Functional biopolymer modification reagents and uses thereof

• US 20050203289A1
• Filed: 09/08/2004
• Published: 09/15/2005
• Est. Priority Date: 03/22/2000
• Status: Active Grant

• Current Assignee: Vector Laboratories
• Original Assignee: Solulink, Incorporated (Maravai Life Sciences Holdings LLC)
• Inventors: Schwartz, David A., Hogrefe, Richard I.
• Granted Patent: US 7,732,628 B2
• Time in Patent Office: Days
• US Class Current: 536/25.400
• CPC Class Codes: A61K 47/61 the organic macromolecular compound being a polysaccharide or a derivative thereof
• CPC Class Codes: A61K 47/62 the modifying agent being a protein, peptide or polyamino acid
• CPC Class Codes: A61K 47/643 Albumins, e.g. HSA, BSA, ovalbumin or a Keyhole Limpet Hemocyanin [KHL]
• CPC Class Codes: A61K 47/645 Polycationic or polyanionic oligopeptides, polypeptides or polyamino acids, e.g. polylysine, polyarginine, polyglutamic acid or peptide TAT
• CPC Class Codes: A61K 47/6801 Drug-antibody or immunoglobulin conjugates defined by the pharmacologically or therapeutically active agent
• CPC Class Codes: C04B 35/632 Organic additives
• CPC Class Codes: C07D 207/452 with hydrocarbon radicals, substituted by hetero atoms, directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 207/46 with hetero atoms directly attached to the ring nitrogen atom
• CPC Class Codes: C07D 213/82 in position 3
• CPC Class Codes: C07D 401/12 linked by a chain containing hetero atoms as chain links
• CPC Class Codes: C07F 7/1804 Compounds having Si-O-C linkages Si-O-acyl linkages C07F7/1896
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• CPC Class Codes: C07F 9/58 Pyridine rings
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C08G 65/329 with organic compounds

Abstract

Hydrazino, oxyamino and carbonyl-based reagents and methods for incorporation into oligonucleotides during their solid phase synthesis are provided. Modified oligonucleotides are provided that incorporate the reagents provided herien. Immobilized oligonucleotides and oligonucleotide conjugates that contain covalent hydrazone or oxime linkages are provided. Methods for preparation of surface bound oligonucleotides are provided. Methods for the preparation of oligonucleotide conjugates are also provided.

Claims

1. A compound that has formula (I):

P¹-M-X

or a derivative thereof, wherein;

P¹ is a phosphorous based coupling group;

X is a protected hydrazino group, a protected oxyamino group, or a carbonyl derivative; and

M is a divalent group having any combination of the following groups, which are combined in any order;

arylene, heteroarylene, cycloalkylene, C(R¹)₂, —

C(R¹)═

C(R¹)—

, >

C═

C(R²)(R³), >

C(R²)(R³), —

C≡

C—

, O, S(A)_a, P(D)_b(R¹), P(D)_b(ER¹), N(R¹), >

N⁺(R²)(R³) and C(E);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

A is O or NR¹;

D is S or O; and

E is S, O or NR¹;

each R¹ is a monovalent group independently selected from hydrogen and M¹-R⁴;

each M¹ is a divalent group independently having any combination of the following groups, which groups are combined in any order;

a direct link, arylene, heteroarylene, cycloalkylene, C(R⁵)₂, —

C(R⁵)═

C(R⁵)—

, >

C═

C(R²)(R³), >

C(R²)(R³), —

C≡

C—

, O, S(A)_a, P(D)_b(R⁵), P(D)_b(ER⁵), N(R⁵), N(COR⁵), >

N⁺(R²)(R³) and C(E);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

A is O or NR⁵;

D is S or O; and

E is S, O or NR⁵;

R⁴ and R⁵ are each independently selected from the group consisting of hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR⁶R⁷R⁸, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR⁹R¹⁰;

R⁹ and R¹⁰ are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R² and R³ are selected from (i) or (ii) as follows;

