Hydrazine-based and carbonyl-based bifunctional crosslinking reagents

• US 20040110730A1
• Filed: 11/24/2003
• Published: 06/10/2004
• 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.
• Granted Patent: US 7,462,689 B2
• Time in Patent Office: Days
• US Class Current: 514/151
• 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: 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: C07K 1/1077 by covalent attachment of residues other than amino acids or peptide residues, e.g. sugars, polyols, fatty acids
• CPC Class Codes: C08G 65/329 with organic compounds
• CPC Class Codes: C08G 65/336 containing silicon
• CPC Class Codes: C08L 2203/02 for biomedical use
• CPC Class Codes: C12N 9/96 Stabilising an enzyme by forming an adduct or a composition; Forming enzyme conjugates
• CPC Class Codes: Y10S 530/81 Carrier - bound or immobilized peptides or proteins and the preparation thereof, e.g. biological cell or cell fragment as carrier

Abstract

Reagents and methods are provided for crosslinking and immobilizing biomolecules, drugs and synthetic polymers. The reagents possess (i) a thiol or amino reactive group; and (ii) a hydrazino or oxyamino moiety. Conjugates and immobilized biomolecules are also provided.

Claims

1. A compound of formula I:

B—

R—

A—

NHNH₂.HX 

 

I

or a derivative thereof, wherein;

A is —

NH(C═

O)—

, —

NH(C═

S)—

, —

NHNH(C═

O)—

, —

NHNH(C═

S)—

, or a direct bond to R;

B is an amino or thiol reactive moiety;

R is an aliphatic divalent group having any combination of the following groups, which are combined in any order;

cycloalkylene, C(R¹⁰)₂, —

C(R¹⁰)═

C(R¹⁰)—

, >

C═

C(R¹²)(R¹³), >

C(R¹²)(R¹³), —

C≡

C—

, O, S(G)_a, P(J)_b(R¹⁰), P(J)_b(LR¹⁰), N(R¹⁰), >

N⁺(R¹²)(R¹³) and C(L);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

G is O or NR¹⁰;

J is S or O; and

L is S, O or NR¹⁰;

each R¹⁰ is a monovalent group independently selected from hydrogen and M¹-R¹⁴;

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(R¹⁵)₂, —

C(R¹⁵)═

C(R¹⁵)—

, >

C═

C(R¹²)(R¹³), >

C(R¹²)(R¹³), —

C≡

C—

, O, S(G¹)_a, P(J)_b(R¹⁵), P(J)_b(LR¹⁵), N(R¹⁵), N(COR¹⁵), >

N⁺(R¹²)(R¹³) and C(L);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

G¹ is O or NR¹⁵;

J is S or O; and

L is S, O or NR¹⁵;

R¹⁴ and R¹⁵ are each independently selected from the group among hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR¹⁶R¹⁷R¹⁸, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR¹⁹R²⁰;

R¹⁹ and R²⁰ are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R¹² and R¹³ are selected from (i) or (ii) as follows;

(i) R¹² and R¹³ are independently selected from among hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl;

or (ii) R¹² and R¹³ together form alkylen, alkenylene or cycloalkylene;

R¹⁶, R¹⁷ and R¹⁸ are each independently a monovalent group sel cted from hydrogen, alkyl, alk nyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR¹⁹R²⁰; and

R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ can be 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)_hR³⁰, NR³⁰R³¹, COOR³⁰, COR³⁰, CONR³⁰R³¹, OC(O)NR³⁰R³¹, N(R³⁰)C(O)R³¹, 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

R³⁰ and R³¹ 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; and

X is a negative counterion.

2. The compound of claim 1, wherein R is, or is a combination of, a saturated straight chain of 1 to 20 carbon atoms, a chain of 2 to 2000 ethyleneoxide moieties, or a saturated or unsaturated carbocyclic moiety of 3 to 20 carbon atoms.

3. The compound of claim 1 that is:

4. The compound of claim 1 that is:

22. The compound of claim 1, wherein X is a halide or trifluoroacetate.

23. The compound of claim 1, wherein B is an amino reactive moiety selected from succininimidyl ester, hydroxybenzotriazolyl ester, or pentafluorophenol ester.

24. The compound of claim 1, wherein B is a thiol reactiv moiety selected from maleimido, α

-bromoacetyl or pyridyidisulfide.

