Photocleavable protecting groups and methods for their use

• US 6,147,205 A
• Filed: 03/05/1997
• Issued: 11/14/2000
• 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: McGall, Glenn H., Rava, Richard P., Nam, Ngo Q.
• Primary Examiner(s): Wilson, James O.
• Application Number:
  US08/812,005
• Time in Patent Office: 1,350 Days
• Field of Search: 536/22.1, 536/23.1, 536/25.32, 536/25.33, 536/25.34, 536/26.26, 536/26.6, 536/27.13, 536/27.6, 536/27.8, 536/27.81, 536/28.4, 536/28.5 , 536/28.53, 536/28.54, 536/25.3, 536/26.71, 536/26.72, 536/26.73, 536/26.74
• US Class Current: 506/32
• 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 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.

2. The compound of claim 1, wherein the chemical fragment X is a nucleic acid or an oligonucleotide, wherein each nucleoside of the the nucleic acid or the oligonucleotide has a base selected from the group consisting of adenine, cytosine, guanine, thymine, uracil or an analog thereof and the base is linked to a ribose, a 2'"'"'-O-alkylribose, a 2'"'"'-O-allylribose, a 2'"'"'-deoxyribose, a 2'"'"'-deoxy-2'"'"'-fluororibose, or a 2'"'"'-deoxy-2'"'"'-bromoribose.

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 the nucleoside is linked to Ar--C(R₁)(R₂)--O--C(O)-- via a 3'"'"' or 5'"'"'-OH of the ribose.

5. The compound of claim 4, wherein the nucleoside is a base-protected deoxyribonucleoside.

6. The compound of claim 4 wherein the nucleoside is a base-protected ribonucleoside.

7. The compound of claim 4, wherein the nucleoside is a base-protected 2'"'"'-O-methylribonucleoside.

8. The compound of claim 2, wherein Ar is 1-pyrenyl or 9-anthracenyl;

and R₁ and R₂ are independently H, methyl, methoxy, dimethoxyphenyl, 9-anthracenyl or 1-pyrenyl.

9. The compound of claim 8, wherein the nucleoside is a base protected deoxyribonucleoside H-phosphonate or a base protected deoxyribonucleoside phosphoramidite.

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

15. The compound of claim 2, wherein the nucleic acid or the oligonucleotide further comprises a polyamide backbone attached to the bases.

16. The compound of claim 15, wherein the oligonucleotide is linked to Ar--C(R₁)(R₂)--O--C(O)-- via a 3'"'"' or 5'"'"'-OH of the ribose.

17. The compound of claim 1, wherein the chemical fragment X is a nucleoside or a nucleotide comprising the nucleoside, wherein the nucleoside has a base selected from the group consisting of adenine, cytosine, guanine, thymine, uracil or an analog thereof and the base is linked to a ribose, a 2'"'"'-O-alkylribose, a 2'"'"'-O-allylribose, a 2'"'"'-deoxyribose, a 2'"'"'-deoxy-2'"'"'-fluororibose, or a 2'"'"'-deoxy-2'"'"'-bromoribose.

18. The compound of claim 17, wherein the nucleoside or the nucleotide is linked to Ar--C(R₁)(R₂)--O--C(O)-- via a 3'"'"' or 5'"'"'-OH of the ribose.

19. The compound of claim 1, wherein the chemical fragment X is an amino acid, a peptide, or a polypeptide.

20. The compound of claim 1, wherein the chemical fragment X is a monosaccharide, oligosaccharide or a polysaccharide.

23. The compound of claim 1, wherein the leaving group X is capable of reacting with a molecule possessing a nucleophilic group, wherein the nucleophilic group is a hydroxy, amino, alkylamino, or thio group.

24. The compound of claim 23, wherein the leaving group X is a halo, oxycarbonyl, imidazolyl, or pentafluorophenoxy group.

25. The compound of claim 1, wherein the solid support X has reacting groups selected from the group consisting of hydroxyl, amino, alkylamino, or thio.

26. The solid support of claim 25, wherein the support is made of material selected from the group consisting of glass, latex, polyethyleneglycol, cross-linked polystyrene, colloidal gold, or a combination thereof.

27. The solid support of claim 25, wherein the support has the characteristic of light-absorption.

28. The solid support of claim 27, wherein the support is made of material selected from the group consisting of:

polymerized Langmuir Blodgett film, functionalized glass, Si, Ge, GaAs, GaP, SiO₂, SiN₄, modified silicon, or a combination thereof.

29. The solid support of claim 28, wherein the support is made of a polymer selected from the group consisting of:

polytetrafluoroethylene, polyvinylidenedifluoride, polystyrene, polycarbonate, or a combination thereof.

30. The solid support of claim 25, wherein the support is a resin or a bead.

11. A method of making a compound of the formula Ar--C(R₁)(R₂)--O--C(O)--N, wherein Ar is 1-pyrenyl;

R₁ and R₂ are H; and

N is a base-protected nucleoside, wherein the nucleoside has a base selected from the group consisting of adenine, cytosine, guanine, thymine, uracil or an analog thereof and the base is linked to a ribose, a 2'"'"'-O-alkylribose, a 2'"'"'-O-allylribose, a 2'"'"'-deoxyribose, a 2'"'"'-deoxy-2'"'"'-fluororibose, or a 2'"'"'-deoxy-2'"'"'-bromoribose;

the method comprising the steps of;

(a) acylating the 5'"'"'-OH of the base-protected nucleoside with pentafluorophenoxy chloroformate to provide a 5'"'"'-O-C(O)--pentafluorophenoxy base-protected nucleoside;

(b) reacting the 5'"'"'-O-C(O)-pentafluorophenoxy base-protected nucleoside with 1-pyrenyl methyl carbinol to provide a compound of the formula Ar--C(R₁)(R₂)--O--C(O)--N.

13. A 5'"'"'-O-pyrenylmethyloxycarbonyl base-protected deoxynucleoside made by the method of claim 11.

21. The method of claim 11, wherein the nucleoside is a deoxynucleoside.

12. A method of making a compound of the formula Ar--C(R₁)(R₂)--O--C(O)--N, wherein Ar is 9-anthracenyl;

R₁ and R₂ are H; and

N is a base-protected nucleoside, wherein the nucleoside has a base selected from the group consisting of adenine, cytosine, guanine, thymine, uracil or an analog thereof and the base is linked to a ribose, a 2'"'"'-O-alkylribose, a 2'"'"'-O-allylribose, a 2'"'"'-deoxyribose, a 2'"'"'-deoxy-2'"'"'-fluororibose, or a 2'"'"'-deoxy-2'"'"'-bromoribose;

the method comprising the steps of;

(a) acylating the 5'"'"'-OH of the base protected nucleoside with pentafluorophenoxy chloroformate to provide a 5'"'"'-O-C(O)-pentafluorophenoxy base-protected nucleoside;

(b) reacting the 5'"'"'-O-C(O)-pentafluorophenoxy base-protected nucleoside with 9-anthracenyl methyl carbinol to provide a compound of the formula Ar--C(R₁)(R₂)--O--C(O)--N.

14. A 5'"'"'-O-anthracenylmethyloxycarbonyl base-protected deoxynucleoside made by the method of claim 12.

22. The method of claim 12, wherein the nucleoside is a deoxynucleoside.