METHODS FOR LABELING A SUBSTRATE USING A HETERO-DIELS-ALDER REACTION

1 . A method for labeling a substrate, the method comprising:

generating an o-quinone methide attached to a surface of a substrate and having the formula:

contacting the o-quinone methide with a polarized olefin having the formula:

under conditions effective to form a hetero-Diels-Alder adduct, wherein:

each R 1 is independently H, halogen, or an organic group, with the proviso that at least one R 1 comprises a linker group attached to the surface of the substrate;

each R 2 is independently H or an organic group;

each R 3 is independently H, halogen, or an organic group;

X is O or NR 4 , wherein R 4 is H or an organic group;

optionally, two or more R 1 groups may be combined to form one or more rings;

optionally, two or more R 2 groups may be combined to form one or more rings;

optionally, two or more R 3 and/or R 4 groups may be combined to form one or more rings; and

with the proviso that at least one R 2 , R 3 , or R 4 comprises a detectable label.

2 . The method of claim 1 wherein the o-quinone methide is an o-naphthoquinone methide having one of the formulas:

3 . The method of claim 1 wherein the substrate comprises a planar surface or a bead.

4 . The method of claim 1 wherein the substrate is selected from the group consisting of glass, quartz, silica, a metal, a semi-conductor, a polymer, a membrane, a liposome, a micelle, a macromolecule, a biomaterial, and combinations thereof.

5 . The method of claim 4 wherein the biomaterial is selected from the group consisting of a virus, a small multicellular organism, DNA, RNA, a peptide, a polypeptide, a protein, a carbohydrate, a lipid, tissue, and combinations thereof.

6 . The method of claim 1 wherein the label is detectable by method selected from the group consisting of fluorescence, phosphorescence, radiation detection, optical methods, electrochemical methods, surface plasmon resonance imaging (SPRi), and combinations thereof.

7 . The method of claim 1 wherein the detectable label comprises a probe.

8 . The method of claim 7 wherein the probe comprises DNA, a peptide, a polypeptide, a protein, or a combination thereof.

9 . The method of claim 1 wherein the conditions effective to form the hetero-Diels-Alder adduct comprise contacting in an aqueous solution, suspension, or dispersion.

10 . The method of claim 1 wherein R 3 or R 4 comprises the detectable label, and the method further comprises removing the detectable label from the surface of the substrate by hydrolyzing the C—X bond.

11 . A method for labeling a substrate, the method comprising:

providing a precursor compound attached to a surface of a substrate and having the formula:

irradiating the precursor compound under conditions effective to form an o-quinone methide having the formula:

contacting the o-quinone methide with a compound having the formula

under conditions effective to form a hetero-Diels-Alder adduct, wherein:

each R 1 is independently H, halogen, or an organic group, with the proviso that at least one R 1 comprises a linker group attached to the surface of the substrate;

each R 2 is independently H or an organic group;

each R 3 is independently H, halogen, or an organic group;

X is O or NR 4 ;

Y is OR 5 , NR 5 2 , NR 5 3 + (Z 1/q ) − wherein Z is an anion having a negative charge of q;

each R 5 is independently H or an organic group,

optionally, two or more R 1 groups may be combined to form one or more rings;

optionally, two or more R 2 groups may be combined to form one or more rings;

optionally, two or more R 3 and/or R 4 groups may be combined to form one or more rings;

optionally, two or more R 5 groups may be combined to form one or more rings; and

with the proviso that at least one R 2 , R 3 , or R 4 comprises a detectable label.

12 . The method of claim 11 wherein the precursor compound has one of the formulas:

wherein irradiating the precursor compound under conditions effective to form an o-quinone methide forms an o-naphthoquinone methide having one of the formulas:

13 . The method of claim 11 wherein the precursor compound is irradiated in the presence of the polarized olefin.

14 . The method of claim 11 wherein irradiating the precursor compound comprises pattern-wise irradiating the substrate to provide a pattern-wise labeled surface of the substrate.

15 . A method for labeling a substrate, the method comprising:

generating an o-quinone methide having the formula:

contacting the o-quinone methide with a polarized olefin attached to a surface of a substrate and having the formula:

under conditions effective to form a hetero-Diels-Alder adduct, wherein:

each R 1 is independently H, halogen, or an organic group, with the proviso that at least one R 1 comprises a detectable label;

each R 2 is independently H or an organic group;

each R 3 is independently H, halogen, or an organic group;

X is O or NR 4 , wherein R 4 is H or an organic group;

optionally, two or more R 1 groups may be combined to form one or more rings;

optionally, two or more R 2 groups may be combined to form one or more rings;

optionally, two or more R 3 and/or R 4 groups may be combined to form one or more rings; and

with the proviso that at least one R 2 , R 3 , or R 4 comprises a linker group attached to the surface of the substrate.