25. A conjugate, comprising the compound of claim 1 bound to a natural or synthetic biological molecule.

26. The conjugate of claim 25, wherein the natural or synthetic molecule is selected from a protein, a glycoprotein, a peptide, an oligonucleotide, an RNA, a DNA and a synthetic polymer.

27. The conjugate of claim 26, wherein the protein is an antibody.

28. A method of immobilizing a natural or synthetic biological molecule, comprising:

(i) preparing the conjugate of claim 25;

and

(ii) applying the conjugate to a surface wherein the surface has at least one carbonyl moiety for a time and under conditions such that the hydrazine moiety of the conjugate reacts with the carbonyl moiety of the surface forming a hydrazone bond to the surface.

29. A method of crosslinking, a natural or synthetic biological molecule, comprising:

(i) preparing the conjugate of claim 25;

and

(ii) applying the conjugate to a surface wherein the surface has at least one amino or one thiol reactive moiety for a time and under conditions such that the conjugate reacts with the amino moiety or thiol moiety of the surface forming a bond to the surface.

30. A method of crosslinking a natural or synthetic biological molecule, comprising:

(i) preparing the conjugate of claim 25;

and

(ii) mixing the conjugate with a natural or synthetic biological molecule wherein the molecule has at least one carbonyl moiety for a time and under conditions such that the hydrazine moiety of the conjugate reacts with the carbonyl moiety of the molecule forming a hydrazone bond to the molecule.

40. The method of claim 28, wherein the surface is selected from glass, polymer, latex and colloidal metal.

41. The method of claim 30, wherein the natural or synthetic biological molecule is selected from a protein, a glycoprotein, a peptide, an oligonucleotide, an RNA and a DNA.

42. The method of claim 41, wher in the protein is an antibody.

43. A surface prepared by the method of claim 28.

44. A composition prepared by the method of claim 30.

5. A compound of formula II:

B—

R—

A—

NHN═

C(R¹R²) 

 

II

or a derivative thereof, wherein;

A is NH(C═

O)—

, NH(C═

S)—

, NHNH(C═

O)—

, or NHNH(C═

S)—

or a direct bond to R;

B is an amino or thiol reactive moiety;

R is an aliphatic divalent group having any combination of the following groups, which are combined in any order;

cycloalkylene, C(R¹⁰)₂, —

C(R¹⁰)═

C(R¹⁰)—

, >

C═

C(R¹²)(R¹³), >

C(R¹²)(R¹³), —

C≡

C—

, O, S(G)_a, P(J)_b(R¹⁰), P(J)_b(LR¹⁰), N(R¹⁰), >

N⁺(R¹²)(R¹³) and C(L);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

G is O or NR¹⁰;

J is S or O; and

L is S, O or NR¹⁰;

each R¹⁰ is a monovalent group independently selected from hydrogen and M¹-R¹⁴;

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(R¹⁵)₂, —

C(R¹⁵)═

C(R¹⁵)—

, >

C═

C(R¹²)(R¹³), >

C(R¹²)(R¹³), —

C≡

C, O—

, S(G¹)_a, P(J)_b(R¹⁵), P(J)_b(LR¹⁵), N(R¹⁵), N(COR¹⁵), >

N⁺(R¹²)(R¹³) and C(L);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

G¹ is O or NR¹⁵;

J is S or O; and

L is S, O or NR¹⁵;

R¹⁴ and R¹⁵ are each independently selected from the group among hydrogen, halo, pseudohalo, cyano, azido, nitr , SiR¹⁶R¹⁷R¹⁸, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR¹⁹R²⁰;

R¹⁹ and R²⁰ are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R¹² and R¹³ are selected from (i) or (ii) as follows;

(i) R¹² and R¹³ are independently selected from among hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl;

or (ii) R¹² and R¹³ together form alkylene, alkenylene or cycloalkylene;

R¹⁶, R¹⁷ and R¹⁸ 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 NR¹⁹R²⁰; and

R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ can be 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)_hR³⁰, NR³⁰R³¹, COOR³⁰, COR³⁰, CONR³⁰R³¹, OC(O)NR³⁰R³¹, N(R³⁰)C(O)R³¹, 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

R³⁰ and R³¹ 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, h t roaryl, h t roaralkyl, heteroaralkenyl, het roaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, het roaralkoxy, amino, amido, alkylamino, dialkylamino, alkylarylamino, diarylamino and arylamino;

R¹ is a saturated straight chain of 3 to 20 carbon atoms, a chain of 2 to 2000 ethyleneoxide moieties, or a saturated or unsaturated carbocyclic moiety of 3 to 20 carbon atoms; and

R² is a saturated straight chain of 3 to 20 carbon atoms, a chain of 2 to 2000 ethyleneoxide moieties, a saturated or unsaturated carbocyclic moiety of 3 to 20 carbon atoms.

