Photocleavable protecting groups and methods for their use

• US 20030144499A1
• Filed: 01/22/2003
• Published: 07/31/2003
• Est. Priority Date: 12/15/1995
• Status: Active Grant

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: McGall, Glenn H., Rava, Richard P., Nam, Ngo Q.
• Granted Patent: US 6,881,836 B2
• Time in Patent Office: Days
• US Class Current: 536/25.3
• CPC Class Codes: B01J 2219/00432 Photolithographic masks
• CPC Class Codes: C07B 2200/11 Compounds covalently bound to a solid support
• CPC Class Codes: C07H 19/10 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/20 with the saccharide radical esterified by phosphoric or polyphosphoric acids
• CPC Class Codes: C07H 19/207 the phosphoric or polyphosphoric acids being esterified by a further hydroxylic compound, e.g. flavine adenine dinucleotide or nicotinamide-adenine dinucleotide
• CPC Class Codes: C07H 21/00 Compounds containing two or more mononucleotide units having separate phosphate or polyphosphate groups linked by saccharide radicals of nucleoside groups, e.g. nucleic acids
• CPC Class Codes: C07K 1/061 using protecting groups
• CPC Class Codes: C40B 40/04 Libraries containing only organic compounds
• CPC Class Codes: C40B 50/14 Solid phase synthesis, i.e. wherein one or more library building blocks are bound to a solid support during library creation; Particular methods of cleavage from the solid support
• CPC Class Codes: C40B 60/14 for creating libraries
• CPC Class Codes: G01N 33/54353 with ligand attached to the carrier via a chemical coupling agent coatings G01N33/54393
• CPC Class Codes: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups

Abstract

Novel compounds are provided which are useful as linking groups in chemical synthesis, preferably in the solid phase synthesis of oligonucleotides and polypeptides. These compounds are generally photolabile and comprise protecting groups which can be removed by photolysis to unmask a reactive group. The protecting group has the general formula Ar—C(R₁)(R₂)—O—C(O)— wherein:

Ar is an optionally substituted fused polycyclic aryl or heteroaromatic group or a vinylogous derivative thereof;

R₁ and R2 are independently H, optionally substituted alkyl, alkenyl or alkynyl, optionally substituted aryl or optionally substituted heteroaromatic, or a vinylogous derivative of the foregoing; and

X is a leaving group, a chemical fragment linked to Ar—C(R₁)(R₂)—O—C(O)— via a heteroatom, or a solid support; provided that when Ar is 1-pyrenyl and R₁ and R₂ are H, X is not linked to Ar—C(R₁)(R₂)—O—C(O)— via a nitrogen atom. Preferred embodiments are those in which Ar is a fused polycyclic aromatic hydrocarbon and in which the substituents on Ar, R₁ and R₂ are electron donating groups. A particularly preferred protecting group is the “PYMOC” protecting group, pyrenylmethyloxycarbonyl, where Ar is pyrenyl and R₁ and R₂ are H.

Also provided is a method of forming, from component molecules, a plurality of compounds on a support, each compound occupying a separate predefined region of the support, using the protected compounds described above.

Claims

1. A compound of the formula Ar—

C(R₁)(R₂)—

O—

C(O)—

X, wherein;

Ar is an optionally substituted fused polycyclic aryl or a vinylogous derivative thereof;

R₁ and R₂ are H, optionally substituted alkyl, alkenyl or alkynyl, optionally substituted aryl or optionally substituted heteroaromatic, or a vinylogous derivative of the foregoing; and

X is a leaving group, a chemical fragment linked to Ar—

C(R₁)(R₂)—

O—

C(O)—

via a heteroatom, or a solid support;

provided that when Ar is 1-pyrenyl and R₁ and R₂ are H, X is not linked to Ar—

C(R₁)(R₂)—

O—

C(O)—

via a nitrogen atom.

2. The compound of claim 1, wherein X is a chemical fragment selected from the group consisting of an amino acid, a nucleic acid or analog thereof, a monosaccharide and a protein.