(i) R² and R³ are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl;

or

(ii) R² and R³ together form alkylene, alkenylene or cycloalkylene;

R⁶, R⁷ and R⁸ are each independently a monovalent group selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR⁹R¹⁰; and

R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are unsubstituted or substituted with one or more substituents each independently selected from Z, wherein Z is selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy, S(O)_hR²⁰, NR²⁰R²¹, COOR²⁰, COR²⁰, CONR²⁰R²¹, OC(O)NR²⁰R²¹, N(R²⁰)C(O)R²¹, alkoxy, aryloxy, heteroaryl, heterocyclyl, heteroaryloxy, heterocyclyloxy, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, aralkoxy, heteroaralkoxy, alkoxycarbonyl, carbamoyl, thiocarbamoyl, alkoxycarbonyl, carboxyaryl, halo, pseudohalo, haloalkyl and carboxamido;

h is 0, 1 or 2;

R²⁰ and R²¹ are each independently selected from among hydrogen, halo, pseudohalo, cyano, azido, nitro, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy, amino, amido, alkylamino, dialkylamino, alkylarylamino, diarylamino and arylamino;

with the proviso that when X is a protected oxyamino group, M is not (CH₂CH₂O)₄.

2. The compound of claim 1, wherein X is a protected hydrazino group.

3. The compound of claim 1, wherein P¹ is selected from the group consisting of a phosphoramidite, an H-phosphionate or a phosphotriester.

4. The compound of claim 1, wherein X is a carbonyl group.

5. The compound of claim 4, wherein X is an aldehyde or ketone group.

6. The compound of claim 1, wherein X is a protected oxyamino group.

7. The compound of claim 1, wherein the protecting group is a salt, an amine protecting group, or a hydrazine protecting group.

8. The compound of claim 1, wherein M comprises 1-50 of any of the following group combined in any order:

arylene, heteroarylene, C(R⁵)₂, O, S(A)_a, N(R⁵), N(COR⁵) and C(E);

where a is 0, 1 or 2;

A is O or NR⁵; and

E is S, O or NR⁵.

9. The compound of claim 1, wherein M comprises 1-50 of any of the following groups combined in any order:

heteroarylene, C(R⁵)₂, N(R⁵) and C(E);

where E is S, O or NR⁵.

10. The compound of claim 1, wherein M comprises 1-50 of any of the following groups combined in any order:

arylene, C(R⁵)₂, N(R⁵) and C(E);

where E is S, O or NR⁵.

11. The compound of claim 1, wherein M comprises a chain of 1-2000 repeating monomer units selected from among ethylene oxide, propylene oxide, methacrylamide and ethylene glycol.

12. The compound of claim 1, wherein M comprises a cleavable linkage selected from among a disulfide, an ester, an enzyme specific peptide, a photocleavable linkage and an acid labile group.

13. The compound of claim 1, wherein X is —

C(O)R³⁰, —

Y—

N(R³¹)—

Y¹—

N(R³²)—

Y² or —

O—

N(R³⁰)—

Y²;

where R³⁰, R³¹ and R³² are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl or cycloalkyl;

Y and Y¹ are selected as in (i) or (ii) as follows;

(i) Y is a direct link, and Y¹ is a direct link, C(O)N(R³⁵), N(R³⁵)C(O)N(R³⁶), C(S)N(R³⁵), N(R³⁵)C(S)N(R³⁶) or C(O)N(R³⁵)N(R³⁶)C(O)N(R³⁷);

or

(ii) Y is C(O) or OC(O), and Y¹ is a direct link;

where R³⁵, R³⁶ and R³⁷ are each independently selected from among hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl and cylcoalkyl; and