16 . The method of claim 15 wherein the o-quinone methide is an o-naphthoquinone methide having one of the formulas:

17 . The method of claim 15 wherein the substrate comprises a planar surface or a bead.

18 . The method of claim 15 wherein the substrate is selected from the group consisting of glass, quartz, silica, a metal, a semi-conductor, a polymer, a membrane, a liposome, a micelle, a macromolecule, a biomaterial, and combinations thereof.

19 . The method of claim 18 wherein the biomaterial is selected from the group consisting of a virus, a small multicellular organism, DNA, RNA, a peptide, a polypeptide, a protein, a carbohydrate, a lipid, tissue, and combinations thereof.

20 . The method of claim 15 wherein the label is detectable by a method selected from the group consisting of fluorescence, phosphorescence, radiation detection, optical and electrochemical methods, surface plasmon resonance imaging (SPR 1 ), and combinations thereof.

21 . The method of claim 15 wherein the detectable label comprises a probe.

22 . The method of claim 21 wherein the probe comprises DNA, a peptide, a polypeptide, a protein, or a combination thereof.

23 . The method of claim 15 wherein the conditions effective to form the hetero-Diels-Alder adduct comprise contacting in an aqueous solution, suspension, or dispersion.

24 . The method of claim 15 further comprising removing the detectable label from the surface of the substrate by hydrolyzing the C—X bond.

25 . A method for labeling a substrate, the method comprising:

providing a precursor compound having the formula:

irradiating the precursor compound under conditions effective to fowl an o-quinone methide having the formula:

contacting the o-quinone methide with a polarized olefin attached to a surface of a substrate and having the formula:

under conditions effective to form a hetero-Diels-Alder adduct, wherein:

each R 1 is independently H, halogen, or an organic group, with the proviso that at least one R 1 comprises a detectable label;

each R 2 is independently H or an organic group;

each R 3 is independently H, halogen, or an organic group;

X is O or NR 4 ;

Y is OR 5 , NR 5 2 , NR 5 3 + (Z 1/q ) − wherein Z is an anion having a negative charge of q;

each R 5 is independently H or an organic group;

optionally, two or more R 1 groups may be combined to form one or more rings;

optionally, two or more R 2 groups may be combined to form one or more rings;

optionally, two or more R 3 and/or R 4 groups may be combined to form one or more rings;

optionally, two or more R 5 groups may be combined to form one or more rings; and

with the proviso that at least one R 2 , R 3 , or R 4 comprises a linker group attached to the surface of the substrate.

26 . The method of claim 25 wherein the precursor compound has one of the formulas:

wherein irradiating the precursor compound under conditions effective to form an o-quinone methide forms an o-naphthoquinone methide having one of the formulas:

27 . The method of claim 25 wherein the precursor compound is irradiated in the presence of the polarized olefin.

28 . The method of claim 27 wherein irradiating the precursor compound comprises pattern-wise irradiating the substrate to provide a pattern-wise labeled surface of the substrate.

29 . The method of claim 25 wherein conditions effective to form the o-quinone methide comprise irradiation of an aqueous solution, suspension, or dispersion of the precursor compound.

30 . A precursor compound having the formula:

wherein:

each R 1 is independently H, halogen, or an organic group;

Y is OR 5 , NR 5 2 , NR 5 3 + (Z 1/q ) − wherein Z is an anion having a negative charge of q;

each R 5 is independently H or an organic group;

optionally, two or more R 1 groups may be combined to form one or more rings;

optionally, two or more R 5 groups may be combined to form one or more rings; and

with the proviso that at least one R 1 comprises a detectable label or a linker group attached to a surface of a substrate.

31 . The precursor compound of claim 30 having one of the formulas:

32 . An o-quinone methide having the formula:

wherein each R 1 is independently H, halogen, or an organic group, with the proviso that at least one R 1 comprises a detectable label or a linker group attached to a surface of a substrate; and wherein optionally two or more R 1 groups may be combined to form one or more rings.

33 . The o-quinone methide of claim 32 wherein the o-quinone methide is an o-naphthoquinone methide of one of the formulas: