Synthesis of oligonucleotide arrays using photocleavable protecting groups

• US 6,022,963 A
• Filed: 04/10/1996
• Issued: 02/08/2000
• Est. Priority Date: 12/15/1995
• Status: Expired due to Term

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: Nam, Ngo Quoc, McGall, Glenn H.
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US08/630,148
• Time in Patent Office: 1,399 Days
• Field of Search: 536/25.3, 536/25.31, 536/25.32, 536/25.33, 536/25.34, 536/25.6, 536/124, 435/6, 435/7.92, 435/7.94, 435/7.95, 436/518, 436/527, 436/528, 436/807
• US Class Current: 506/32
• 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: 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: Y02P 20/55 Design of synthesis routes, e.g. reducing the use of auxiliary or protecting groups
• CPC Class Codes: Y10S 436/807 Apparatus included in process claim, e.g. physical support structures

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 =R₂ =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=pyrenyl and R₁ =R₂ =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 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.

2. The method of claim 1, wherein the binding step in (b) forms a covalent linkage.

3. The method of claim 2, 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.

4. The method of claim 3, further comprising repeating steps (a) and (b) with a succession of molecules to provide a chain of molecules immobilized on the support.

5. The method of claim 4, wherein the molecules are deoxynucleosides.

6. The method of claim 5, wherein the support is a glass or silica substrate.

7. The method of claim 1, wherein Ar is independently 1-pyrenyl or 9-anthracenyl, R₁ =H and R₂ is independently H, methyl, p-methoxyphenyl, 3,5-dimethoxyphenyl, 9-anthracenyl or 1-pyrenyl.

8. The method of claim 7, wherein Ar is 1-pyrenyl and R₁ and R₂ are H.

9. The method of claim 5, wherein the deoxynucleosides are linked to the photolabile group via a 5'"'"'--OH.

10. The method of claim 1, wherein the photolabile group is removed by irradiation at a wavelength of greater than 350 nm.

11. The method of claim 10, wherein the wavelength is about 365 nm.

12. 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.

13. The method of claim 12, wherein the binding steps form covalent linkages.

14. The method of claim 13, wherein the molecules are deoxynucleosides.

15. The method of claim 14, wherein the support is a glass or silica substrate.

16. Th e method of claim 12, wherein Ar is independently 1-pyrenyl or 9-anthracenyl, R₁ =H and R₂ is independently H, methyl, p-methoxyphenyl, 3,5-dimethoxyphenyl, 9-anthracenyl or 1-pyrenyl.

17. The method of claim 16, wherein Ar is 1-pyrenyl and R₁ and R₂ are H.

18. The method of claim 14, wherein the deoxynucleosides are linked to the photolabile group via a 5'"'"'--OH.

19. The method of claim 12, wherein the photolabile group is removed by irradiation at a wavelength of greater than 350 nm.

20. The method of claim 19, wherein the wavelength is about 365 nm.

21. The method of claim 14, wherein at least 106 chains a re immobilized on the support.

22. The method of claim 12, wherein each of the regions has an area of between about 1 μ

m² and 10,000 μ

m².

23. The method of claim 12, further comprising:

(g) 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) or is an unmasked reactive site on a molecule on the support as formed in step (d);

(h) 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

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

29. The method of claim 23 wherein the binding steps form covalent linkages.

24. 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.

25. 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.

26. 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.

27. 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.

28. The method of claim 27, wherein the binding steps from covalent linkages.