6. The compound of claim 5, wherein R is, or is a combination of, a saturated straight chain of 1 to 20 carbon atoms, a chain of 2 to 2000 ethyleneoxide moieties, or a saturated or unsaturated carbocyclic moiety of 3 to 20 carbon atoms.

7. The compound of claim 5 that is:

49. The compound of claim 5, wherein B is an amino reactive moiety selected from succininimidyl ester, hydroxybenzotriazolyl ester, or pentafluorophenol ester.

52. The compound of claim 5, wherein B is a thiol reactive moiety selected from maleimido, α

-bromoacetyl or pyridyidisulfide.

8. A compound of formula III:

B—

R—

(C═

O)—

NHNH₂.HX 

 

III

or a derivative thereof, wherein;

B is an amino reactive moiety;

R is an aliphatic divalent group having any combination of the following groups, which are combined in any order;

cycloalkylene, C(R¹⁰)₂, —

C(R¹⁰)═

C(R¹⁰)—

;

>

C═

C(R¹²)(R¹³), >

C(R¹²)(R¹³), —

C≡

C—

, O, S(G)_a, P(J)_b(R¹⁰), P(J)_b(LR¹⁰), N(R¹⁰), >

N⁺(R¹²)(R¹³) and C(L);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

G is O or NR¹⁰;

J is S or O; and

L is S, O or NR¹⁰;

each R¹⁰ is a monovalent group independently selected from hydrogen and M¹-R¹⁴;

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

a direct link, arylene, het roarylene, cycloalkylene, C(R¹⁵)₂, —

C(R¹⁵)═

C(R¹⁵)—

, >

C═

C(R¹²)(R¹³), >

C(R¹²)(R¹³), —

C≡

C, O, S(G¹)_a, P(J)_b(R¹⁵), P(J)_b(LR¹⁵), N(R¹⁵), N(COR¹⁵), >

N⁺(R¹²)(R¹³) and C(L);

wh re a is 0, 1 or 2;

b is 0, 1, 2 or 3;

G¹ is O or NR¹⁵;

J is S or O; and

L is S, O or NR¹⁵;

R¹⁴ and R¹⁵ are each independently selected from the group among hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR¹⁶R¹⁷R¹⁸, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR¹⁹R²⁰;

R¹⁹ and R²⁰ are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R¹² and R¹³ are selected from (i) or (ii) as follows;

(i) R¹² and R¹³ are independently selected from among hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl;

or (ii) R¹² and R¹³ together form alkylene, alkenylene or cycloalkylene;

R¹⁶, R¹⁷ and R¹⁸ 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 NR¹⁹R²⁰; and

R¹¹, R^{12, R13}, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ can be 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)_hR³⁰, NR³⁰R³¹, COOR³⁰, COR³⁰, CONR³⁰R³¹, OC(O)NR³⁰R³¹, N(R³⁰)C(O)R³¹, 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

R³⁰ and R³¹ are each independ ntly selected from among hydrogen, halo, ps udohalo, 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; and

X is a negative counterion.

9. The compound of claim 8, wherein R is, or is a combination of, a saturated straight chain of 1 to 20 carbon atoms, a chain of 2 to 2000 ethyleneoxide moieties or a saturated or unsaturated carbocyclic moiety of 3 to 20 carbon atoms.

45. The compound of claim 8, wherein X is a halide or trifluoroacetate.

50. The compound of claim 8, wherein B is an amino reactive moiety selected from succininimidyl ester, hydroxybenzotriazolyl ester, or pentafluorophenol ester.