3. The compound of claim 2, wherein X is a chemical fragment comprising an oxygen or nitrogen atom linking X to Ar—

C(R₁)(R₂)—

O—

C(O)—

.

4. The compound of claim 2, wherein X is a nucleoside or analog thereof linked to Ar—

C(R₁)(R₂)—

O—

C(O)—

via a 3′

or 5′

-OH of the nucleoside.

5. The compound of claim 4, wherein X comprises a base-protected deoxyribonucleoside, wherein the deoxyribonucleoside is a deoxyadenosine, a deoxycytidine, a thymidine or a deoxyguanosine.

6. The compound of claim 4 wherein X comprises a base-protected ribonucleoside.

7. The compound of claim 4, wherein X comprises a base-protected 2′

-O-methylribonucleoside.

8. The compound of claim 4, wherein Ar is independently 1-pyrenyl or 9-anthracenyl, R₁ is H and R₂ is H, methyl, substituted phenyl, 9-anthracenyl or 1-pyrenyl.

9. The compound of claim 8, wherein X is selected from the group consisting of base protected deoxyribonucleoside H-phosphonates and base protected deoxyribonucleoside phosphoramidites.

10. The compound of claim 4, wherein Ar is 1-pyrenyl and R₁ and R₂ are H.

17. The method of claim 16, further comprising repeating steps (a) and (b) of claim 10 with a succession of molecules to provide a chain of molecules immobilized on the support.

18. The method of claim 17, wherein the molecules are selected from ribonucleosides, deoxyribonucleosides and 2′

-O-methylribonucleosides.

19. The method of claim 18, wherein the support is a glass or silica substrate.

23. The method of claim 18, wherein the deoxynucleosides are linked to the photolabile group via a 3′

or 5′

-OH.

38. The method of claim 27, further comprising:

(a) covalently binding a second molecule comprising a masked reactive site linked to a chemically labile protecting group to a reactive site, wherein the reactive site is either on an activated region of the support as formed in step (a) of claim 19 or is an unmasked reactive site on a molecule on the support as formed in step (d) of claim 19;

(b) replacing the chemically labile protecting group with the photolabile protecting group to provide a region of the support having a molecule with the photolabile protecting group; and

(c) repeating steps (d)-(f) of claim 19 as desired.

11. A method of attaching a molecule with a reactive site to a support comprising the steps of:

(a) providing a support with a reactive site;

(b) binding a molecule to the reactive site, said first molecule comprising a masked reactive site attached to a photolabile protecting group of the formula Ar—

C(R₁)(R₂)—

O—

C(O)—

, wherein;

Ar is an optionally substituted fused polycyclic aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing;

R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl or alkynyl, or optionally substituted aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing;

to produce a derivatized support having immobilized thereon the molecule attached to the photolabile protecting group; and

(c) removing the photolabile protecting group to provide a derivatized support comprising the molecule with an unmasked reactive site immobilized thereon.

12. The method of claim 11, wherein the binding step in (b) is covalent.

13. The method of claim 11, wherein said removing step is carried out in the presence of a nucleophilic solvent.

14. The method of claim 13, wherein said nucleophilic solvent is selected from water, an alcohol, a water:

alcohol mixture and a water;

organic solvent mixture.

15. The method of claim 14, wherein said alcohol is selected from methanol and ethanol.

16. The method of claim 11, further comprising:

(a) coupling a second molecule to the unmasked reactive site, which second molecule comprises a second masked reactive site attached to the photolabile protecting group to produce a derivatized support having immobilized thereon a chain of the first and second molecules; and

(b) removing the photolabile protecting group to provide a derivatized support with a chain of the first and second molecules with a second unmasked reactive site immobilized thereon.

20. The method of claim 11, wherein Ar is independently 1-pyrenyl or 9-anthracenyl, R₁ is H and R₂ is independently H, methyl, substituted phenyl, 9-anthracenyl or 1-pyrenyl.

21. The method of claim 20, wherein Ar is 1-pyrenyl and R₁ and R₂ are H.

22. The method of claim 20, wherein Ar is 9-anthracenyl, R₁ is H and R₂ is H.

24. The method of claim 11, wherein the photolabile group is removed by irradiation at a wavelength of greater than 340 nm.

25. The method of claim 24, wherein the wavelength is about 365 nm.

26. A method of forming, from component molecules, a plurality of compounds on a support, each compound occupying a separate predefined region of the support, said method comprising the steps of:

(a) activating a region of the support;

(b) binding a molecule to the first region, said molecule comprising a masked reactive site linked to a photolabile protecting group of the formula Ar—

C(R₁)(R₂)—

O—

C(O)—

, wherein;

Ar is an optionally substituted fused polycyclic aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing;

R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl or alkynyl, or optionally substituted aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing;

(c) repeating steps (a) and (b) on other regions of the support whereby each of said other regions has bound thereto another molecule comprising a masked reactive site linked to the photolabile protecting group, wherein said another molecules may be the same or different from that used in step (b);

(d) removing the photolabile protecting group from one of the molecules bound to one of the regions of the support to provide a region bearing a molecule with an unmasked reactive site;

(e) binding an additional molecule to the molecule with an unmasked reactive site;

(f) repeating steps (d) and (e) on regions of the support until a desired plurality of compounds is formed from the component molecules, each compound occupying separate regions of the support.

27. The method of claim 26, wherein the binding steps are covalent.

28. The method of claim 27, wherein the molecules are selected from ribonucleosides, deoxyribonucleosides and 2′

-O-methylribonucleosides.

29. The method of claim 28, wherein the support is a glass or silica substrate.

30. The method of claim 26, wherein Ar is independently 1-pyrenyl or 9-anthracenyl, R₁ is H and R₂ is independently H, methyl, substituted phenyl, 9-anthracenyl or 1-pyrenyl.

31. The method of claim 30, wherein Ar is 1-pyrenyl and R₁ and R₂ are H.

32. The method of claim 30, wherein Ar is 9-anthracenyl, R₁ is H and R₂ is H.

33. The method of claim 28, wherein the deoxyribonucleosides are linked to the photolabile group via a 3′

or 5′

-OH.

34. The method of claim 26, wherein the photolabile group is removed by irradiation at a wavelength of greater than 340 nm.

35. The method of claim 26, wherein the wavelength is about 365 nm.

36. The method of claim 26, wherein at least 10⁶ chains are immobilized on the support.

37. The method of claim 26, wherein each of the regions has an area of between about 1 μ

m² and 10,000 μ

m².

39. A method of protecting and deprotecting a reactive group in a compound, comprising:

coupling the reactive group with a second compound of the formula Ar—

C(R₁)(R₂)—

O—

C(O)—

X, wherein;

Ar is an optionally substituted fused polycyclic aryl or heteroaromatic group or a vinylogously substituted species of the foregoing;

R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl or alkynyl, or optionally substituted aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing; and

X is a leaving group;

provided that when Ar is 1-pyrenyl and R₁ and R₂ are H, the reactive group being protected is not an amine, to provide a protected compound with its reactive group protected by linkage to Ar—

C(R₁)(R₂)—

O—

C(O)—

; and

irradiating the protected compound to provide a deprotected compound.

57. The method of claim 39, wherein Ar is independently 1-pyrenyl or 9-anthracenyl, R₁ is hydrogen and R₂ is independently hydrogen, methyl, substituted phenyl, 9-anthracenyl or 1-pyrenyl.

40. A method of attaching a molecule with a reactive site to a support comprising the steps of:

(a) providing a support with a reactive site;

(b) binding a molecule to the reactive site, said molecule comprising a masked reactive site attached to a photolabile protecting group of the formula Ar—

C(R₁)(R₂)—

, wherein;

Ar is an optionally substituted fused polycyclic aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing;

R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl or alkynyl, or optionally substituted aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing;

to produce a derivatized support having immobilized thereon the molecule attached to the photolabile protecting group; and

(c) removing, by irradiation, the photolabile protecting group to provide a derivatized support comprising the molecule with an unmasked reactive site immobilized thereon.

