Chemoselective Fluorgenic Molecular Linkers and Methods for Their Preparation and Use

• US 20070224695A1
• Filed: 04/15/2005
• Published: 09/27/2007
• Est. Priority Date: 04/16/2004
• Status: Active Grant

• Current Assignee: University of South Carolina
• Original Assignee: University of South Carolina
• Inventors: Wang, Qian, Sivakumar, Krishnamoorthy
• Granted Patent: US 7,745,229 B2
• Time in Patent Office: Days
• US Class Current: 436/172
• CPC Class Codes: C07D 405/04 directly linked by a ring-member-to-ring-member bond
• CPC Class Codes: C07D 405/14 containing three or more hetero rings
• CPC Class Codes: C07D 491/14 Ortho-condensed systems
• CPC Class Codes: C07K 1/13 Labelling of peptides
• CPC Class Codes: C12Q 1/6818 involving interaction of two or more labels, e.g. resonant energy transfer
• CPC Class Codes: G01N 33/533 with fluorescent label
• CPC Class Codes: G01N 33/542 with steric inhibition or signal modification, e.g. fluorescent quenching
• CPC Class Codes: Y10S 436/80 Fluorescent dyes, e.g. rhodamine
• CPC Class Codes: Y10T 436/13 Tracers or tags

Abstract

The present invention describes a bioconjugation strategy and compounds that are useful therein in which a fluorescent signal is produced when two molecular or supramolecular entities are linked by chemoselective combination of one linker having an azido or halide substituent group with another linker having a cyano or an alkyne substituent group. A kit is also provided.

Claims

1. A method of forming a linkage complex having a fluorescent signal, the method comprising:

contacting a first linkage compound having the structure A-(Y)_y—

N₃, or A-(Y)_y—

X;

with a second linkage compound having the structure E-(Z)_z-C≡

CR^E, or E-(Z)_z-C≡

N;

to form a linkage complex having a fluorescent signal that is different than the fluorescent signal of the first linkage compound and the fluorescent signal of the second linkage compound;

where;

at least one of A and E is a fluorophore;

X is halo, and when the first linkage compound is A-(Y)_y—

X, the first linkage compound is contacted with the second linkage compound in the presence of an azide salt of lithium, sodium, or potassium;

R^E is selected from any chemically possible substituent group;

y and z are each independently an integer from 0 to 20; and

Y and Z are each independently a branched or unbranched alkyl, alkenyl, or alkynyl, which can be substituted or unsubstituted.

2. The method according to claim 1, wherein the linkage complex comprises a triazole or a tetrazole.

3. The method according to claim 1, wherein the fluorophore is selected from fluorescein, eosin, erythrosin, Oregon-green, naphthofluorescein, rhodol, rhodamine, Texas Red, polymethine dyes, cyanine, hemicyanine, streptocyanine, sulfo and aza derivatives of rhodamine, resorufin, boron dipyrromethene derivatives (BODIPY), connexin, azo dyes, malachite green, isosulfan blue, naphthalene derivatives, dansyl dyes, anthracene derivatives, pyrene derivatives pyridyloxazoles, or coumarin.

4. The method according to claim 1, wherein the fluorophore is a substituted or unsubstituted coumarin, rhodamine, or fluorescein.

5. The method according to claim 1, wherein the fluorophore is a substituted or unsubstituted coumarin.

6. The method according to claim 1, wherein the first linkage compound comprises a compound having the structure:

7. The method according to claim 6, wherein:

at least one of R² and R³ is —

(Y)_y—

N₃, or —

(Y)_y—

X, and the other is any R¹ group; and

n is an integer from 0 to 4;

m is 0 or 1; and

each R¹ is the same or different and is selected from hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, halo, carboxyl, carbonyl, alkyloxyl, alkoxyl, alkoxyalkyl, amino, alkylamino, azido, hydroxyalkyl, sulfonyl, sulfinyl, phospho, phosphino, or optionally two or more R¹ groups join to form one or more substituted or unsubstituted rings.

8. The method according to claim 7, wherein at least one of R² and R³ is —

(Y)_y—

N₃.

9. The method according to claim 7, wherein L^A is an organic substituent group having at least one reactive group that is selected from carboxylic acid esters, sulfonyl halide, acyl halide, aldehydes, reactive ketones, aromatic halides, bromoacetamides, idoacetamides, maleimides, disulfides, hydroxyl, amines, diazonium compounds, active alkylating reagents, active acylating reagents, or diketones.

10. The method according to claim 7, wherein L^A is an organic substituent group that is selected from branched or unbranched C₁-C₁₂ alkyl, C₁-C₁₂ alkenyl, C₁-C₁₂ alkynyl, or aryl, heteroaryl, or heterocyclyl, and which has at least one substituent reactive group that is selected from carboxylic acid esters, sulfonyl halide, acyl halide, aldehydes, reactive ketones, aromatic halides, bromoacetamides, idoacetamides, maleimides, disulfides, hydroxyl, amines, diazonium compounds, active alkylating reagents, active acylating reagents, or diketones.