10. A compound that has one of formulae VII or VIII:

B—

R—

ONH₂.HX 

 

VII;

orB—

R—

ON═

C(R¹R²) 

 

VIII

or a derivative thereof, wherein;

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

arylene, heteroarylene, cycloalkylene, C(R¹⁰)₂, —

C(R¹⁰)═

C(R¹⁰)—

, >

C═

C(R¹²)(R¹³), >

C(R¹²)(R¹³), —

C≡

C—

, O, S(G)_a, P(J)_b(R¹⁰), P(J)_b(LR¹⁰), N(R¹⁰), >

N⁺(R¹²)(R¹³) and C(L);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

G is O or NR¹⁰;

J is S or O; and

L is S, O or NR¹⁰;

each R¹⁰ 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(R¹⁵)₂, —

C(R¹⁵)═

C(R¹⁵)—

, >

C═

C(R¹²)(R¹³), >

C(R¹²)(R¹³), —

C≡

C—

, O, S(G¹)_a, P(J)_b(R¹⁵), P(J)_b(LR¹⁵), N(R¹⁵), N(COR¹⁵), >

N⁺(R¹²)(R¹³) and C(L);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

G¹ is O or NR¹⁵;

J is S or O; and

L is S, O or NR¹⁵;

R¹⁴ and R¹⁵ are each independently selected from the group among hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR¹⁶R¹⁷R¹⁸, alkyl, alk nyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, h teroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalk nyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, h teroaralkoxy and NR¹⁹R²⁰;

R¹⁹ and R²⁰ are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R¹² and R¹³ are selected from (i) or (ii) as follows;

(i) R¹² and R¹³ are independently selected from among hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl;

or (ii) R¹² and R¹³ together form alkylene, alkenylene or cycloalkylene;

R¹⁶, R¹⁷ and R¹⁸ 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 NR¹⁹R²⁰; and

R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹, R¹⁸, R¹⁹ and R²⁰ can be 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)_hR³⁰, NR³⁰R³¹, COOR³⁰, COR³⁰, CONR³⁰R³¹, OC(O)NR³⁰R³¹, N(R³⁰)C(O)R³¹, 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

R³⁰ and R³¹ 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;

B is an amino or thiol reactive moiety;

R¹ is H or a saturated straight chain of 3 to 20 carbon atoms, a chain of 2 to 2000 ethyleneoxide moieties, a saturated or unsaturated carbocyclic moiety of 3 to 20 carbon atoms;

R² is a saturated straight chain of 3 to 20 carbon atoms, a chain of 2 to 2000 ethyleneoxide moieties, a saturated or unsaturated carbocyclic moiety of 3 to 20 carbon atoms; and

X is a negative counterion.

11. The compound of claim 10, wherein R is a straight chain, branched or cyclic aliphatic moiety, a aromatic, heteroaromatic, polyaromatic or polyheteroaromatic moiety, a saturated straight chain of 2 to 20 carbon atoms, a chain of 2 to 2000 ethyleneoxide moieties, a saturated or unsaturated carbocyclic moiety of 3 to 20 carbon atoms, or or a combination thereof.

12. The compound or claim 10, wherein R is a divalent aliphatic group.

13. The compound of claim 10, selected from:

46. The compound of claim 10, wherein X is a halide or trifluoroacetate.

51. The compound of claim 10, wherein B is an amino reactive moiety selected from succininimidyl ester, hydroxybenzotriazolyl ester, or pentafluorophenol ester.

53. The compound of claim 10, wherein B is a thiol reactive moiety selected from maleimido, α

-bromoacetyl or pyridyidisulfide.

14. A compound that has any of formulae XI:

15. The compound of claim 14, wherein R is a straight chain, branched or cyclic alkyl group of 2-15 carbons, a polyethyleneglycol moiety of 2-2000 monomers or an aromatic group, or a combination thereof.

16. The compound of claim 14 that is:

17. A compound that has one of formulae XII:

18. The compound of claim 17, wherein R is a straight chain, branched or cyclic alkyl group of 2-15 carbons, a polyethyleneglycol moiety of 2-2000 monomers or an aromatic group, or a combination thereof.

19. A compound that has any of formulae XIII:

R³ S—

R—

A—

NHNH₂.HX,R³⁰S—

R—

A—

NHN═

CR¹R²,(S—

R—

A—

NHNH₂.HX)₂, or(S—

R—

A—

NHN═

CR¹R²)₂;

or a derivative thereof, wherein

R¹ is H or a saturated straight chain of 3 to 20 carbon atoms, a chain of 2 to 2000 ethyleneoxide moieties, or a saturated or unsaturated carbocyclic moiety of 3 to 20 carbon atoms;

R² is a saturated straight chain of 3 to 20 carbon atoms, a chain of 2 to 2000 ethyleneoxide moieties, or a saturated or unsaturated carbocyclic moiety of 3 to 20 carbon atoms;

X is a negative counterion; and

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

arylene, heteroarylene, cycloalkylene, C(R¹⁰)₂, —

C(R¹⁰)═

C(R¹⁰)—

, >

C═

C(R¹²)(R¹³), >

C(R¹²)(R¹³), —

C≡

C—

, O, S(G)_a, P(J)_b(R¹⁰), P(J)_b(LR¹⁰), N(R¹⁰), >

N⁺(R¹²)(R¹³) and C(L);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

G is O or NR¹⁰;

J is S or O; and

L is S, O, or NR¹⁰;

each R¹⁰ is a monovalent group independently selected from hydrogen and M¹-R¹⁴;

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(R¹⁵)₂, —

C(R¹⁵)═

C(R¹⁵)—

, >

C═

C(R¹²)(R¹³), >

C(R¹²)(R¹³), —

C≡

C—

, O, S(G¹)_a, P(J)_b(R¹⁵), P(J)_b(LR¹⁵), N(R¹⁵), N(COR¹⁵), >

N⁺(R¹²(R¹³) and C(L);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

G¹ is O or NR¹⁵;

J is S or O; and

L is S, O, or NR¹⁵;

R¹⁴ and R¹⁵ are each independently selected from the group among hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR¹⁶R¹⁷R¹⁸, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, he teroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR¹⁹R²⁰;

R¹⁹ and R²⁰ are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R¹² and R¹³ are selected from (i) or (ii) as follows;

(i) R¹² and R¹³ are independently selected from among hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl;

or (ii) R¹² and R¹³ together form alkylene, alkenylene or cycloalkylene;

R16, R¹⁷ and R¹⁸ 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 NR¹⁹R²⁰; and

R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ can be 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)_hR³⁰, NR³⁰R³¹, COOR³⁰, COR³⁰, CONR³⁰R³¹, OC(O)NR³⁰R³¹, N(R³⁰)C(O)R³¹, 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

R³⁰ and R³¹ 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;

A is a direct link, C═

O, C═

S, NH(C═

O), NH(C═

S), NHNH(C═

O), or NHNH(C═

S); and

R³⁰ is hydrogen or a thiol protecting group.

20. The compound of claim 19 that is:

47. The compound of claim 19, wherein X is a halide or trifluoroacetate.

21. A compound that has one of formulae XII:

R³⁰S—

R—

ONH₂.HX,R³⁰S—

R—

ON═

CR¹R²,(S—

R—

ONH₂.HX)₂, or(S—

R—

ON═

CR¹R²)₂;

or a derivative thereof, wher in

R¹ is H or a saturated straight chain of 3 to 20 carbon atoms, a chain of 2 to 2000 ethyleneoxide moieties, or a saturated or unsaturated carbocyclic moiety of 3 to 20 carbon atoms;

R² is a saturated straight chain of 3 to 20 carbon atoms, a chain of 2 to 2000 ethyleneoxide moieties, or a saturated or unsaturated carbocyclic moiety of 3 to 20 carbon atoms;

X is a negative counterion; and

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

arylene, heteroarylene, cycloalkylene, C(R¹⁰)₂, —

C(R¹⁰)═

C(R¹⁰)—

, >

C═

C(R¹²)(R¹³), >

C(R¹²)(R¹³), —

C≡

C—

, O, S(G)_a, P(J)_b(R¹⁰), P(J)_b(LR¹⁰), N(R¹⁰), >

N⁺(R¹²)(R¹³) and C(L);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

G is O or NR¹⁰;

J is S or O; and

L is S, O, or NR¹⁰;

each R¹⁰ is a monovalent group independently selected from hydrogen and M¹-R¹⁴;

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(R¹⁵)₂, —

C(R¹⁵)═

C(R¹⁵)—

, >

C═

C(R¹²)(R¹³), >

C(R¹²)(R¹³), —

C≡

C—

, O, S(G¹)_a, P(J)_b(R¹⁵), P(J)_b(LR¹⁵), N(R¹⁵), N(COR¹⁵), >

N⁺(R¹²)(R¹³), and C(L);

where a is 0, 1 or 2;

b is 0, 1, 2 or 3;

G¹ is O or NR¹⁵;

J is S or O; and

L is S, O, or NR¹⁵;

R¹⁴ and R¹⁵ are each independently selected from the group among hydrogen, halo, pseudohalo, cyano, azido, nitro, SiR¹⁶R¹⁷R¹⁸, alkyl, alkenyl, alkynyl, haloalkyl, haloalkoxy, aryl, aralkyl, aralkenyl, aralkynyl, heteroaryl, heteroaralkyl, heteroaralkenyl, heteroaralkynyl, heterocyclyl, heterocyclylalkyl, heterocyclylalkenyl, heterocyclylalkynyl, hydroxy, alkoxy, aryloxy, aralkoxy, heteroaralkoxy and NR¹⁹R²⁰;

R¹⁹ and R²⁰ are each independently selected from hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl, aralkyl, heteroaryl, heteroaralkyl and heterocyclyl;

R¹² and R¹³ are selected from (i) or (ii) as follows;

(i) R¹² and R¹³ are independently s lected from among hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, aryl and heteroaryl;

or (ii) R¹² and R¹³ together form alkylene, alkenylene or cycloalkylene;

R¹⁶, R¹⁷ and R¹⁸ 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 NR¹⁹R²⁰; and

R¹¹, R¹², R¹³, R¹⁴, R¹⁵, R¹⁶, R¹⁷, R¹⁸, R¹⁹ and R²⁰ can be 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)_hR³⁰, NR³⁰R³¹, COOR³⁰, COR³⁰, CONR³⁰R³¹, OC(O)NR³⁰R³¹, N(R³⁰)C(O)R³¹, 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

R³⁰ and R³¹ 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; and

R³⁰ is hydrogen or a thiol protecting group.

48. The compound of claim 21, wherein X is a halide or trifluoroacetate.

31. A method of crosslinking a natural or synthetic biological molecule, comprising:

(i) preparing a conjugate of formula IVa;

 

or a derivative thereof, wherein;

A is NH(C═

O), NH(C═

S), NH(C═

NH), NHNH(C═

O), NHNH(C═

S), NHNH(C═

NH) or a direct bond;

B is a natural or synthetic biological molecule;

D is a carbon or nitrogen atom;

E is a carbon or nitrogen atom; and

X is a negative counter ion, oxygen, sulfur or —

NH; and

(ii) applying the conjugate to a surface wherein the surface has at least one carbonyl moiety for a time and under conditions such that the hydrazine moiety of the conjugate reacts with the carbonyl moiety of the surface forming a hydrazone bond to the surface.

32. A method of crosslinking a natural or synthetic biological molecule, comprising:

(i) preparing a conjugate of formula Va;

 

or a derivative thereof, wherein;

A is NH(C═

O), NH(C═

S), NH(C═

NH), NHNH(C═

O), NHNH(C═

S), NHNH(C═

NH) or a direct bond;

B is a natural or synthetic biological molecule;

D is a carbon or nitrogen atom;

E is a carbon or nitrogen atom;

R¹ is hydrogen or a saturated straight chain of 1 to 12 carbon atoms; and

R² is hydrogen or a saturated straight chain of 1 to 12 carbon atoms; and

(ii) applying the conjugate to a surface wherein the surface has at least one amino or one thiol reactive moiety for a time and under conditions such that the conjugate reacts with the amino or thiol reactive moiety of the surface forming a bond to the surface.

33. A method of crosslinking a natural or synthetic biological molecule, comprising:

(i) preparing a conjugate of the formula VIa;

 

or a derivative thereof, wherein;

B is a natural or synthetic biological molecule;

D is a carbon or nitrogen atom;

E is a carbon or nitrogen atom; and

X is a negative counter ion, oxygen, sulfur or —

NH; and

(ii) applying the conjugate to a surface wherein the surface has at least one carbonyl moiety for a time and under conditions such that the hydrazine moiety of the conjugate reacts with the carbonyl moiety of the surface forming a hydrazone bond to the surface.