41. A method of forming, from component molecules, a plurality of compounds on a support, each compound occupying a separate predefined region of the support, said method comprising the steps of:

(a) activating a region of the support;

(b) binding a molecule to the first region, said molecule comprising a masked reactive site linked to a photolabile protecting group of the formula Ar—

C(R₁)(R₂)—

, wherein;

Ar is an optionally substituted fused polycyclic aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing;

R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl or alkynyl, or optionally substituted aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing;

(c) repeating steps (a) and (b) on other regions of the support whereby each of said other regions has bound thereto another molecule comprising a masked reactive site linked to the photolabile protecting group, wherein said another molecules may be the same or different from that used in step (b);

(d) removing, by irradiation, the photolabile protecting group from one of the molecules bound to one of the regions of the support to provide a region bearing a molecule with an unmasked reactive site;

(e) binding an additional molecule to the molecule with an unmasked reactive site;

(f) repeating steps (d) and (e) on regions of the support until a desired plurality of compounds is formed from the component molecules, each compound occupying separate regions of the support.

42. A method of protecting and deprotecting a reactive group in a compound, comprising:

coupling the reactive group with a second compound of the formula Ar—

C(R₁)(R₂)—

X, wherein;

Ar is an optionally substituted fused polycyclic aryl or heteroaromatic group or a vinylogously substituted species of the foregoing;

R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl or alkynyl, or optionally substituted aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing; and

X is a leaving group, to provide a protected compound with its reactive group protected by linkage to Ar—

C(R₁)(R₂)—

O—

C(O)—

; and

irradiating the protected compound to provide a deprotected compound.

58. The method of claim 42, wherein Ar is independently 1-pyrenyl or 9-anthracenyl, R₁ is hydrogen and R₂ is independently hydrogen, methyl, substituted phenyl, 9-anthracenyl or 1-pyrenyl.

59. The method of claim 58, wherein Ar is 1-pyrenyl and R₁ and R₂ are hydrogen.

60. The method of claim 58, wherein Ar is 9-anthracenyl, R₁ is hydrogen and R₂ is hydrogen.

61. The method of claim 42, wherein the compound is a ribonucleoside, deoxyribonucleoside or 2′

-O-methylribonucleoside.

62. The method of claim 61, wherein the deoxyribonucleoside is linked to the Ar—

C(R₁)(R₂)—

O—

C(O)—

group via a 3′

or 5′

-OH.

63. The method of claim 42, wherein the Ar—

C(R₁)(R₂)—

O—

C(O)—

group is removed by irradiation with light

64. The method of claim 63, wherein the wavelength is greater than 340 nm.

65. The method of claim 64, wherein the wavelength is about 365 nm.

43. A method of attaching to a solid support a molecule with a masked reactive site linked to a photolabile protecting group having a formula Ar—

C(R₁)(R₂)—

O—

C(O)—

, wherein;

Ar is an optionally substituted fused polycyclic aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing; and

R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl or alkynyl, or optionally substituted aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing;

said method comprising the steps of;

(a) activating a region of the support;

(b) binding a molecule to the first region, said molecule comprising a masked reactive site linked to a protecting group;

(c) removing the protecting group to provide a region bearing a molecule with an unmasked reactive site;

(d) protecting the unmasked reactive site by exposing it to a reagent of the formula Ar—

C(R₁)(R₂)—

O—

C(O)—

X, wherein X is a leaving group, to provide a support with a molecule with a masked reactive site linked to a photolabile protecting group.

