C 7 -C 30 aralkyl;

(b) reacting the product of step (a) with a morpholino subunit of structure (XXXI) wherein Z is halo;

(c) repeating n-2 iterations of steps (a) and (b), wherein n is 10 to 40; and

(d) cleaving the product of steps (a), (b), and (c) from the solid-phase support,

wherein the oligomer is an oligomer comprising a backbone, the backbone comprising a sequence of morpholino ring structures joined by intersubunit linkages, the intersubunit linkages joining a 3′-end of one morpholino ring structure to a 5′-end of an adjacent morpholino ring structure, wherein each morpholino ring structure is bound to a base-pairing moiety, such that the oligomer can bind in a sequence-specific manner to a target nucleic acid, wherein the intersubunit linkages have the following general structure (I):

or a salt, stereoisomer or tautomer thereof, and wherein each of the intersubunit linkages (I) are independently linkage (A) or linkage (B):

wherein for linkage (A):

W is, at each occurrence, independently S or O;

X is, at each occurrence, independently —N(CH 3 ) 2 , —NR 1 R 2 , —OR 3 or

Y is, at each occurrence, independently O or —NR 2 ;

R 1 is, at each occurrence, independently hydrogen or methyl;

R 2 is, at each occurrence, independently hydrogen or -LNR 4 R 5 R 7 ;

R 3 is, at each occurrence, independently hydrogen or C 1 -C 6 alkyl;

R 4 is, at each occurrence, independently hydrogen, methyl, —C(═NH)NH 2 , -Z-L-NHC(═NH)NH 2 or -[C(O)CHR′NH m ]H, where Z is carbonyl(C(O)) or a direct bond, R′ is a side chain of a naturally occurring amino acid or a one- or two-carbon homolog thereof, and m is 1 to 6;

R 5 is, at each occurrence, independently hydrogen, methyl or an electron pair;

R 6 is, at each occurrence, independently hydrogen or methyl;

R 7 is, at each occurrence, independently hydrogen C 1 -C 6 alkyl or C 1 -C 6 alkoxyalkyl;

L is an optional linker up to 18 atoms in length comprising alkyl, alkoxy or alkylamino groups, or combinations thereof; and

wherein for linkage (B):

W is, at each occurrence, independently S or O;

X is, at each occurrence, independently —NR 8 R 9 or —OR 3 ; and

Y is, at each occurrence, independently O or —NR 10 ,

R 8 is, at each occurrence, independently hydrogen or C 2 -C 12 alkyl;

R 9 is, at each occurrence, independently hydrogen, C 1 -C 12 alkyl, C 1 -C 12 aralkyl or aryl;

R 10 is, at each occurrence, independently hydrogen, C 1 -C 12 alkyl or -LNR 4 R 5 R 7 ;

wherein R 8 and R 9 may join to form a 5-18 membered mono or bicyclic heterocycle or R 8 , R 9 or R 3 may join with R 10 to form a 5-7 membered heterocycle, and wherein when X is 4-piperazinyl, X has the following structure (III):

wherein:

R 11 is, at each occurrence, independently C 7 -C 12 alkyl, C 1 -C 12 aminoalkyl, C 1 -C 12 alkylcarbonyl, wherein alkyl is not substituted with amidinyl or guanidinyl, aryl, heteroaryl or heterocyclyl; and

R is, at each occurrence, independently an electron pair, hydrogen or C 1 -C 12 alkyl; and

R 12 is, at each occurrence, independently, hydrogen, C 1 -C 12 alkyl, C 1 -C 12 aminoalkyl, —NH 2 , —CONH 2 , —NR 13 R 14 , —NR 13 R 14 R 15 , C 1 -C 12 alkylcarbonyl, oxo, —CN, trifluoromethyl, amidyl, amidinyl, amidinylalkyl, amidinylalkylcarbonyl guanidinyl, guanidinylalkyl, guanidinylalkylcarbonyl, cholate, deoxycholate, aryl, heteroaryl, heterocycle, —SR 13 or C 1 -C 12 alkoxy, wherein R 13 , R 14 and R 15 are, at each occurrence, independently C 1 -C 12 alkyl; and

wherein at least one of the intersubunit linkages is linkage (B) wherein: i) R 8 and R 9 join to form a 5-18 membered mono or bicyclic heterocycle; or ii) R 8 , R 9 or R 3 join with R 10 to form a 5-7 membered heterocycle.

