Spatially-addressable immobilization of anti-ligands on surfaces

• US 5,451,683 A
• Filed: 04/23/1993
• Issued: 09/19/1995
• Est. Priority Date: 11/13/1989
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymax Incorporated
• Inventors: Barrett, Ronald W., Pirrung, Michael C., Holmes, Christopher P., Sundberg, Steven A., Stryer, Lubert
• Primary Examiner(s): Lee, Mary C.
• Assistant Examiner(s): LUTZ, LAURA REBEKAH
• Application Number:
  US08/053,124
• Time in Patent Office: 879 Days
• Field of Search: 548/303.7, 548/304.1, 548/303.1, 548/302.7, 548/323.5, 548/324.5, 548/316.4
• US Class Current: 548/302.7
• CPC Class Codes: B01J 2219/00608 DNA chips
• CPC Class Codes: B01J 2219/0061 The surface being organic
• CPC Class Codes: B01J 2219/00612 the surface being inorganic
• CPC Class Codes: B01J 2219/00617 by chemical means
• CPC Class Codes: B01J 2219/00626 Covalent
• CPC Class Codes: B01J 2219/0063 Other, e.g. van der Waals forces, hydrogen bonding
• CPC Class Codes: B01J 2219/00659 Two-dimensional arrays
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: C07D 233/38 with acyl radicals or hetero atoms directly attached to ring nitrogen atoms
• CPC Class Codes: C07D 495/04 Ortho-condensed systems
• CPC Class Codes: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: G01N 33/54306 Solid-phase reaction mechanisms
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: G01N 33/552 Glass or silica
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Methods and compositions are described for immobilizing anti-ligands, such as antibodies or antigens, hormones or hormone receptors, oligonucleotides, and polysaccharides on surfaces of solid substrates for various uses. The methods provide surfaces covered with caged binding members which comprise protecting groups capable of being removed upon application of a suitable energy source. Spatially addressed irradiation of predefined regions on the surface permits immobilization of anti-ligands at the activated regions on the surface. Cycles of irradiation on different regions of the surface and immobilization of different anti-ligands allows formation of an immobilized matrix of anti-ligands at defined sites on the surface. The immobilized matrix of anti-ligands permits simultaneous screenings of a liquid sample for ligands having high affinities for certain anti-ligands of the matrix. A preferred embodiment of the invention involves attaching photoactivatable biotin derivatives to a surface. Photolytic activation of the biotin derivatives forms biotin analogs having strong binding affinity for avidin. Biotinylated anti-ligands can be immobilized on activated regions of the surface previously treated with avidin.

Claims

1. A caged binding member compound having the formula:

##STR30## wherein X and Z are selected from the group consisting of (a) hydrogen,

(b) oxycarbonyls of lower alkyl, aryl, and benzyl groups,

(c) a nitroveratryloxycarbonyl group,

(d) a nitropiperonyloxycarbonyl group where Z is hydrogen and R is p-nitrophenyl formate,

(e) ##STR31## wherein R₁ and R₂ are hydrogen, lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, sulfonate, formamido or phosphido groups,

(f) ##STR32## wherein R₁ and R₂ are hydrogen, lower alkyl, aryl, benzyl, pyrenyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido or phosphido groups, and R₃ and R₄ are hydrogen, lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido or phosphido groups,

(g) ##STR33## wherein R₁ and R₂ are hydrogen, lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido or phosphido groups, and

(h) ##STR34## wherein R₁, R₂, R₃ and R₄ are hydrogen, lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido or phosphido groups, provided that when X is hydrogen, Z is not hydrogen or methyloxycarbonyl and provided that when Z is hydrogen, X is not hydrogen or methyloxycarbonyl;

R is selected from the group consisting of hydrogen, lower alkyl, aryl, carboxylate lower alkyl formate, aryl formate, methyl formate, p-nitrophenyl formate, formamide, N-alkylformamide, N-succinimidyl, hydroxyl, alkoxyl, thiol, thioether, disulfide, hydrazide and amine groups, provided that when X or Z is methyloxycarbonyl, R is not methyl formate;

U is O, S or NH;

Y is selected from the group consisting of sulfur, oxygen, methylene, carbonyl, sulfinyl and sulfonyl groups, or Y represents two hydrogens attached to the respective carbons;

n=0-7;

or acid addition salts of the compound.