44. A method of forming, from component molecules, a plurality of compounds on a support, each compound occupying a separate predefined region of the support, said method comprising the steps of:

(a) activating a region of the support;

(b) binding a molecule to the first region, said molecule comprising a masked reactive site linked to a protecting group;

(c) repeating steps (a) and (b) on other regions of the support whereby each of said other regions has bound thereto another molecule comprising a masked reactive site linked to a protecting group, wherein said another molecules and protecting groups can be the same or different 12

to each other;

(d) removing the protecting group from one of the molecules bound to one of the regions of the support to provide a region bearing a molecule with an unmasked reactive site;

(e) binding an additional molecule to the molecule with an unmasked reactive site;

(f) repeating steps (d) and (e) on regions of the support, until a desired plurality of compounds is formed from the component molecules, each compound occupying separate regions of the support, with the proviso that at least one of the protecting groups used in steps (a)-(f) is a photolabile protecting group of the formula Ar—

C(R₁)(R₂)—

O—

C(O)—

, wherein;

Ar is an optionally substituted fused polycyclic aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing;

R₁ and R₂ are independently H, optionally substituted alkyl, alkenyl or alkynyl, or optionally substituted aryl or heteroaromatic group or a vinylogously substituted derivative of the foregoing.

45. The method of claim 44, wherein the binding steps are covalent.

46. A method of making a compound of a formula Ar—

C(R₁)(R₂)—

O—

C(O)—

N, where Ar is 1-pyrenyl, R₁ and R₂ are H and N is a base-protected deoxynucleoside, the method comprising the steps of;

(a) acylating a 5′

-OH of a base protected deoxynucleoside with pentafluorophenoxy chloroformate to provide a 5′

-O—

C(O)-pentafluorophenoxy base-protected deoxynucleoside;

(b) reacting the 5′

-O—

C(O)-pentafluorophenoxy base-protected deoxynucleoside with 1-pyrenyl methyl carbinol in the presence of a base to provide compound of a formula Ar—

C(R₁)(R₂)—

O—

C(O)—

N.

48. A 5′

-O-pyrenylmethyloxycarbonyl base-protected deoxynucleoside made by the process of claim 46.

47. A method of making a compound of a formula Ar—

C(R₁)(R₂)—

O—

C(O)—

N, where Ar is 9-anthracenyl, R₁ and R₂ are H and N is a base-protected deoxynucleoside, the method comprising the steps of;

(a) acylating a 5′

-OH of a base protected deoxynucleoside with pentafluorophenoxy chloroformate to provide a 5′

-O—

C(O)-pentafluorophenoxy base-protected deoxynucleoside;

(b) reacting the 5′

-O—

C(O)-pentafluorophenoxy base-protected deoxynucleoside with 9-anthracenyl methyl carbinol in the presence of a base to provide compound of a formula Ar—

C(R₁)(R₂)—

O—

C(O)—

N.

49. A 5′

-O-anthracenylmethyloxycarbonyl base-protected deoxynucleoside made by the process of claim 47.

50. A method of performing chemical reactions on a surface, comprising:

providing at least one chemical reactant on said surface;

applying a coating to said surface, wherein said coating provides an environment that is favorable to reaction of said at least first chemical reactant;

reacting said at least first chemical reactant.

51. A method of activating a functional group on a surface of a substrate, wherein said functional group is protected by a protecting group having the formula Ar—

C(R₁)(R₂)—

O—

C(O)—

X, wherein Ar is an optionally substituted fused polycyclic aryl or a vinylogous derivative thereof;

R₁ and R2 are H, optionally substituted alkyl, alkenyl or alkynyl, optionally substituted aryl or optionally substituted heteroaromatic, or a vinylogous derivative of the foregoing; and

X is a leaving group, a chemical fragment linked to Ar—

C(R₁)(R₂)—

O—

C(O)—

via a heteroatom, or a solid support;

provided that when Ar is 1-pyrenyl and R₁ and R₂ are H, X is not linked to Ar—

C(R₁)(R₂)—

O—

C(O)—

via a nitrogen atom, the method comprising;

applying a nucleophilic coating to said surface; and

exposing said surface to light to remove said protecting group.

52. The method of claim 51, wherein said nucleophilic coating comprises a surfactant.

53. The method of claim 51, wherein said surfactant is present at a concentration of from about 0.01% to about 10% v/v.

54. The method of claim 51, wherein said coating comprises a poly-alcohol.

55. The method of claim 54, wherein said poly-alcohol is selected from glycerol, ethylene glycol, and propylene glycol.

56. The method of claim 51, wherein said coating is spin coated onto said substrate surface.