11. The method according to claim 6, wherein at least one of R² and R³ is —

(Y)_y—

X.

12. The method according to claim 11, wherein:

X is selected from Br, or Cl;

the coumarin is substituted at carbon 7 with a group selected from hydrogen, hydroxyl, amino, alkyl, alkenyl, alkoxyl, or alkylamine; and

the R² or R³ that is not —

(Y)_y—

X is selected from hydrogen, amino, nitro, carboxyl, or halo.

13. The method according to claim 1, wherein the first linkage compound is chemically bonded to a biomaterial.

14. The method according to claim 13, wherein the biomaterial is selected from small molecules, drugs, ligands, catalysts, peptides, proteins, nucleic acids, oligonucleotides, saccharides, viruses, cells, organic polymers, inorganic polymers, nanoparticles, or solid surfaces.

15. The method according to claim 6, wherein E is selected from hydrogen, hydroxyl, carboxyl, amino, alkylamino, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxyl, alkoxyalkyl, alkoxyalkenyl, carboxyalkyl, amino, aminoalkyl, aryl, heteroaryl, or heterocyclyl, and if substituted, at least one substituent is optionally L^B, where L^B is a linking group.

16. The method according to claim 1, wherein the second linkage compound comprises a compound having the structure:

17. The method according to claim 16, wherein:

at least one of R² and R³ is -(Z)_z-C≡

CR^E, or -(Z)_z-C≡

N, and the other is any R¹ group; and

n is an integer from 0 to 4;

m is 0 or 1; and

each R¹ is the same or different and is selected from hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, halo, carboxyl, carbonyl, alkyloxyl, alkoxyl, alkoxyalkyl, amino, alkylamino, azido, hydroxyalkyl, sulfonyl, sulfinyl, phospho, phosphino, or optionally two or more R¹ groups join to form one or more substituted or unsubstituted rings.

18. The method according to claim 16, wherein R^E is selected from —

H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkyl-R¹¹, C₂-C₆ alkenyl-R¹¹, C₂-C₆ alkynyl-R¹¹, C₁-C₆ alkyl-(R¹¹)₂, C₂-C₆ alkenyl-(R¹¹)₂, CSR¹¹, N═

NR⁷, amino, CONHR¹¹, NHR⁷, NR⁸R⁹, N(R⁷)—

N(R⁸)(R⁹), C(R¹¹)═

N—

N(R⁸)(R⁹), N═

N(R⁷), N(R⁷)—

N═

C(R⁸), C(R¹¹)═

N—

O(R¹⁰), ON═

C(R¹¹), C₁-C₆ alkyl-NHR⁷, C₁-C₆ alkyl-NR⁸R⁹, (C₁-C₄)alkyl-N(R⁷)—

N(R⁸)(R⁹), (C₁-C₄)alkylC(R¹¹)═

N—

N(R⁸)(R⁹), (C₁-C₄)alkyl-N═

N(R⁷), (C₁-C₄)alkyl-N(R⁷)—

N═

C(R⁸), nitro, cyano, CO₂R¹¹, O—

R¹⁰, C₁-C₄ alkyl-OR¹⁰, COR¹¹, SR¹⁰, SSR¹⁰, SOR¹¹, SO₂R¹¹, C₁-C₆ alkyl-COR¹¹, C₁-C₆ alkyl-SR¹⁰, C₁-C₆ alkyl-SOR¹¹, C₁-C₆ alkyl-SO₂R¹¹, halo, Si(R¹¹)₃, halo C₁-C₄ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R¹²;

R⁷R⁸ and R⁹ are each independently selected from —

H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₄ alkyl-R¹¹, C₁-C₆ alkyl-NHR¹³, C₁-C₆ alkyl-NR¹³R¹⁴, O—

R¹⁵, C₁-C₄ alkyl-OR¹⁵, CO₂R¹⁵, C(S)OR¹⁵, C(O)SR¹⁵, C(O)R¹⁷, C(S)R¹⁷, CONHR¹⁶, C(S)NHR¹⁶, CON(R¹⁶)₂, C(S)N(R¹⁶)₂, SR¹⁵, SOR¹⁷, SO₂R¹⁷, C₁-C₆ alkyl-CO₂R¹⁵, C₁-C₆ alkyl-C(S)OR¹⁵, C₁-C₆ alkyl-C(O)SR¹⁵, C₁-C₆ alkyl-COR¹⁷, C₁-C₆ alkyl-C(S)R¹⁷, C₁-C₆ alkyl-CONHR¹⁶, C₁-C₆ alkyl-C(S)NHR¹⁶, C₁-C₆ alkyl-CON(R¹⁶)₂, C₁-C₆ alkyl-C(S)N(R¹⁶)₂, C₁-C₆ alkyl-SR¹⁵, C₁-C₆ alkyl-SOR⁷, C₁-C₆ alkyl-SO₂R¹⁷, halo C₁-C₄ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R¹⁸;

R¹⁰ is selected from —

H, C₁-C₁₀ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkyl-NHR¹³, C₁-C₆ alkyl-NR¹³R¹⁴, C₁-C₄ alkyl-OR¹⁵, CSR¹¹, CO₂R¹⁵, C(S)OR¹⁵, C(O)SR¹⁵, COR¹⁷, C(S)R¹⁷, CONHR¹⁶, C₁-C₄ alkyl-R¹¹, C₁-C₄ alkyl-NH₂R¹³, C(S)NHR¹⁶, O—

R¹⁵, CON(R¹⁶)₂, C(S)N(R¹⁶)₂, SOR¹⁷, SO₂R¹⁷, C₁-C₆ alkyl-CO₂R¹⁵, C₁-C₆ alkyl-C(S)OR¹⁵, C₁-C₆ alkyl-C(O)SR¹⁵, C₁-C₆ alkyl-COR¹⁷, C₁-C₆ alkyl-C(S)R¹⁷, C₁-C₆ alkyl-CONHR¹⁶, C₁-C₆ alkyl-C(S)NHR¹⁶, C₁-C₆ alkyl-CON(R¹⁶)₂, Si(R¹³)₂R¹⁷, C₁-C₆ alkyl-C(S)N(R¹⁶)₂, C₁-C₆ alkyl-SR¹⁵, C₁-C₆ alkyl-SOR¹⁷, C₁-C₆ alkyl-SO₂R¹⁷, halo C₁-C₄ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R¹⁸;

R¹¹ is selected from —

H, C₁-C₆ alkyl, C₁-C₆ alkoxy, C₂-C₆ alkenyl, C₂-C₆ alkynyl, amino, NHR¹³, NR¹³R¹⁴, N═

NR¹³, C₁-C₆ alkyl-NHR¹³, C₁-C₆ alkyl-NR¹³R¹⁴, O—

R¹⁵, C₁-C₄ alkyl-OR¹⁵, SR¹⁵, COR¹³, CO₂R¹⁷, C₁-C₆ alkyl-CO₂R¹⁵, C₁-C₆ alkyl-C(S)OR¹⁵, C₁-C₆ alkyl-C(O)SR¹⁵, C₁-C₆ alkyl-COR¹⁷, C₁-C₆ alkyl-C(S)R¹⁷, C₁-C₆ alkyl-CONHR¹⁵, C₁-C₆ alkyl-C(S)NHR¹⁶, C₁-C₆ alkyl-CON(R¹⁶)₂, C₁-C₆ alkyl-C(S)N(R¹⁶)₂, C₁-C₆ alkyl-SR¹⁵, C₁-C₆ alkyl-SOR¹⁷, C₁-C₆ alkyl-SO₂R¹⁷, halo, halo C₁-C₄ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R¹⁸;

R¹² is selected from —

H, OH, oxo, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkyl-R¹¹, C₂-C₁₀ alkenyl-R¹¹, C₂-C₁₀ alkynyl-R¹¹, C₁-C₁₀ alkyl-(R¹¹)₂, C₂-C₁₀ alkenyl-(R¹¹)₂, CSR¹¹, N═

NR⁷, hydroxyl C₁-C₆ alkyl-R¹¹, amino C₁-C₄ alkyl-R⁷, amino, NHR⁷, NR⁸R⁹, N(R⁷)—

N(R⁸)(R⁹), C(R¹¹)═

N—

N(R⁸)(R⁹), N═

N(R⁷), N(R⁷)—

N═

C(R⁸), C(R¹¹)═

N—

O(R¹⁰), ON═

C(R¹¹), C₁-C₁₀ alkyl-NHR⁷, C₁-C₁₀ alkyl-NR⁸R⁹, (C₁-C₁₀)alkyl-N(R⁷)—

N(R⁸)(R⁹), (C₁-C₁₀)alkylC(R¹¹)═

N—

N(R⁸)(R⁹), (C₁-C₁₀)alkyl-N═

N(R⁷), (C₁-C₁₀)alkyl-N(R⁷)—

N═

C(R⁸), SCN, NCS, C₁-C₁₀ alkyl SCN, C₁-C₁₀ alkyl NCS, nitro, cyano, O—

R¹⁰, C₁-C₁₀ alkyl-OR¹⁰, COR¹¹, CO₂R¹¹, SR¹⁰, SSR¹⁰, SOR¹¹, SO₂R¹¹, C₁-C₁₀ alkyl-COR¹¹, C₁-C₁₀ alkyl-SR¹⁰, C₁-C₁₀ alkyl-SOR¹¹, C₁-C₁₀ alkyl-SO₂R¹¹, halo, Si(R¹¹)₃, halo C₁-C₁₀ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R¹⁸;

R¹³ and R¹⁴ are each independently selected from —

H, oxo, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₄ alkyl-R²³, C₁-C₆ alkyl-NHR¹⁹, C₁-C₆ alkyl-NR¹⁹R²⁰, O—

R²¹, C₁-C₄ alkyl-OR²¹, CO₂R²¹, COR²¹, C(S)OR²¹, C(O)SR²¹, C(O)R²³, C(S)R²³, CONHR²², C(S)NHR²², CON(R²²)₂, C(S)N(R²²)₂, SR²¹, SOR²³, SO₂R²³, C₁-C₆ alkyl-CO₂R²¹, C₁-C₆ alkyl-C(S)OR²¹, C₁-C₆ alkyl-C(O)SR²¹, C₁-C₆ alkyl-COR²³, C₁-C₆ alkyl-C(S)R²³, C1-C₆ alkyl-CONHR²², C1-C₆ alkyl-C(S)NHR²², C₁-C₆ alkyl-CON(R²²)₂, C₁-C₆ alkyl-C(S)N(R²²)₂, C₁-C₆ alkyl-SR²¹, C₁-C₆ alkyl-SOR²³, C₁-C₆ alkyl-SO₂R²³, halo C₁-C₄ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R²⁴;

R¹⁵ and R¹⁶ are independently selected from —

H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkyl-NHR¹⁹, C₁-C₆ alkyl-NR¹⁹R²⁰, C₁-C₄ alkyl-OR²¹, CSR¹¹, CO₂R²², COR²³, CONHR²², CON(R²²)₂, SOR²³, SO₂R²³, C₁-C₆ alkyl-CO₂R²², C₁-C₆ alkyl-COR²³, C₁-C₆ alkyl-CONHR²², C₁-C₆ alkyl-CON(R²²)₂, C₁-C₆ alkyl-SR²¹, C₁-C₆ alkyl-SOR²³, C₁-C₆ alkyl-SO₂R²³, halo C₁-C₄ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R²⁴;

R¹⁷ is selected from —

H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkenyl-R¹⁹, C₁-C₆ alkyl-R¹⁹, C₂-C₆ alkynyl, amino, NHR¹⁹, NR¹⁹R²⁰, C₁-C₆ alkyl-NHR¹⁹, C₁-C₆ alkyl-NR¹⁹R²⁰, O—

R²¹, C₁-C₄ alkyl-OR²¹, SR²¹, C₁-C₆ alkyl-CO₂R²¹, C₁-C₆ alkyl-C(S)OR²¹, C₁-C₆ alkyl-C(O)SR²¹, C₁-C₆ alkyl-COR²³, C₁-C₆ alkyl-C(S)R²³, C₁-C₆ alkyl-CONHR²², C₁-C₆ alkyl-C(S)NHR²², C₁-C₆ alkyl-CON(R²²)₂, C₁-C₆ alkyl-C(S)N(R²²)₂, C₁-C₆ alkyl-SR²¹, C₁-C₆ alkyl-SOR²³, C₁-C₆ alkyl-SO₂R²³, halo C₁-C₄ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R²⁴;

R¹⁸ is selected from —

H, oxo, OH, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkyl-R²³, C₂-C₁₀ alkenyl-R²³, C₂-C₁₀ alkynyl-R²³, C₁-C₁₀ alkyl-(R²³)₂, C₂-C₁₀ alkenyl-(R²³)₂, CSR²³, N═

NR¹⁹, amino, NHR¹⁹, NR²⁰R²⁰, N(R¹⁹)—

N(R²⁰)(R²⁰), C(R²³)═

N—

N(R²⁰)(R²⁰), N═

N(R¹⁹), N(R¹⁹)—

N═

C(R²⁰), C(R²³)═

N—

O(R²¹), ON═

C(R²³), C₁-C₁₀ alkyl-NHR¹⁹, C₁-C₁₀ alkyl-NR²⁰R²⁰, (C₁-C₁₀)alkyl-N(R¹⁹)—

N(R²⁰)(R²⁰), (C₁-C₁₀)alkylC(R²³)═

N—

N(R²⁰)(R²⁰), (C₁-C₁₀)alkyl-N═

N(R¹⁹), (C₁-C₁₀)alkyl-N(R¹⁹)—

N═

C(R²⁰), SCN, NCS, C₁-C₁₀ alkyl SCN, C₁-C₁₀ alkyl NCS, nitro, cyano, O—

R²¹, C₁-C₁₀ alkyl-OR²¹, COR²³, CO₂R²³, SR²¹, SSR²¹, SOR²³, SO₂R²³, C₁-C₁₀ alkyl-COR²³, C₁-C₁₀ alkyl-SR²¹, C₁-C₁₀ alkyl-SOR²³, C₁-C₁₀ alkyl-SO₂R²³, halo, Si(R²³)₃, halo C₁-C₁₀ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R²⁴;

R¹⁹ and R²⁰ are each independently selected from —

H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₄ alkyl-R²⁹, C₁-C₆ alkyl-NHR²⁵, C₁-C₆ alkyl-NR²⁵R²⁶, O—