34. A method of crosslinking a natural or synthetic biological molecule, comprising:

(i) preparing a conjugate of formula IVa;

 

or a derivative thereof, wherein;

A is NH(C═

O), NH(C═

S), NH(C═

NH), NHNH(C═

O), NHNH(C═

S), NHNH(C═

NH) or a direct bond;

B is a natural or synthetic biological molecule;

D is a carbon or nitrogen atom;

E is a carbon or nitrogen atom; and

X is a negative counter ion, oxygen, sulfur or —

NH; and

(ii) applying the conjugate to a surface wherein the surface has at least one amino or one thiol reactive moiety for a time and under conditions such that the conjugate reacts with the amino or thiol reactive moiety of the surface forming a bond to the surface.

35. A method of crosslinking a natural or synthetic biological molecule, comprising:

(i) preparing a conjugate of formula Va;

 

or a derivative thereof, wherein;

A is NH(C═

O), NH(C═

S), NH(C═

NH), NHNH(C═

O), NHNH(C═

S), NHNH(C═

NH) or a direct bond;

B is a natural or synthetic biological molecule;

D is a carbon or nitrog n atom;

E is a carbon or nitrogen atom;

R¹ is hydrogen or a saturated straight chain of 1 to 12 carbon atoms; and

R² is hydrogen or a saturated straight chain of 1 to 12 carbon atoms; and

(ii) applying the conjugate to a surface wherein the surface has at least one amino or one thiol reactive moiety for a time and under conditions such that the conjugate reacts with the amino or thiol reactive moiety of the surface forming a bond to the surface.

36. A method of crosslinking a natural or synthetic biological molecule, comprising:

(i) preparing a conjugate of formula VIa;

 

or a derivative thereof, wherein;

B is a natural or synthetic biological molecule;

D is a carbon or nitrogen atom;

E is a carbon or nitrogen atom; and

X is a negative counter ion, oxygen, sulfur or —

NH; and

(ii) applying the conjugate to a surface wherein the surface has at least one amino or one thiol reactive moiety for a time and under conditions such that the conjugate reacts with the amino or thiol reactive moiety of the surface forming a bond to the surface.

37. A method of crosslinking a natural or synthetic biological molecule, comprising:

(i) preparing a conjugate of formula IVa;

 

or a derivative thereof, wherein;

A is NH(C═

O), NH(C═

S), NH(C═

NH), NHNH(C═

O), NHNH(C═

S), NHNH(C═

NH) or a direct bond;

B is a natural or synthetic biological molecule;

D is a carbon or nitrogen atom;

E is a carbon or nitrogen atom; and

X is a negative counter ion, oxygen, sulfur or —

NH; and

(ii) mixing the conjugate to a natural or synthetic biological molecule, wherein the molecule has at least one carbonyl moiety, for a time and under conditions such that the hydrazine moiety of the conjugate reacts with the carbonyl moiety of the molecule forming a hydrazone bond to the molecule.

38. A method of crosslinking a natural or synthetic biological molecule, comprising:

(i) preparing a conjugate of formula Va;

 

or a derivative thereof, wherein;

A is NH(C═

O), NH(C═

S), NH(C═

NH), NHNH(C═

O), NHNH(C═

S), NHNH(C═

NH) or a direct bond;

B is a natural or synthetic biological molecule;

D is a carbon or nitrogen atom;

E is a carbon or nitrogen atom;

R¹ is hydrogen or a saturated straight chain of 1 to 12 carbon atoms; and

R² is hydrogen or a saturated straight chain of 1 to 12 carbon atoms; and

(ii) mixing the conjugate with a natural or synthetic biological molecule, wherein the molecule has at least one carbonyl moiety, for a time and under conditions such that the hydrazine moiety of the conjugate reacts with the carbonyl moiety of the molecule forming a hydrazone bond to the molecule.

39. A method of crosslinking a natural or synthetic biological molecule, comprising:

(i) preparing a conjugate of formula VIa;

 

or a derivative thereof, wherein;

B is a natural or synthetic biological molecule;

D is a carbon or nitrogen atom;

E is a carbon or nitrogen atom; and

X is a negative counter ion, oxygen, sulfur or —

NH; and

(ii) mixing the conjugate with a natural or synthetic biological molecule, wherein the molecule has at least one carbonyl moiety for a time and under conditions such that the hydrazine moiety of the conjugate reacts with the carbonyl moiety of the molecule forming a hydrazone bond to the molecule.