2. A compound as in claim 1 wherein U is O, Y is sulfur, and n=4.

3. A compound as in claim 1 wherein one of X and Z is a nitroveratryloxycarbonyl group.

4. A compound as in claim 1 wherein one of X and Z has the formula:

##STR35## wherein R₁ and R₂ are hydrogen, lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, sulfonate, formamido or phosphido groups.

5. A compound as in claim 1 wherein X is 6-nitroveratryloxycarbonyl;

Z is hydrogen; and

R is methyl formate.

6. A compound as in claim 1 wherein X is 6-nitroveratryloxycarbonyl;

Z is hydrogen; and

R is p-nitrophenyl formate.

7. A compound as in claim 1 wherein X is 6-nitropiperonyloxycarbonyl, Z is hydrogen;

and R is p-nitrophenyl formate.

8. A compound as in claim 1 wherein one of X and Z is ##STR36## wherein R₁ and R₂ are hydrogen, lower alkyl, aryl, benzyl, pyrenyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido or phosphido groups, and R₃ and R₄ are hydrogen, lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido or phosphido groups.

20. A compound as in claim 8 wherein R₁ and R₂ are methoxy.

21. A compound as in claim 8 wherein R₃ and R₄ are methyl.

9. A caged binding member compound having the formula selected from the group consisting of:

##STR37## wherein X is selected from the group consisting of (a) hydrogen,

(b) lower alkyl,

(c) aryl,

(d) benzyl,

(e) substituted benzyl selected from the group consisting of ##STR38## wherein R₁, R₂, R₃ and R₄ are selected from the group consisting of hydrogen, lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido and phosphido groups provided that R₁, R₂, R₃ and R₄ are not all hydrogen, and ##STR39## wherein R₁ and R₂ are selected from the group consisting of hydrogen, lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido and phosphido groups.

(f) nitroveratryl,

(g) 6-nitroveratryl,

(h) 6-nitroveratryloxycarbonyl, and

(i) 6-nitropiperonyloxycarbonyl where U or W is hydrogen;

U and W are selected from the group consisting of hydrogen, lower alkyl, aryl, benzyl and substituted benzyl selected from the group consisting of ##STR40## wherein R₁, R₂, R₃ and R₄ are selected from the group consisting of hydrogen, lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido and phosphido groups provided that R₁, R₂, R₃ and R₄ are not all hydrogen, and ##STR41## wherein R₁ and R₂ are selected from the group consisting of hydrogen, lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido and phosphido groups, provided that only one of U or W is present;

R is selected from the group consisting of hydrogen, lower alkyl, aryl, carboxylate, alkyl formate, aryl formate, formamide, N-alkyl formamide, N-succinimidyl, hydroxyl, alkoxyl, thiol, thioether, disulfide, hydrazide and amine groups;

Y is selected from the group consisting of sulfur, oxygen, methylene, carbonyl, sulfinyl and sulfonyl groups, or Y represents two hydrogens attached to the respective carbons; and

n=0-7;

or acid addition salts of the compound.

10. A compound as in claim 9 wherein Y is sulfur and n=4.

11. A compound as in claim 9 wherein one of X, U or W has the formula:

##STR42## wherein R₁, R₂, R₃ and R₄ are selected from the group consisting of hydrogen, lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido and phosphido groups provided that R₁, R₂, R₄ are not all hydrogen.

12. A compound as in claim 11 wherein R₁ and R₂ are methoxy.

13. A compound as in claim 11 wherein R₃ and R₄ are methyl.

14. A compound as in claim 9 wherein X is a nitroveratryl group.

15. A compound as in claim 9, wherein X has the formula:

##STR43## wherein R₁ and R₂ are selected from the group consisting of hydrogen, lower alkyl, aryl, benzyl, halogen, hydroxyl, alkoxyl, thiol, thioether, amino, nitro, carboxyl, formate, formamido and phosphido groups.

16. A compound as in claim 9 wherein X is 6-nitroveratryl;

U or W is hydrogen; and

R is methyl formate.

17. A compound as in claim 9 wherein X is 6-nitroveratryloxycarbonyl hydrogen;

and R is p-nitrophenyl formate.

18. A compound as in claim 9 wherein X is 6-nitropiperonyloxycarbonyl and U or W is hydrogen.

19. A compound as in claim 9 wherein X is a substituted benzyl group, and R is methyl formate or p-nitrophenyl formate.