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1. WO2016109733 - DÉTECTION HAUTEMENT SENSIBLE DE BIOMOLÉCULES PAR DES RÉACTIONS BIOORTHOGONALES INDUITES PAR LA PROXIMITÉ

Note: Texte fondé sur des processus automatiques de reconnaissance optique de caractères. Seule la version PDF a une valeur juridique

[ EN ]

WHAT IS CLAIMED IS:

1. A method of detecting binding of a first affinity ligand and a second affinity ligand, the method comprising

(i) contacting a tetrazine-containing compound with a dienophile-containing compound, said tetrazine-containing compound comprising a first affinity ligand covalently attached to a tetrazine moiety and said dienophile-containing comprising a second affinity ligand covalently attached to a dienophile moiety;

(ii) allowing said first affinity ligand to bind to said second affinity ligand or allowing said first affinity ligand and said second affinity ligand to bind to a third affinity ligand; (iii) allowing said tetrazine moiety to react with said dienophile moiety to form a pyridazine moiety within a detectable compound, said detectable compound comprising a fluorescent moiety rendered detectable upon formation of said pyridazine moiety.

2. The method of claim 1, wherein said fluorescent moiety is quenched upon formation of said pyridazine moiety.

3. The method of claim 1, wherein said fluorescent moiety is activated upon formation of said pyridazine moiety.

4. The method of claim 1, wherein said dienophile-containing compound comprises a fluorescent moiety and said dienophile moiety is a vinyl ether functional group.

5. The method of claim 4, wherein said fluorescent moiety is a xanthene, a coumarin, or a cyanine group.

6. The method of claim 5, wherein said xanthene group is a fluorescein or a rhodamine group.

7. The method of claim 4, wherein said dienophile-containing compound has a structure according to formula (III),


(III), wherein

X2 is O or NRX2RX2’;

R11 is hydrogen, substituted or unsubsituted alkyl, substituted or unsubstituted heteroalkyl, or–L3-R14;

R12 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, --CI3, -CN, -OR12A, -NR12AR12B, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2,

-NHNH2, - ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or–L4-R15;

R13 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, --CI3, -CN, -OR13A, -NR13AR13B, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, - ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or–L4-R15;

R14 is hydrogen or–L4-R15;

L3 is a bond, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

L4 is independently a bond, -NRL4-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

R15 is independently said second affinity ligand;

each R12A, R12B, R13A, R13B, RL4, RX2, andRX2’ is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and

wherein one and only one of R12, R13, and R14 is–L4-R15.

8. The method of claim 7, wherein R11 is hydrogen, substituted or unsubsituted alkyl, or substituted or unsubstituted heteroalkyl.

9. The method of claim 7 or 8, wherein R13 is–L4-R15.

10. The method of claim 7 or 8, wherein R12 and R13 are independently hydrogen, F, Br, I, or SO3H.

11. The method of claim 7, wherein the compound of formula (III) has the following structure,

12. The method of claim 11, wherein the compound of formula (III) has the


(IIIA-1),

wherein

R11A is independently hydrogen, substituted or unsubstituted alkyl, -CO2R11C, or–L4-R15; R11B is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, - -CI3, -CN, -OR11D, -NR11DR11E, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, - ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or–L4-R15; and

each R11C, R11D, and R11E is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

13. The method of claim 11, wherein the compound of formula (III) has the following


(IIIA-2),

wherein

R11A is independently hydrogen, substituted or unsubstituted alkyl, -CO2R11C, or–L4-R15; R11B is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, - -CI3, -CN, -OR11D, -NR11DR11E, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, - ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or–L4-R15;

each R11C, R11D, and R11E is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and

each RX2 andRX2’ is independently hydrogen or unsubstituted alkyl.

14. The method of any one of claims 11 to 13, wherein R12 and R13 are independently hydrogen, F, Br, I, or SO3H.

15. The method of claim 4, wherein said dienophile-containing compound has a structure according to formula (IV),

wherein

X3 is O or NRX3RX3’;

R16 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, -CI3, -CN, -OR16A, -NR16AR16B, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, - ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -OCH=CH2, or–L4-R15;

R17 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, -CI3, -CN, -OR17A, -NR17AR17B, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, - ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -OCH=CH2, or–L4-R15;

R18 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, -CI3, -CN, -OR18A, -NR18AR18B, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, - ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -OCH=CH2, or–L4-R15;

R19 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, -CI3, -CN, -OR19A, -NR19AR19B, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, - ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, -OCH=CH2, or–L4-R15;

R20 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, -CI3, -CN, -OR20A, -NR20AR20B, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2,

-NHNH2, - ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or–L4-R15;

R21 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, -CI3, -CN, -OR21A, -NR21AR21B, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, - ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or–L4-R15;

each R16A, R16B, R17A, R17B, R18A, R18B, R19A, R19B, R20A, R20B, R21A, and R21B is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

each RX3 and RX3’ is independently hydrogen or substituted or unsubstituted alkyl; L4 is independently a bond, -NRL4-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

R15 is independently said second affinity ligand; and

wherein one and only one of R16, R17, R18, and R19 is -OCH=CH2; and

wherein one and only one of R16, R17, R18, R19, R20, and R21 is–L4-R15.

16. The method of claim 4, wherein said dienophile-containing compound has a structure according to formula (V),

wherein

R21 is independently hydrogen, OR21A, NR21AR21B, substituted or unsubstituted alkyl, or– L4-R15;

R22 and R23 are independently hydrogen, substituted or unsubstituted alkyl, or–L4-R15; and

each R24, R25, R26, R27, R28, R29, R30, and R31 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, -CI3, -CN, -OH, -NH2, -NMe2, -NEt2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -OCH3, -NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or

unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or–L4-R15;

each R21A and R21B is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

L4 is independently a bond, -NRL4-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

R15 is independently said second affinity ligand; and

wherein one and only one of R21, R22, R23, R24, R25, R26, R27, R28, R29, R30, and R31 is– L4-R15.

17. The method of claim 16, wherein R22 and R23 are independently substituted or unsubstituted alkyl.

18. The method of claim 17, wherein

R22 is independently methyl, -(CH2)n22SO3H, or -(CH2)n22CO2H;

R23 is independently methyl, -(CH2)n23SO3H, or -(CH2)n23CO2H; and

n22 and n23 are independently an integer from 1 to 5.

19. The method of any one of claims 16 to 18, wherein each R24, R25, R26, R27, R28, R29, R30, and R31 is independently hydrogen or–SO3H.

20. The method of any one of claims 16 to 19, wherein R21 is OR21A, NR21AR21B, -(CH2)n21NR21CR21D, -(CH2)n21COR21C, or -(CH2)n21CO2R21C, wherein each R21C and R21D is independently hydrogen or substituted or unsubstituted alkyl, and n21 is an integer from 1 to 5.

21. The method of claim 4, wherein said dienophile-containing compound has a structure according to formula (VI-a) or (VI-b),

wherein

R11A is independently hydrogen, substituted or unsubstituted alkyl, -CO C

2R11 , or–L4-R15;

each R32 and R33 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, -CI3, -CN, -OH, -NH2, -NMe2, -NEt2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or–L4-R15;

R11C is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.L4 is independently a bond, -NRL4-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted

heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

R15 is independently said second affinity ligand;

n33 is 0, 1, or 2; and

wherein one and only one or R32 and R33 is–L4-R15.

22. The method of any one of claims 4 to 21, wherein said tetrazine-containing compound has a structure according to the following formula (VII),


wherein

R34 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, -CI3, -CN, -OR34A, -NR34AR34B, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, - ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

L5 is independently a bond, -NRL5-, O, S, substituted or unsubstituted alkylene, substituted or unsubstituted alkenylene, substituted or unsubstituted alkynylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

R35 is independently said first affinity ligand; and

each R34A, R34B, and RL5 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

23. The method of claim 1, wherein said tetrazine-containing compound has a structure according to formula (I),


L1 is independently a bond, -NRA-, O, S, substituted or unsubstituted alkylene, substituted or unsubstituted alkenylene, substituted or unsubstituted alkynylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted

heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

R1 is independently a binding-detecting group comprising a fluorescent moiety;

R2 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, --CI3, -CN, -OH, -NH2, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and

each RA and RB is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl.

24. The method of claim 23, wherein R2 is independently hydrogen or substituted or unsubstituted alkyl.

25. The method of claim 23 or 24, wherein L1 is independently substituted or unsubstituted alkenylene.

26. The method of any one of claims 23 to 25, wherein said fluorescent moiety is a xanthene, cyanine, or a boron-dipyrromethene (BODIPY) group.

27. The method of claim 26, wherein said fluorescent moiety is a fluorescein or a rhodamine group.

28. The method of claim 23, wherein the compound of formula (I) has the following structure,

wherein

X1A is independently O, SiMe2, GeMe2, SnMe2, or TeO;

X1B is independently O or NR1BR1B’;

X1C is independently O or NR1C’;

R1A is independently hydrogen or substituted or unsubstituted alkyl;

each of R1B, R1B’, R1C, and R1C’ is independently hydrogen or substituted or unsubstituted alkyl;

each of R1D and R1E is independently hydrogen, halogen, -SO3H, or -LR1-X1;

LR1 is independently a bond, -NRB-, O, S, substituted or unsubstituted alkylene, substituted or unsubstituted alkenylene, substituted or unsubstituted alkynylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

X1 is said first affinity ligand; and

wherein one and only one of R1D and R1E is -LR1-X1.

29. The method of claim 28, wherein the compound of formula (I) has the following structure,

30. The method of claim 28, wherein the compound of formula (I) has the following structure,

31. The method of claim 28, wherein the compound of formula (I) has the following structure,

32. The method of claim 28, wherein the compound of formula (I) has the following structure,


(IA-4b).

33. The method of claim 28, wherein the compound of formula (I) has the following structure,


(IA-5b).

34. The method of any one of claims 29 to 33, wherein R1D and R1E are independently hydrogen, F, Br, I, SO3H, or -LR1-X1.

35. The method of any one of claims 29 to 34, wherein X1A is O.

.

36. The method of claim 23, wherein the compound of formula (I) has the following structure,


(IE), wherein

each R1F, R1G, R1H, R1I, R1J, and R1K is independently hydrogen, halogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or -LR1-X1;

LR1 is independently a bond, -NRB-, O, S, substituted or unsubstituted alkylene, substituted or unsubstituted alkenylene, substituted or unsubstituted alkynylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

X1 is said first affinity ligand; and

wherein one and only one of R1F, R1G, R1H, R1I, R1J, and R1K is -LR1-X1.

37. The method of claim 23, wherein the compound of formula (I) has the following structure,

wherein

each R1F, R1G, and R1H is independently hydrogen, substituted or unsubstituted alkyl,

substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl,

;

each R1I, R1J, and R1K is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or -LR1-X1;

LR1 is independently a bond, -NRB-, O, S, substituted or unsubstituted alkylene, substituted or unsubstituted alkenylene, substituted or unsubstituted alkynylene, substituted or

unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

X1 is said first affinity ligand; and

wherein one and only one of R1F, R1G, R1H, R1I, R1J, and R1K is -LR1-X1; and

wherein one and only one of R1F, R1G, and R1H is
.

38. The method of claim 23, wherein the compound of formula (I) has the following structure,

wherein

LR1 is independently a bond, -NRB-, O, S, substituted or unsubstituted alkylene, substituted or unsubstituted alkenylene, substituted or unsubstituted alkynylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

X1 is said first affinity ligand; and

n is 1, 2, or 3.

39. The method of claim 23, wherein the compound of formula (I) has the following structure,

wherein

LR1 is independently a bond, -NRB-, O, S, substituted or unsubstituted alkylene, substituted or unsubstituted alkenylene, substituted or unsubstituted alkynylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

X1 is said first affinity ligand; and

n is 1, 2, or 3.

40. The method of claim 23, wherein the compound of formula (I) has the following structure,


(IJ), wherein

each R1L, R1M, R1N, R1O, and R1P is independently hydrogen, -LR1-X1, or

R1Q is independently OR1Q’ or NR1Q’R1Q” , and

each R1Q’ and R1Q” is independently hydrogen or substituted or unsubstituted alkyl; LR1 is independently a bond, -NRB-, O, S, substituted or unsubstituted alkylene, substituted or unsubstituted alkenylene, substituted or unsubstituted alkynylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

X1 is said first affinity ligand; and

wherein one and only one of R1L, R1M, R1N, R1O, and R1P is -LR1-X1; and

41. The method of claim 23, wherein said tetrazine-containing compound has a structure accordin to formula I


(IC-1b), wherein

X1A is independently O, SiMe2, GeMe2, SnMe2, or TeO;

R2 is–LR1-X1;

R1A is independently hydrogen or substituted or unsubstituted alkyl;

LR1 is independently a bond, -NRB-, O, S, substituted or unsubstituted alkylene, substituted or unsubstituted alkenylene, substituted or unsubstituted alkynylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

X1 is said first affinity ligand.

42. The method of claim 41, wherein X1A is O, and R1A is hydrogen.

43. The method of any one of claims 1 to 42, wherein said dienophile-containing compound has the following structure,


X is independently O, S, NRXA, or CRXBRXC;

each of R3 and R4 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, or–L2-R9;

R5 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, --CI3, -CN, -OR5A, -NR5AR5B, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or–L2-R9;

R6 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, --CI3, -CN, -OR6A, -NR6AR6B, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or–L2-R9;

R7 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, --CI 7A

3, -CN, -OR7A, -NR R7B, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or–L2-R9;

R8 is independently hydrogen, halogen, -N3, -NO2, -CF3, -CCl3, -CBr3, --CI3, -CN, -OR8A, -NR8AR8B, -COOH, -CONH2, -NO2, -SH, -SO2Cl, -SO3H, -SO4H, -SO2NH2, -NHNH2, -ONH2, -OCH3, - NHCNHNH2, substituted or unsubstituted alkyl, substituted or unsubstituted

heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or–L2-R9;

L2 is independently a bond, -NRL2-, substituted or unsubstituted alkylene, substituted or unsubstituted heteroalkylene, substituted or unsubstituted cycloalkylene, substituted or unsubstituted heterocycloalkylene, substituted or unsubstituted arylene, or substituted or unsubstituted heteroarylene;

R9 is independently said second affinity ligand;

R10 is independently hydrogen, -OR10A, -NR10AR10B, or substituted or unsubstituted alkyl; each R5A, R5B, R6A, R6B, R7A, R7B, R8A, R8B, R10A, and R10B is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl;

each RXA, RXB, and RXC is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, or–L2-R9;

RL2 is independently hydrogen, substituted or unsubstituted alkyl, substituted or unsubstituted heteroalkyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted heterocycloalkyl, substituted or unsubstituted aryl, or substituted or unsubstituted heteroaryl; and

wherein one and only one of R3, R4, R5, R6, R7, R8, RXA, RXB, and RXC is -L2-R9.

44. The method of claim 43, wherein R3, R4, and R10 are hydrogen.

45. The method of claim 43 or 44, wherein R5, R6, R7 and R8 are each hydrogen.

46. The method of claim 43 or 44, wherein one of R5, R6, R7, and R8 is -L2-R9, and the others are hydrogen.

47. The method of any one of claims 43 to 46, wherein X is NRXA.

48. The method of claim 47, wherein RXA is -L2-R9.

49. The method of any one of claims 1 to 48, wherein the tetrazine-containing compound comprises a first affinity ligand that isa biomolecule or nanomaterial.

50. The method of claim 49, wherein said biomolecule is a nucleic acid, peptide, small molecule, protein, lipid, or sugar.

51. The method of claim 50, wherein said protein is an antibody or wherein said nucleic acid is RNA or DNA.

52. The method of any one of claims 1 to 51, wherein the dienophile-containing compound comprises a second affinity ligand that is a biomolecule or nanomaterial.

53. The method of claim 52, wherein said biomolecule is a nucleic acid, peptide, small molecule, protein, lipid, or sugar.

54. The method of claim 53, wherein said protein is an antibody or wherein said nucleic acid is RNA or DNA.

55. The method of any one of claims 1 to 54, further comprising the detection of a biomolecule.

56. The method of claim 55, wherein said biomolecule is a nucleic acid, protein, lipid, or sugar.

57. The method of claim 56, wherein said biomolecule is DNA,RNA, or an antibody.

58. The method of claim 55, wherein said biomolecule is microRNA, telomeres, genomic loci, non-coding RNA, mRNA, a disease associated antibody, a protein of diagnostic utility, or a glycan.

59. The method of claim 58, wherein said protein of diagnostic utility is Hemoglobin A1c, Glucagon, Leptin, Haptoglobin, histidine rich protein II, pLDH, C reactive protein, or Epo.

60. A method of synthesizing a compound having a structure according to formula (I’),

according to formula (I),

(I),

with a compound having a structure according to formula (II),


(II).

156

61. The method of claim 60, wherein the compound of formula (I’) has a structure according to the following formula,


62. The method of claim 60, wherein the compound of formula (I’) has a structure according to the following formula,


63. The method of claim 60, wherein the compound of formula (I’) has a structure according to the f ll in f rm l


(ID’) or (IE’).

64. The method of claim 60, wherein the compound of formula (I’) has a structure according to the following formula,


65. The method of claim 60, wherein the compound of formula (I’) has a structure according to the following formula,

66. The method of claim 60, wherein the compound of formula (I’) has a structure according to the following formula,

67. The method of claim 60, wherein the compound of formula (I’) has a structure according to the following formula,

(IJ’), wherein one and only one of R1L, R1M, R1N, R1O, and R1P is


68. The method of any one of claims 60 to 67, wherein said contacting is in a cell.

69. The method of any one of claims 60 to 68, further comprising detecting the compound of formula (I’).

159

70. A method of synthesizing a compound having a structure according to formula (III’),


(III’), comprising contacting a compound having a structure according to formula III


(III),

with a compound having a structure according to formula (VII),

(VII).

71. The method of claim 70, wherein the compound of formula (III’) has the following structure


(IIIA’).

72. The method of claim 70, wherein the compound of formula (III’) has the following structure,

160

73. The method of claim 70, wherein the compound of formula (III’) has the following stru


(IIIA-2’).

74. The method of any one of claims 70 to 73, wherein said contacting is in a cell.

75. The method of any one of claims 70 to 74, further comprising detecting the compound of formula (III’).

76. A method of synthesizing a compound having a structure according to formula (IV’),


(IV’), wherein one and only one of R16, R17, R18, and R19 is -OH; comprising contacting a compound having a structure according to formula (III),


(IV), wherein one and only one of R16, R17, R18, and R19 is -OCH=CH2;

with a compound having a structure according to formula (VII),


77. The method of claim 76, wherein said contacting is in a cell.

78. The method of claim 76 or 77, further comprising detecting the compound of formula (IV’).

79. A method of synthesizing a compound having a structure according to formula (V’),

comprising contacting a compound having a structure according to formula (V),

with a compound having a structure according to formula (VII),

(VII).

80. The method of claim 79, wherein said contacting is in a cell.

81. The method of claim 79 or 80, further comprising detecting the compound of formula (V’).

82. A method of synthesizing a compound having a structure according to the formula,

comprising contacting a compound having a structure according to formula (VI-a),

162

with a compound having a structure according to formula (VII),


83. A method of synthesizing a compound having a structure according to the formula,


comprising contacting a compound having a structure according to formula (VI),


with a compound having a structure according to formula (VII),


84. The method of claim 82 or 83, wherein said contacting is in a cell.

85. The method of any one of claims 82 to 84, further comprising detecting the compound of formula (VI’).

86. A compound having a structure according to formula (I).

87. The compound of claim 86, wherein the compound has a structure according to f rm l IA- r IA-


88. The compound of claim 86, wherein the compound has a structure according to formula (IB-a) or (IB-b),


89. The compound of claim 86, wherein the compound has a structure according to formula (IA-1a) or (IA-1b),


90. The compound of claim 86, wherein the compound has a structure according to 2 formula (IC-1a) or (IC-1b),

3


1 91. The compound of claim 86, wherein the compound has a structure according to 2 formula (IF),

3


92. The compound of claim 86, wherein the compound has a structure according to formula (IG),

93. The compound of claim 86, wherein the compound has a structure according to formula (IH),

94. The compound of claim 86, wherein the compound has a structure according to formula (IJ),

95. A compound having a structure according to formula (III),

96. The compound of claim 95, wherein the compound has a structure according to formula (IIIA),

97. The compound of claim 95, wherein the compound has a structure according to formula (IIIA-1),

166

98. The compound of claim 95, wherein the compound has a structure according to formula (IIIA-2),


99. A compound having a structure according to formula (IV),

100. A compound having a structure according to formula (V),


101. A compound having a structure according to formula (VI-a) or formula (VI-b),

12 A m n h in a structure according to formula (I’),


103. The compound of claim 102, wherein the compound has a structure according to f rml IA-’ r IA-’


104. The compound of claim 102, wherein the compound has a structure according to formula (IB-a’) or (IB-b’),


105. The compound of claim 102, wherein the compound has a structure according to 2 formula (IC-1a’) or (IC-1b’),

3


1 106. The compound of claim 102, wherein the compound has a structure according to 2 formula (ID’),

3

107. The compound of claim 102, wherein the compound has a structure according to formula (IE’),


108. The compound of claim 102, wherein the compound has a structure according to one of the following formulas,

3

4


1 109. The compound of claim 102, wherein the compound has a structure according to 2 formula (IG’),

3


1 110. The compound of claim 102, wherein the compound has a structure according to 2 formula (IH’),

3

111. The compound of claim 102, wherein the compound has a structure according to formula (IJ’),

112. A compound having a structure according to formula (III’),

113. The compound of claim 112, wherein the compound has a structure according to formula (IIIA’),

114. The compound of claim 112, wherein the compound has a structure according to formula (IIIA-1’),

115. The compound of claim 112, wherein the compound has a structure according to formula (IIIA-2’),


IIIA-2’).

171

116. A compound having a structure according to formula (IV’),
(IV’).

117. A compound having a structure according to formula (V’),
(V’).

118. A compound having a structure according to formula (VI-a’) or (VI-b’),


(VI-b’).

172