2. The process of claim 1 , wherein Z of step (b) is chloro.

3. The process of claim 1 , wherein the PG is trityl or methoxy trityl.

4. The process of 1 , wherein at least one of the intersubunit linkages is linkage (A), and wherein X is —N(CH 3 ) 2 at each occurrence of linkage (A).

5. The process of claim 1 , wherein at least one linkage (B) has the following structure (IV):

wherein Z represents a 5-18 membered mono or bicyclic heterocycle.

6. The process of claim 1 , wherein the oligomer has the following structure (XVII):

or a salt, stereoisomer or tautomer thereof, wherein:

R 17 is, at each occurrence, independently absent, hydrogen or C 1 -C 6 alkyl;

R 18 and R 19 are, at each occurrence, independently absent, hydrogen, a cell-penetrating peptide, a natural or non-natural amino acid, C 2 -C 30 alkylcarbonyl, —C(═O)OR 21 or R 20 ;

R 20 is, at each occurrence, independently guanidinyl, heterocyclyl, C 1 -C 30 alkyl, C 3 -C 8 cycloalkyl; C 6 -C 30 aryl, C 7 -C 30 aralkyl, C 3 -C 30 alkylcarbonyl, C 3 -C 08 cycloalkylcarbonyl, C 3 -C 08 cycloalkylalkylcarbonyl, C 7 -C 30 arylcarbonyl, C 7 -C 30 aralkylcarbonyl, C 2 -C 30 alkyloxycarbonyl, C 3 -C 8 cycloalkyloxycarbonyl, C 7 -C 30 aryloxycarbonyl, C 8 -C 30 aralkyloxycarbonyl, or —P(═O)(R 22 ) 2 ;

R 21 is C 1 -C 30 alkyl comprising one or more oxygen or hydroxyl moieties or combinations thereof;

each R 22 is independently C 6 -C 12 aryloxy

B is a base-pairing moiety;

L 1 is an optional linker up to 18 atoms in length comprising bonds selected from alkyl, hydroxyl, alkoxy, alkylamino, amide, ester, carbonyl, carbamate, phosphorodiamidate, phosphoroamidate, phosphorothioate and phosphodiester;

x is an integer of 0 or greater; and

provided that both of R 17 and R 18 are not absent.

7. The process of claim 6 , wherein at least one of R 18 or R 19 is R 20 .

8. The process of claim 6 , wherein R 19 is piperizinyl or

9. The process of claim 1 , wherein the oligomer is an oligomer comprising a backbone, the backbone comprising a sequence of morpholino ring structures joined by intersubunit linkages of type (A), (B), or combinations thereof, wherein each morpholino ring structure supports a base-pairing moiety, such that the oligomer compound can bind in a sequence-specific manner to a target nucleic acid, and wherein the oligomer comprises a 3′ terminus, a 5′ terminus and has the following structure (XVII):

or a salt, stereoisomer or tautomer thereof, and

wherein for linkage (A):

W is, at each occurrence, independently S or O;

X is, at each occurrence, independently —N(CH 3 ) 2 , —NR 1 R 2 , —OR 3 or;

Y is, at each occurrence, independently O or —NR 2 ,

R 1 is, at each occurrence, independently hydrogen or methyl;

R 2 is, at each occurrence, independently hydrogen or -LNR 4 R 5 R 7 ;