Y² is a salt of the hydrazino or oxyamino group, or any amino or hydrazino protecting group;

where R³⁰, R³¹, R³², R³⁵, R³⁶, R³⁷ and Y² are unsubsituted or substituted with one or more substituents each independently selected from Z, wherein Z is selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy, S(O)_hR²⁰, NR²⁰R²¹, COOR²⁰, COR²⁰, CONR²⁰R²¹, OC(O)NR²⁰R²¹, N(R²⁰)C(O)R²¹, alkoxy, aryloxy, heteroaryl, heterocyclyl, heteroaryloxy, heterocyclyloxy, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, aralkoxy, heteroaralkoxy, alkoxycarbonyl, carbamoyl, thiocarbamoyl, alkoxycarbonyl, carboxyaryl, halo, pseudohalo, haloalkyl and carboxamido;

h is 0, 1 or 2; and

R²⁰ and R²¹ are each independently selected from among hydrogen, halo, pseudohalo, cyano, azido, nitro, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy, amino, amido, alkylamino, dialkylamino, alkylarylamino, diarylamino and arylamino.

14. The compound of claim 13, wherein Y² is selected from monomethoxytrityl (MMT), dimethoxytrityl (DMT), 9-fluorenylmethoxycarbonyl (FMOC), acetyl, trifluroracetyl (TFA), benzoyl, or a lower aliphatic hydrazone or oxime;

with the proviso that when X is a protected oxyamino group, the protecting group is not a phthalimidyl group.

15. The compound of claim 1, wherein X has the formula:

—

Y—

N(R³¹)—

Y¹—

N(R³²)—

Y²,

wherein;

Y and Y¹ are selected as in (i) or (ii) as follows;

(i) Y is a direct link, and Y¹ is a direct link, C(O)N(R³⁵), N(R³⁵)C(O)N(R³⁶), C(S)N(R³⁵), N(R³⁵)C(S)N(R³⁶) or C(O)N(R³⁵)N(R³⁶)C(O)N(R³⁷);

or

(ii) Y is C(O) or OC(O), and Y¹ is a direct link;

where R³⁵, R³⁶ and R³⁷ are each independently selected from among hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl and cylcoalkyl;

Y² is a salt, or any amino or hydrazino protecting group; and

R³¹ and R³² are each independently hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl or cycloalkyl;

where R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰, R³¹, R³², R³⁵, R³⁶, R³⁷ and Y² are unsubstituted or substituted with one or more substituents each independently selected from Z.

16. The compound of claim 15, wherein X has the formula:

—

Y—

NH—

Y¹—

NH—

Y²;

wherein Y and Y¹ are selected from (i) or (ii) as follows;

(i) Y is a direct link, and Y¹ is a direct link, C(O)NH, NHC(O)NH, C(S) NH, NHC(S)NH or C(O)NHNHC(O)NH;

or

(ii) Y is C(O) or OC(O), and Y¹ is a direct link.

17. The compound of claim 1, wherein M has 2,5-pyridylene attached to X;

and comprises 1-9 of any of the following groups combined in any order;

CH₂, NH and C(O).

18. The compound of claim 1, wherein M has the formula:

19. The compound of claim 1, selected from the group consisting of N-dimethoxytrityl-N′

-{5-[8-(2-cyanoethyl-N,N-diisopropylphosphoramidyloxy)-2-aza-1-oxooct-1-yl]pyrid-2-yl}hydrazine and N-(9-fluorenylmethoxycarbonyl)-N′

-[8-(2-cyanoethyl-N,N-diisopropylphosphoramidyloxy)-2-aza-1-thiooct-1-yl]hydrazine, having the structures;

20. The compound of claim 1 that has formula (V):

P¹-M-C(O)R³⁰,

or a derivative thereof, wherein;

R³⁰ is hydrogen, alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl or cycloalkyl;

where R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and R³⁰ are unsubstituted or substituted with one or more substituents each independently selected from Z.

21. The compound of claim 20, wherein R³⁰ is hydrogen or lower alkyl.

22. The compound of claim 20, wherein R³⁰ is hydrogen or methyl.

23. The compound of claim 1 that has either of formulae (VI):

P¹-M-CHO or P¹-M-C(O)Me,

or a derivative thereof.

24. The compound of claim 20, wherein M has phenylene attached to C(O)R³⁰, and comprises 1-9 any of the following groups combined in any order:

CH₂, NH and C(O).

25. The compound of claim 20, wherein M has the formula:

26. The compound of claim 20 selected from the group consisting of 4-[8-(2-cyanoethyl-N,N-diisopropylphosphoramidyloxy)-2-aza-1-oxooct-1-yl]benzaldehyde, 4-[8-(2-cyanoethyl-N,N-diisopropylphosphoramidyloxy)-2-aza-1-oxooct-1-yl]acetophenone and N-[6-(2-cyanoethyl-N,N-diisopropylphosphoramidyloxy)hexyl]4-ketopentanamide, having the structures:

27. The compound of claim 1 that has formula (VII):

P¹-M-O—

NH—

Y²,

or a derivative thereof, wherein;

Y² is a salt, or any amino protecting group;

where R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹, R¹⁰ and Y² are unsubstituted or substituted with one or more substituents each independently selected from Z;

with the proviso that M is not (CH₂CH₂O)₄.

28. The compound of claim 27, wherein M has pyridylene attached to the oxygen of the oxyamino group, and comprises 1-9 any of the following groups combined in any order:

CH₂, NH and C(O).

29. The compound of claim 27, wherein M has the formula:

30. The compound of claim 27 that is N-monomethoxytrityl-O-{5-[8-(2-cyanoethyl-N,N-diisopropylphosphoramidyloxy)-2-aza-1-oxooct-1-yl]pyrid-2-yl}oxyamine, having the structure:

31. The compound of claim 1 that is covalently coupled to an oligonucleotide.

54. The compound of claim 1, wherein P¹ is a phosphoramidite group.

56. A methods of preparation of a modified oligonucleotide, comprising the steps of:

(i) synthesizing an oligonucleotide on a solid phase synthesizer wherein at least one of the monomers is a phosphoramidite of claim 1;

(ii) removing the hydrazino or oxyamino protecting group, of present; and

(iii) cleaving the modified oligonucleotide from the solid surface;

wherein steps (ii) and (iii) are performed in either order.

32. A compound that has formula (II):

O¹—

P²-M-X,

or a derivative thereof, wherein;

O¹ is a first component selected from an oligonucleotide or analog thereof;

P² is a phosphorous based linking group;

X is a protected or unprotected hydrazino group, protected or unprotected oxyamino group, or a carbonyl group; and

M is a divalent group comprising any combination of the following groups, which are combined in any order;

arylene, heteroarylene, cycloalkylene, C(R¹)₂, —

C(R¹)═

C(R¹)—

, >

C═

C(R²)(R³), >

C(R²)(R³), —

C≡

C—

, O, S(A)_a, P(D)_b(R¹), P(D)_b(ER¹), N(R¹), >

N⁺(R²)(R³) and C(E);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

A is O or NR¹;

D is S or O; and

E is S, O or NR¹;

each R¹ is a monovalent group independently selected from hydrogen and M¹-R⁴;

each M¹ is a divalent group reaching independently comprising any combination of the following groups, which groups are combined in any order;

a direct link, arylene, heteroarylene, cycloalkylene, C(R⁵)₂, —

C(R⁵)═

C(R⁵)—

, >

C═

C(R²)(R³), >

C(R²)(R³), —

C≡

C—

, O, S(A)_a, P(D)_b(R⁵), P(D)_b(ER⁵), N(R⁵), N(COR⁵), >

N⁺(R²)(R³) and C(E);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

A is O or NR⁵;

D is S or O; and

E is S, O or NR⁵;

R⁴ and R⁵ are each independently selected from the group consisting of hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR⁶R⁷R⁸, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR⁹R¹⁰;

R⁹ and R¹⁰ are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R² and R³ are selected from (i) or (ii) as follows;

(i) R² and R³ are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl;

or

(ii) R² and R³ together form alkylene, alkenylene or cycloalkylene;

R⁶, R⁷ and R⁸ are each independently a monovalent group selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR⁹R¹⁰; and

each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ is optionally substituted with one or more substituents each independently selected from Z, wherein Z is selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy, S(O)_hR²⁰, NR²⁰R²¹, COOR²⁰, COR²⁰, CONR²⁰R²¹, OC(O)NR²⁰R²¹, N(R²⁰)C(O)R²¹, alkoxy, aryloxy, heteroaryl, heterocyclyl, heteroaryloxy, heterocyclyloxy, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, aralkoxy, heteroaralkoxy, alkoxycarbonyl, carbamoyl, thiocarbamoyl, alkoxycarbonyl, carboxyaryl, halo, pseudohalo, haloalkyl and carboxamido;

h is 0, 1 or 2;

R²⁰ and R²¹ are each independently selected from among hydrogen, halo, pseudohalo, cyano, azido, nitro, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy, amino, amido, alkylamino, dialkylamino, alkylarylamino, diarylamino and arylamino;

with the provisos that (i) if X is CHO, then M is not undecylenecarbonylaminomethylene (n-C₁₁H₂₂C(O)NHCH₂) or C_1-20alkylene;

(ii) X is not a hydrazide (—

C(O)—

NHNH₂) group;

(iii) if X is —

NHNH₂, then M is not —

CH(CH₂OH)(CH₂)₆NHCO-5-pyrid-2-ylene; and

(iv) P² is not a phosphoramidate group.

33. The compound of claim 32 that is:

34. The compound of claim 32 that is immobilized on a solid surface.

35. The compound of claim 32 conjugated to a second component.

36. A method for immobilizing oligonucleotides on a surface, comprising the step of:

reacting the compound of claim 32 or a plurality of said compounds with a surface, whereby the oligonucleotide compound is immobilized on the surface, wherein the compound and the surface comprise complementary reactive groups.

37. An immobilized oligonucleotide prepared by the method of claim 36.

38. A method for formation of an oligonucleotide conjugate, comprising the step of:

reacting the compound of claim 32 with a second component, whereby the oligonucleotide compound is conjugated to the second component to form a conjugate, wherein the compound and the second component comprise complementary reactive groups.

39. An oligonucleotide conjugate prepared by the method of claim 38.

40. A compound of formula:

X-M-S—

S-M-X,

or a derivative thereof, wherein;

each S is sulfur;

each X is independently a hydrazino group; and

each M is independently a divalent group comprising any combination of the following groups, which are combined in any order;

arylene, heteroarylene, cycloalkylene, C(R¹)₂, —

C(R¹)═

C(R¹)—

, >

C═

C(R²)(R³), >

C(R²)(R³), —

C≡

C—

, O, S(A)_a, P(D)_b(R¹), P(D)_b(ER¹), N(R¹), >

N⁺(R²)(R³) and C(E);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

A is O or NR¹;

D is S or O; and

E is S, O or NR¹;

each R¹ is a monovalent group independently selected from hydrogen and M¹-R⁴;

each M¹ is a divalent group each independently comprising any combination of the following groups, which groups are combined in any order;

a direct link, arylene, heteroarylene cycloalkylene, C(R⁵)₂, —

C(R⁵)═

C(R⁵)—

, >

C═

C(R²)(R³), >

C(R²)(R³), —

C≡

C—

, O, S(A)_a, P(D)_b(R⁵), P(D)_b(ER⁵), N(R⁵), N(COR⁵), >

N⁺(R²)(R³) and C(E);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

A is O or NR⁵;

D is S or O; and

E is S, O or NR⁵;

R⁴ and R⁵ are each independently selected from the group consisting of hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR⁶R⁷R⁸, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR⁹R¹⁰;

R⁹ and R¹⁰ are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R² and R³ are selected from (i) or (ii) as follows;

(i) R² and R³ are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl;

or

(ii) R² and R³ together form alkylene, alkenylene or cycloalkylene;