R²⁷, C₁-C₄ alkyl-OR²⁷, CO₂R²⁷, C(S)OR²⁷, C(O)SR²⁷, C(O)R²⁹, C(S)R²⁹, CONHR²⁸, C(S)NHR²⁸, CON(R²⁸)₂, C(S)N(R²⁸)₂, SR²⁷, SOR²⁹, SO₂R²⁹, C₁-C₆ alkyl-CO₂R²⁷, C₁-C₆ alkyl C(S)OR²⁷, C₁-C₆ alkyl-C(O)SR²⁷, C₁-C₆ alkyl-COR²⁹, C₁-C₆ alkyl-C(S)R²⁹, C₁-C₆ alkyl-CONHR²⁸, C₁-C₆ alkyl-C(S)NHR²⁸, C₁-C₆ alkyl-CON(R²⁸)₂, C₁-C₆ alkyl-C(S)N(R²⁸)₂, C₁-C₆ alkyl-SR²⁷, C₁-C₆ alkyl-SOR²⁹, C₁-C₆ alkyl-SO₂R²⁹, halo C₁-C₄ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R³⁰;

R²¹ and R²² are independently selected from —

H, C₁-C₁₀ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkyl-NHR²⁵, C₁-C₆ alkyl-NR²⁵R²⁶, C₁-C₄ alkyl-OR²⁷, CSR¹¹, CO₂R²⁸, COR²⁹, CONHR²⁸, CON(R²⁸)₂, SOR²⁹, SO₂R²⁹, C₁-C₆ alkyl-CO₂R²⁸, C₁-C₆ alkyl-COR²⁹, C₁-C₆ alkyl-CONHR²⁸, C₁-C₆ alkyl-CON(R²⁸)₂, C₁-C₆ alkyl-SR²⁷, C₁-C₆ alkyl-SOR²⁹, C₁-C₆ alkyl-SO₂R²⁹, halo C₁-C₄ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R³⁰;

R²³ is selected from —

H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkenyl-R²⁵, C₁-C₆ alkyl-R²⁵, C₂-C₆ alkynyl, amino, NHR²⁵, NR²⁵R²⁶, C₁-C₆ alkyl-NHR²⁵, C₁-C₆ alkyl-NR²⁵R²⁶, O—

R²⁷, C₁-C₄ alkyl-OR²⁷, SR²⁷, C₁-C₆ alkyl-CO₂R²⁷, C₁-C₆ alkyl-C(S)OR²⁷, C₁-C₆ alkyl-C(O)SR²⁷, C₁-C₆ alkyl-COR²⁹, C₁-C₆ alkyl-C(S)R²⁹, C₁-C₆ alkyl-CONHR²⁸, C₁-C₆ alkyl-C(S)NHR²⁸, C₁-C₆ alkyl-CON(R²⁸)₂, C₁-C₆ alkyl-C(S)N(R²⁸)₂, C₁-C₆ alkyl-SR²⁷, C₁-C₆ alkyl-SOR²⁹, C₁-C₆ alkyl-SO₂R²⁹, halo C₁-C₄ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R³⁰;

R²⁴ is selected from —

H, OH, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkyl-R²⁹, C₂-C₁₀ alkenyl-R²⁹, C₂-C₁₀ alkynyl-R²⁹, C₁-C₁₀ alkyl-(R²⁹)₂, C₂-C₁₀ alkenyl-(R²⁹)₂, CSR²⁹, N═

NR²⁵, amino, NHR²⁵, NR²⁶R²⁶, N(R²⁵)—

N(R²⁶)(R²⁶), C(R²⁹)═

N—

N(R²⁶)(R²⁶), N═

N(R²⁵), N(R²⁵)—

N═

C(R²⁶), C(R²⁹)═

N—

O(R²⁷), ON═

C(R²⁹), C₁-C₁₀ alkyl-NHR²¹, C₁-C₁₀ alkyl-NR²⁶R²⁶, (C₁-C₁₀)alkyl-N(R²⁵)—

N(R²⁶)(R²⁶), (C₁-C₁₀)alkylC(R²⁹)═

N—

N(R²⁶)(R²⁶), (C₁-C₁₀)alkyl-N═

N(R²⁵), (C₁-C₁₀)alkyl-N(R²⁵)—

N═

C(R²⁶), SCN, NCS, C₁-C₁₀ alkyl SCN, C₁-C₁₀ alkyl NCS, nitro, cyano, O—

R²⁷, C₁-C₁₀ alkyl-OR²⁷, CO₂R²⁹, COR²⁹, SR²⁷, SSR²⁷, SOR²⁹, SO₂R²⁹, C₁-C₁₀ alkyl-COR²⁹, C₁-C₁₀ alkyl-SR²⁷, C₁-C₁₀ alkyl-SOR²⁹, C₁-C₁₀ alkyl-SO₂R²⁹, halo, Si(R²⁹)₃, halo C₁-C₁₀ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R³⁰;

R²⁵ and R²⁶ are each independently selected from —

H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₄ alkyl-R³⁵, C₁-C₆ alkyl-NHR³¹, C₁-C₆ alkyl-NR³¹R³², O—

R³³, C₁-C₄ alkyl-OR³³, CO₂R³³, C(S)OR³³, C(O)SR³³, C(O)R³⁵, C(S)R³⁵, CONHR³⁴, C(S)NHR³, CON(R³⁴)₂, C(S)N(R³⁴)₂, SR³³, SOR³⁵, SO₂R³⁵, C₁-C₆ alkyl-CO₂R³³, C₁-C₆ alkyl C(S)OR³³, C₁-C₆ alkyl-C(O)SR³³, C₁-C₆ alkyl-COR³⁵, C₁-C₆ alkyl-C(S)R³⁵, C₁-C₆ alkyl-CONHR³⁴, C₁-C₆ alkyl-C(S)NHR³⁴, C₁-C₆ alkyl-CON(R³⁴)₂, C₁-C₆ alkyl-C(S)N(R³⁴)₂, C₁-C₆ alkyl-SR³³, C₁-C₆ alkyl-SOR³⁵, C₁-C₆ alkyl-SO₂R³⁵, halo C₁-C₄ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R³⁶;

R²⁷ and R²⁸ are independently selected from —

H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkynyl, C₁-C₆ alkyl-NHR³¹, C₁-C₆ alkyl-NR³¹R³², C₁-C₄ alkyl-OR³³, CSR¹¹, CO₂R³⁴, COR³⁵, CONHR³⁴, CON(R³⁴)₂, SOR³⁵, SO₂R³⁵, C₁-C₆ alkyl-CO₂R³⁴, C₁-C₆ alkyl-COR³⁵, C₁-C₆ alkyl-CONHR³⁴, C₁-C₆ alkyl-CON(R³⁴)₂, C₁-C₆ alkyl-SR³³, C₁-C₆ alkyl-SOR³⁵, C₁-C₆ alkyl-SO₂R³⁵, halo C₁-C₄ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R³⁶;

R²⁹ is selected from —

H, C₁-C₆ alkyl, C₂-C₆ alkenyl, C₂-C₆ alkenyl-R³¹, C₁-C₆ alkyl-R³¹, C₂-C₆ alkynyl, amino, NHR³¹, NR³¹R³², C₁-C₆ alkyl-NHR³¹, C₁-C₆ alkyl-NR³¹R³², O—

R³³, C₁-C₄ alkyl-OR³³, SR³³, C₁-C₆ alkyl-CO₂R³³, C₁-C₆ alkyl-C(S)OR³³, C₁-C₆ alkyl-C(O)SR³³, C₁-C₆ alkyl-COR³⁵, C₁-C₆ alkyl-C(S)R³⁵, C₁-C₆ alkyl-CONHR³⁴, C₁-C₆ alkyl-C(S)NHR³⁴, C₁-C₆ alkyl-CON(R³⁴)₂, C₁-C₆ alkyl-C(S)N(R³⁴)₂, C₁-C₆ alkyl-SR³³, C₁-C₆ alkyl-SOR³⁵, C₁-C₆ alkyl-SO₂R³⁵, halo C₁-C₄ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R³⁶;

R³⁰ is selected from —

H, OH, C₁-C₁₀ alkyl, C₂-C₁₀ alkenyl, C₂-C₁₀ alkynyl, C₁-C₁₀ alkyl-R³⁵, C₂-C₁₀ alkenyl-R³⁵, C₂-C₁₀ alkynyl-R³⁵, C₁-C₁₀ alkyl-(R³⁵)₂, C₂-C₁₀ alkenyl-(R³⁵)₂, CSR³⁵, N═

NR³¹, amino, NHR³¹, NR³²R³², N(R³¹)—

N(R³²)(R³²), C(R³⁵)═

N—

N(R³²)(R³²), N═

N(R³¹), N(R³¹)—

N═

C(R³²), C(R³⁵)═

N—

O(R³³), ON═

C(R³⁵), C₁-C₁₀ alkyl-NHR³¹, C₁-C₁₀ alkyl-NR³²R³², (C₁-C₁₀)alkyl-N(R³¹)—

N(R³²)(R³²), (C₁-C₁₀)alkylC(R³⁵)═

N—

N(R³²)(R³²), (C₁-C₁₀)alkyl-N═

N(R³¹), (C₁-C₁₀)alkyl-N(R³¹)—

N═

C(R³²), SCN, NCS, C₁-C₁₀ alkyl SCN, C₁-C₁₀ alkyl NCS, nitro, cyano, O—

R³³, C₁-C₁₀ alkyl-OR³³, COR³⁵, SR³³, SSR³³, SOR³⁵, SO₂R³⁵, C₁-C₁₀ alkyl-COR³⁵, C₁-C₁₀ alkyl-SR³³, C₁-C₁₀ alkyl-SOR³⁵, C₁-C₁₀ alkyl-SO₂R³⁵, halo, Si(R³⁵)₃, halo C₁-C₁₀ alkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R³⁶;

R³¹, R³², R³³ and R³⁴ are each independently selected from —

H, alkyl, alkenyl, alkynyl, aminoalkyl, hydroxyalkyl, alkylamino alkyl, dialkylaminoalkyl, alkoxyalkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R³⁶;

R³⁵ is selected from —

H, alkyl, alkenyl, alkynyl, aminoalkyl, OH, alkoxy, amino, alkylamino, dialkylamino, hydroxyalkyl, alkylamino alkyl, dialkylaminoalkyl, alkoxyalkyl, aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl, wherein aryl, heteroaryl, heterocyclyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heteroarylalkyl, heterocyclylalkyl, and C₁-C₁₀ mono- and bicyclic cycloalkyl are optionally substituted with one or more of the groups defined by R³⁶; and

R³⁶ is selected from —

H, alkyl, alkenyl, alkynyl, aminoalkyl, OH, alkoxy, amino, nitro, cyano, halo, alkylamino, dialkylamino, hydroxyalkyl, alkylamino alkyl, dialkylaminoalkyl, alkoxyalkyl, aryl, heteroaryl, heterocyclyl, cycloalkyl, alkylaryl, alkylheterocyclyl, alkylheteroaryl, arylalkyl, heterocyclylalkyl, and heteroarylalkyl.

19. The method according to claim 17, wherein at least one of R² and R³ is -(Z)_z-C≡

CH.

20. The method according to claim 17, wherein L^B is an organic substituent group having at least one reactive group that is selected from carboxylic acid esters, sulfonyl halide, acyl halide, aldehydes, reactive ketones, aromatic halides, bromoacetamides, idoacetamides, maleimides, disulfides, hydroxyl, amines, diazonium compounds, active alkylating reagents, active acylating reagents, or diketones.

21. The method according to claim 17, wherein L^B is an organic substituent group that is selected from branched or unbranched C₁-C₁₂ alkyl, C₁-C₁₂ alkenyl, C₁-C₁₂ alkynyl, or aryl, heteroaryl, or heterocyclyl, and which has at least one substituent reactive group that is selected from carboxylic acid esters, sulfonyl halide, acyl halide, aldehydes, reactive ketones, aromatic halides, bromoacetamides, idoacetamides, maleimides, disulfides, hydroxyl, amines, diazonium compounds, active alkylating reagents, active acylating reagents, or diketones.

22. The method according to claim 16, wherein at least one of R² and R³ is -(Z)_z-C≡

N.

23. The method according to claim 1, wherein the second linkage compound is chemically bonded to a biomaterial.

24. The method according to claim 23, wherein the biomaterial is selected from small molecules, drugs, ligands, catalysts, peptides, proteins, nucleic acids, oligonucleotides, saccharides, viruses, cells, organic polymers, inorganic polymers, nanoparticles, or solid surfaces.

25. The method according to claim 16, wherein A is selected from hydrogen, hydroxyl, carboxyl, amino, alkylamino, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxyl, alkoxyalkyl, alkoxyalkenyl, carboxyalkyl, amino, aminoalkyl, aryl, heteroaryl, or heterocyclyl, and if substituted, at least one substituent is optionally L^A, where L^A is a linking group.

26. The method according to claim 2, wherein the linkage complex having a fluorescent signal comprises the structure:

27. The method according to claim 2, wherein the linkage complex having a fluorescent signal comprises the structure

28. The method according to claim 27, wherein each of SA and SB is selected from small molecules, drugs, ligands, catalysts, peptides, proteins, nucleic acids, oligonucleotides, saccharides, viruses, cells, organic polymers, inorganic polymers, nanoparticles, solid surfaces.

29. A method of detecting the formation of a linkage between a first material and a second material, the method comprising contacting a first linkage compound having the structure

A-(Y)_y—

N₃, or A-(Y)_y—

X;

with a second linkage compound having the structure

E-(Z)_z-C≡

CR^E, or E-(Z)_z-C≡

N;

to form a linkage complex having a fluorescent signal that is different than the fluorescent signal of the first linkage compound and the fluorescent signal of the second linkage compound;

where;

at least one of A and E is a fluorophore, and any A or E that is not a fluorophore is an organic or inorganic group;

X is halo, and when the first linkage compound is A-(Y)_y—

X, the first linkage compound is contacted with the second linkage compound in the presence of an azide salt of lithium, sodium, or potassium;

R^E is selected from any chemically possible substituent group;

y and z are each independently an integer from 0 to 20;

Y and Z are each independently a branched or unbranched alkyl, alkenyl, or alkynyl, which can be substituted or unsubstituted; and

measuring the fluorescent signal of the linkage complex.

30. A kit for forming a linkage complex having a fluorescent signal, the kit comprising a first linkage compound having the structure

A-(Y)_y—

N₃, or A-(Y)_y—

X;

and a second linkage compound having the structure

E-(Z)_z-C≡

CR^E, or E-(Z)_z-C≡

N;

where;

at least one of A and E is a fluorophore, and any A or E that is not a fluorophore is an organic or inorganic group;

X is halo, and when the first linkage compound is A-(Y)_y—

X, the first linkage compound is contacted with the second linkage compound in the presence of an azide salt of lithium, sodium, or potassium;

R^E is selected from any chemically possible substituent group;

y and z are each independently an integer from 0 to 20;

Y and Z are each independently a branched or unbranched alkyl, alkenyl, or alkynyl, which can be substituted or unsubstituted.