R 3 is, at each occurrence, independently hydrogen or C 1 -C 6 alkyl;

R 4 is, at each occurrence, independently hydrogen, methyl, —C(═NH)NH 2 , -Z-L-NHC(═NH)NH 2 or -[C(O)CHR′NH] m H, where Z is carbonyl(C(O)) or a direct bond, R′ is a side chain of a naturally occurring amino acid or a one- or two-carbon homolog thereof, and m is 1 to 6;

R 6 is, at each occurrence, independently hydrogen, methyl or an electron pair;

R 6 is, at each occurrence, independently hydrogen or methyl;

R 7 is, at each occurrence, independently hydrogen C 1 -C 6 alkyl or C 1 -C 6 alkoxyalkyl;

L is an optional linker up to 18 atoms in length comprising alkyl, alkoxy or alkylamino groups, or combinations thereof; and

wherein for linkage (B):

W is, at each occurrence, independently S or O;

X is, at each occurrence, independently —NR 8 R 9 or —OR 3 ; and

Y is, at each occurrence, independently O or -NR 10 ,

R 8 is, at each occurrence, independently hydrogen or C 2 -C 12 alkyl;

R 9 is, at each occurrence, independently hydrogen, C 1 -C 12 alkyl, C 1 -C 12 aralkyl or aryl;

R 10 is, at each occurrence, independently hydrogen, C 1 -C 12 alkyl or -LNR 4 R 5 R 7 ;

wherein R 8 and R 9 may join to form a 5-18 membered mono or bicyclic heterocycle or R 8 , R 9 or R 3 may join with R 10 to form a 5-7 membered heterocycle, and wherein when X is 4-piperazinyl, X has the following structure (Ill):

wherein:

R 10 is, at each occurrence, independently C 2 -C 12 alkyl, C 1 -C 12 aminoalkyl, C 1 -C 12 alkylcarbonyl, aryl, heteroaryl or heterocyclyl; and

R 11 is, at each occurrence, independently an electron pair, hydrogen or C 1 -C 12 alkyl;

R 12 is, at each occurrence, independently, hydrogen, C 1 -C 12 alkyl, C 1 -C 12 aminoalkyl, —NH 2 , —CONH 2 , —NR 13 R 14 , —NR 13 R 14 R 15 , C 1 -C 12 alkylcarbonyl, —CN, trifluoromethyl, amidyl, amidinyl, amidinylalkyl, amidinylalkylcarbonyl, guanidinyl, guanidinylalkyl, guanidinylalkylcarbonyl, cholate, deoxycholate, aryl, heteroaryl, heterocycle, —SR 13 or C 1 -C 12 alkoxy, wherein R 13 , R 14 and R 15 are, at each occurrence, independently C 1 -C 12 alkyl; and

R 17 is, at each occurrence, independently absent, hydrogen or C 1 -C 6 alkyl;

R 18 and R 19 are, at each occurrence, independently absent, hydrogen, a cell-penetrating peptide, a natural or non-natural amino acid, C 2 -C 30 alkylcarbonyl, —C(═O)OR 21 or R 20 ;

R 20 is, at each occurrence, independently guanidinyl, heterocyclyl, C 1 -C 30 alkyl, C 3 -C 8 cycloalkyl; C 6 -C 30 aryl, C 7 -C 30 aralkyl, C 3 -C 30 alkylcarbonyl, C 3 -C 8 cycloalkylcarbonyl, C 3 -C 8 cycloalkylalkylcarbonyl, C 7 -C 30 arylcarbonyl, C 7 -C 30 aralkylcarbonyl, C 2 -C 30 alkyloxycarbonyl, C 3 -C 8 cycloalkyloxycarbonyl, C 7 -C 30 aryloxycarbonyl, C 8 -C 30 aralkyloxycarbonyl, or —P(═O)(R 22 ) 2 ;

R 21 is C 1 -C 30 alkyl comprising one or more oxygen or hydroxyl moieties or combinations thereof;

each R 22 is independently C 6 -C 12 aryloxy;

B is a base-pairing moiety;

L 1 is an optional linker up to 18 atoms in length comprising bonds selected from alkyl, hydroxyl, alkoxy, alkylamino, amide, ester, disulfide, carbonyl, carbamate, phosphorodiamidate, phosphoroamidate, phosphorothioate, piperazine and phosphodiester;

x is an integer of 0 or greater; and

wherein at least one of R 18 or R 19 is R 20 and provided that both of R 17 and R 18 are not absent, and

wherein if R 18 is R 20 , then R 18 is not trityl, 3-methoxytrityl, or 4-methoxytrityl.

10. The process of claim 9 , wherein R 19 has the following structure:

11. The process of claim 9 , wherein R 18 is a cell-penetrating peptide and R 19 is R 20 .

12. The process of claim 9 , wherein R 19 is a cell-penetrating peptide and R 18 is R 20 .

13. The process of claim 9 , wherein the cell-penetrating peptide is an arginine-rich peptide.

14. The process of any of claim 9 , wherein the cell-penetrating peptide comprises 6 to 16 subunits selected from X′ subunits, Y′ subunits, and Z subunits,

wherein;

(a) each X′ subunit independently represents lysine, arginine or an arginine analog, said analog being a cationic a-amino acid comprising a side chain of the structure R 33 N═C(NH 2 )R 34 , where R 33 is H or R; R 34 is R 35 , NH 2 , NHR, or NR 34 , where R 35 is lower alkyl or lower alkenyl and may further include oxygen or nitrogen; R 3 and R 34 may together form a ring; and the side chain is linked to said amino acid via R 3 or R 34 ;

(b) each Y′ subunit independently represents a neutral amino acid —C(O)—(CHR) n —NH—, where n is 2 to 7 and each R is independently H or methyl; and

(c) each Z′ subunit independently represents an a-amino acid having a neutral aralkyl side chain;

wherein the peptide comprises a sequence represented by one of (X′Y′X′) p , (X′Y′) m , and (X′Z′Z′) p , where p is 2 to 5 and m is 2 to 8.

15. The process of claim 14 , wherein X′ is guanidyl (HN═C(NH 2 )NH—), amidinyl (HN═C(NH 2 )C—), 2-aminodihydropyrimidyl, 2-aminotetrahydropyrimidyl, 2-aminopyridinyl, and 2-aminopyrimidonyl.

16. The process of claim 14 , wherein the carrier peptide is represented by the sequence (RY′R) 4 and (RRY′) 4 , where R is arginine and Y′ is 6-aminohexanoic acid.

17. The process of claim 14 , wherein the cell-penetrating peptide is linked at its C-terminus to the 5′ end of the oligomer through a one- or two-amino acid linker.

18. The process of claim 14 , wherein the linker is AhxB, where Ahx is a 6-aminohexanoic acid subunit and B is a -alanine subunit.

19. The process of claim 14 , wherein the X′ subunit is guanidyl, as in the amino acid subunit arginine (Arg).

20. The process of claim 14 , wherein the Y′ subunit is —CO—(CH 2 ) n —CHR—NH—, where n is 2 to 7 and R is H.

21. The process of claim 14 , wherein the Y′ subunit is a 6-aminohexanoic acid subunit (Ahx).

22. The process of claim 14 , wherein the cell penetrating peptide has the formula (RY′R) p or the formula (RRY′) p ; wherein Y′ is a 6-aminohexanoic acid subunit, R is arginine and p is 4.

23. The process of claim 14 , wherein the Z′ subunit is phenylalanine.

24. The process of claim 14 , wherein the cell-penetrating peptide is linked to a terminus of the oligomer via a linker Ahx-B, wherein Ahx is a 6-aminohexanoic acid subunit and B is a 13-alanine subunit.