R⁶, R⁷ and R⁸ are each independently a monovalent group selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR⁹R¹⁰; and

each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are optionally substituted with one or more substituents each independently selected from Z, wherein Z is selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy S(O)_hR²⁰, NR²⁰R²¹, COOR²⁰, COR²⁰, CONR²⁰R²¹, OC(O)NR²⁰R²¹, N(R²⁰)C(O)R²¹, alkoxy, aryloxy, heteroaryl, heterocyclyl, heteroaryloxy, heterocyclyloxy, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, aralkoxy, heteroaralkoxy, alkoxycarbonyl, carbamoyl, thiocarbamoyl, alkoxycarbonyl, carboxyaryl, halo, pseudohalo, haloalkyl and carboxamido;

h is 0, 1 or 2;

R²⁰ and R²¹ are each independently selected from among hydrogen, halo, pseudohalo, cyano, azido, nitro, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy, amino, amido, alkylamino, dialkylamino, alkylarylamino, diarylamino and arylamino.

41. The compound of claim 40 that has the formula:

42. A compound that has the formula:

X-M-X,

or a derivative thereof, wherein;

each X is independently a hydrazino group or an oxyamino group; and

M is independently a divalent group comprising any combination of the following groups, which are combined in any order;

arylene, heteroarylene, cycloalkylene, C(R¹)₂, —

C(R¹)═

C(R¹)—

, >

C═

C(R²)(R³), >

C(R²)(R³), —

C≡

C—

, O, S(A)_a, P(D)_b(R¹), P(D)_b(ER¹), N(R¹), >

N⁺(R²)(R³) and C(E);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

A is O or NR¹;

D is S or 0; and

E is S, O or NR¹;

each R¹ is a monovalent group independently selected from hydrogen and M¹-R⁴;

each M¹ is a divalent group each independently comprising any combination of the following groups, which groups are combined in any order;

a direct link, arylene, heteroarylene, cycloalkylene, C(R⁵)₂, —

C(R⁵)═

C(R⁵)—

, >

C═

C(R²)(R³), >

C(R²)(R³), —

C≡

C—

, O, S(A)_a, P(D)_b(R⁵), P(D)_b(ER⁵), N(R⁵), N(COR⁵), >

N⁺(R²)(R³) and C(E);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

A is O or NR⁵;

D is S or O; and

E is S, O or NR⁵;

R⁴ and R⁵ are each independently selected from the group consisting of hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR⁶R⁷R⁸, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR⁹R¹⁰;

R⁹ and R¹⁰ are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R² and R³ are selected from (i) or (ii) as follows;

(i) R² and R³ are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl;

or

(ii) R² and R³ together form alkylene, alkenylene or cycloalkylene;

R⁶, R⁷ and R⁸ are each independently a monovalent group selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR⁹R¹⁰; and

each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ are optionally substituted with one or more substituents each independently selected from Z, wherein Z is selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy S(O)_hR²⁰, NR²⁰R²¹, COOR²⁰, COR²⁰, CONR²⁰R²¹, OC(O)NR²⁰R²¹, N(R²⁰)C(O)R²¹, alkoxy, aryloxy, heteroaryl, heterocyclyl, heteroaryloxy, heterocyclyloxy, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, aralkoxy, heteroaralkoxy, alkoxycarbonyl, carbamoyl, thiocarbamoyl, alkoxycarbonyl, carboxyaryl, halo, pseudohalo, haloalkyl and carboxamido;

h is 0, 1 or 2;

R²⁰ and R²¹ are each independently selected from among hydrogen, halo, pseudohalo, cyano, azido, nitro, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy, amino, amido, alkylamino, dialkylamino, alkylarylamino, diarylamino and arylamino.

43. The compound of claim 42 that has formula:

44. A compound that has formula (VIII):

45. The compound of claim 44 that has the formula:

49. The compound of claim 44 covalently coupled to an oligonucleotide.

46. A compound that has formula (X):

P¹—

S¹—

B¹-M-X,

or a derivative thereof, wherein;

P¹ is a phosphorous based coupling group;

M is a divalent group comprising any combination of the following groups, which are combined in any order;

arylene, heteroarylene, cycloalkylene, C(R¹)₂, —

C(R¹)═

C(R¹)—

, >

C═

C(R²)(R³), >

C(R²)(R³), —

C≡

C—

, O, S(A)_a, P(D)_b(R¹), P(D)_b(ER¹), N(R¹), >

N⁺(R²)(R³) and C(E);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

A is O or NR¹;

D is S or O; and

E is S, O or NR¹;

each R¹ is a monovalent group independently selected from hydrogen and M¹-R⁴;

each M¹ is a divalent group each independently comprising any combination of the following groups, which groups are combined in any order;

a direct link, arylene, heteroarylene, cycloalkylene, C(R⁵)₂, —

C(R⁵)═

C(R⁵)—

, >

C═

C(R²)(R³), >

C(R²)(R³), —

C≡

C—

, O, S(A)_a, P(D)_b(R⁵), P(D)_b(ER⁵), N(R⁵), N(COR⁵), >

N⁺(R²)(R³) and C(E);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

A is O or NR⁵;

D is S or O; and

E is S, O or NR⁵;

R⁴ and R⁵ are each independently selected from the group consisting of hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR⁶R⁷R⁸, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR⁹R¹⁰;

R⁹ and R¹⁰ are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R² and R³ are selected from (i) or (ii) as follows;

(i) R² and R³ are independently selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl;

or

(ii) R² and R³ together form alkylene, alkenylene or cycloalkylene;

R⁶, R⁷ and R⁸ are each independently a monovalent group selected from hydrogen, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR⁹R¹⁰;

each of R¹, R², R³, R⁴, R⁵, R⁶, R⁷, R⁸, R⁹ and R¹⁰ is optionally substituted with one or more substituents each independently selected from Z, wherein Z is selected from alkyl, alkenyl, alkynyl, aryl, cycloalkyl, cycloalkenyl, hydroxy, S(O)_hR²⁰, NR²⁰R²¹, COOR², COR²⁰, CONR²⁰R²¹, OC(O)NR²⁰R²¹, N(R²⁰)C(O)R²¹, alkoxy, aryloxy, heteroaryl, heterocyclyl, heteroaryloxy, heterocyclyloxy, aralkyl, aralkenyl, aralkynyl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, aralkoxy, heteroaralkoxy, alkoxycarbonyl, carbamoyl, thiocarbamoyl, alkoxycarbonyl, carboxyaryl, halo, pseudohalo, haloalkyl and carboxamido;

h is 0, 1 or 2;

R²⁰ and R²¹ are each independently selected from among hydrogen, halo, pseudohalo, cyano, azido, nitro, trialkylsilyl, dialkylarylsilyl, alkyldiarylsilyl, triarylsilyl, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy, amino, amido, alkylamino, dialkylamino, alkylarylamino, diarylamino and arylamino;

X is a protected hydrazino group, a protected oxyamino group, or a carbonyl derivative;

S¹ is a ribose, a deoxyribose or a dideoxyribose; and

B¹ is a nucleobase.

47. The compound of claim 46 that has either of the formula:

48. The compound of claim 46 covalently coupled to an oligonucleotide.

50. A compound that has formula (IX):

51. The compound of claim 50 that has either of the formula:

52. A compound that has formula (XI):

53. The compound of claim 52 that has either of the formula:

55. A method of preparing a carbonyl, hydrazino or oxyamino phosphoramidite, comprising the steps of:

(i) derivatizing a carboxylic acid selected from an ω

-carbonyl, an ω

-protected hydrazino, and an ω

-protected oxyamino substituted carboxylic acid as the corresponding active ester;

(ii) reacting the resulting active ester with an α

,ω

-aminoalcohol to provide an ω

-hydroxyamide; and

(iii) derivatizing the ω

-hydroxy group as the corresponding phosphoramidite.