31. The kit according to claim 30, wherein the fluorophore is selected from fluorescein, eosin, erythrosin, Oregon-green, naphthofluorescein, rhodol, rhodamine, Texas Red, polymethine dyes, cyanine, hemicyanine, streptocyanine, sulfo and aza derivatives of rhodamine, resorufin, boron dipyrromethene derivatives (BODIPY), connexin, azo dyes, malachite green, isosulfan blue, naphthalene derivatives, dansyl dyes, anthracene derivatives, pyrene derivatives pyridyloxazoles, or coumarin.

32. The kit according to claim 30, wherein the fluorophore is a substituted or unsubstituted coumarin, rhodamine, or fluorescein.

33. The kit according to claim 30, wherein the fluorophore is a substituted or unsubstituted coumarin.

34. The kit according to claim 30, wherein the first linkage compound comprises a compound having the structure:

35. The kit according to claim 34, wherein:

at least one of R² and R³ is —

(Y)_y—

N₃, or —

Y)_y—

X, and the other is any R¹ group; and

n is an integer from 0 to 4;

m is 0 or 1; and

each R¹ is the same or different and is selected from hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, halo, carboxyl, carbonyl, alkyloxyl, alkoxyl, alkoxyalkyl, amino, alkylamino, azido, hydroxyalkyl, sulfonyl, sulfinyl, phospho, phosphino, or optionally two or more R¹ groups join to form one or more substituted or unsubstituted rings.

36. The kit according to claim 34, wherein at least one of R² and R³ is —

(Y)_y—

N₃.

37. The kit according to claim 34, wherein L^A is an organic substituent group having at least one reactive group that is selected from carboxylic acid esters, sulfonyl halide, acyl halide, aldehydes, reactive ketones, aromatic halides, bromoacetamides, idoacetamides, maleimides, disulfides, hydroxyl, amines, diazonium compounds, active alkylating reagents, active acylating reagents, or diketones.

38. The kit according to claim 34, wherein at least one of R² and R³ is —

(Y)_y—

X.

39. The kit according to claim 38, wherein:

X is selected from Br, or Cl;

the coumarin is substituted at carbon 7 with a group selected from hydrogen, hydroxyl, amino, alkyl, alkenyl, alkoxyl, or alkylamine; and

the R² or R³ that is not —

Y)_y—

X is selected from hydrogen, amino, nitro, carboxyl, or halo.

40. The kit according to claim 30, wherein the first linkage compound is chemically bonded to a biomaterial.

41. The kit according to claim 40, wherein the biomaterial is selected from small molecules, drugs, ligands, catalysts, peptides, proteins, nucleic acids, oligonucleotides, saccharides, viruses, cells, organic polymers, inorganic polymers, nanoparticles, or solid surfaces.

42. The kit according to claim 30, wherein E is selected from hydrogen, hydroxyl, carboxyl, amino, alkylamino, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxyl, alkoxyalkyl, alkoxyalkenyl, carboxyalkyl, amino, aminoalkyl, aryl, heteroaryl, or heterocyclyl, and if substituted, at least one substituent is optionally L^B, where L^B is a linking group.

43. The kit according to claim 30, wherein the second linkage compound comprises a compound having the structure:

44. The kit according to claim 43, wherein:

at least one of R² and R³ is -(Z)_z-C≡

CR^E, or -(Z)_z-C≡

N, and the other is any R¹ group; and

n is an integer from 0 to 4;

m is 0 or 1; and

each R¹ is the same or different and is selected from hydrogen, hydroxyl, alkyl, alkenyl, alkynyl, halo, carboxyl, carbonyl, alkyloxyl, alkoxyl, alkoxyalkyl, amino, alkylamino, azido, hydroxyalkyl, sulfonyl, sulfinyl, phospho, phosphino, or optionally two or more R¹ groups join to form one or more substituted or unsubstituted rings.

45. The kit according to claim 43, wherein at least one of R² and R³ is -(Z)_z-C≡

CH.

46. The kit according to claim 43, wherein L^B is an organic substituent group having at least one reactive group that is selected from carboxylic acid esters, sulfonyl halide, acyl halide, aldehydes, reactive ketones, aromatic halides, bromoacetamides, idoacetamides, maleimides, disulfides, hydroxyl, amines, diazonium compounds, active alkylating reagents, active acylating reagents, or diketones.

47. The kit according to claim 43, wherein at least one of R² and R³ is -(Z)_z-C≡

N.

48. The kit according to claim 30, wherein the second linkage compound is chemically bonded to a biomaterial.

49. The kit according to claim 48, wherein the biomaterial is selected from small molecules, drugs, ligands, catalysts, peptides, proteins, nucleic acids, oligonucleotides, saccharides, viruses, cells, organic polymers, inorganic polymers, nanoparticles, or solid surfaces.

50. The kit according to claim 30, wherein A is selected from hydrogen, hydroxyl, carboxyl, amino, alkylamino, or a substituted or unsubstituted alkyl, alkenyl, alkynyl, alkoxyl, alkoxyalkyl, alkoxyalkenyl, carboxyalkyl, amino, aminoalkyl, aryl, heteroaryl, or heterocyclyl, and if substituted, at least one substituent is optionally L^A, where L^A is a linking group.