Reagents and methods for solid phase synthesis and display

• US 20040106728A1
• Filed: 07/14/2003
• Published: 06/03/2004
• Est. Priority Date: 06/22/1998
• Status: Abandoned Application

• Current Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Original Assignee: Affymetrix, Inc. (Thermo Fisher Scientific Incorporated)
• Inventors: McGall, Glenn H., Barone, Anthony D., Diggelmann, Martin
• Application Number:
  US10/619,799
• Publication Number:
  US 20040106728A1
• Time in Patent Office: Days
• US Class Current: 525/54.1
• CPC Class Codes: B01J 2219/00722 Nucleotides
• CPC Class Codes: B01J 2219/00725 Peptides
• CPC Class Codes: C07D 493/10 Spiro-condensed systems
• CPC Class Codes: C07F 9/2408 of hydroxyalkyl compounds
• 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: C40B 40/06 Libraries containing nucleotides or polynucleotides, or derivatives thereof
• CPC Class Codes: C40B 40/10 Libraries containing peptides or polypeptides, or derivatives thereof

Abstract

New compounds, compositions and methods which find application in solid phase synthesis including the preparation of high-density arrays of diverse polymer sequences such as diverse peptides and oligonucleotides as well as in preparation of arrays of small ligand molecules. The compounds of the present invention are those which are typically referred to as linking groups, linkers or spacers and include unsymmetrical disulfide linking groups, and 1,3-diol derivatives capable of providing a triggered release of an attached compound from a solid support under mild conditions. Additional new compounds are labels which can be incorporated into either the 3′ or 5′ terminus of a DNA oligomer.

Claims

1. A compound having the formula:

P¹—

X¹—

(W¹)_n—

S—

S—

(W²)_m—

X²—

P² 

 

(I)

wherein

P¹ and P² are each members independently selected from the group consisting of a hydrogen atom, an activating group and a protecting group;

X¹ and X² are each independently selected from the group consisting of a bond, —

O—

, —

NH—

, —

NR— and

—

CO₂—

, wherein R is a lower alkyl group having one to four carbon atoms;

W¹ and W² are each independently selected from the group consisting of methylene, oxyethylene and oxypropylene; and

n and m are each independently integers of from 2 to 12 with the proviso that n and m are not the same when W¹ and W² are the same, and with the further proviso that P¹ and P² are not both hydrogen atoms.

2. A compound in accordance with claim 1, wherein P² is an activating group selected from the group consisting of a phosphoramidite, a trialkylammonium H-phosphonate and a phosphate triester.

3. A compound in accordance with claim 1, wherein P² is a phosphoramidite, P¹ is a protecting group selected from the group consisting of acid labile protecting groups, W¹ and W² are both methylene, X¹ and X² are both —

O—

, and n and m are each integers of from 2 to 8.

4. A compound in accordance with claim 1, wherein P² is a phosphoramidite, P¹ is DMT, W¹ and W² are both methylene, X¹ and X² are both —

O—

, and n and m are each integers of from 3 to 8.

5. A modified substrate for use in solid phase chemical synthesis, said substrate having the formula:

A¹—

B¹—

L¹ 

 

(II)

wherein A¹ is a solid support, B¹ is a bond or a spacer group, and L¹ is a linking group having the formula;

P¹—

X¹—

(W¹)_n—

S—

S—

(W²)_m—

X²—

 

 

(IIa)

wherein,

P¹ is a protecting group;

X¹ and X² are each independently selected from the group consisting of a bond, —

O—

, —

NH—

, —

NR— and

—

CO₂—

, wherein R is a lower alkyl group having one to four carbon atoms;

W¹ and W² are each independently selected from the group consisting of methylene, oxyethylene and oxypropylene; and

n and m are each independently integers of from 2 to 12 with the proviso that n and m are not the same when W¹ and W² are the same.

6. A substrate in accordance with claim 5, wherein P¹ is a photolabile protecting group.

7. A substrate in accordance with claim 5, wherein P¹ is a photolabile protecting group, W¹ and W² are both methylene, and X¹ and X² are both —

O—

.

8. A substrate in accordance with claim 5, wherein P¹ is a photolabile protecting group, X¹ and X² are both —

O—

, and n and m are each integers of from 2 to 8.

9. A substrate in accordance with claim 5, wherein P¹ is DMT, X¹ and X² are both —

O—

, W¹ and W² are both methylene, and n and m are each integers of from 2 to 8.

10. A method of synthesizing small ligand molecules on a solid support having optional spacers, said small ligand molecules being removable therefrom upon treatment with a suitable disulfide cleaving reagent, said method comprising:

(a) contacting a solid support an unsymmetrical disulfide linking group of formula;

P¹—

X¹—

(W¹)_n—

S—

S—

(W²)_m—

X²—

P² 

 

(IIb)

 

wherein,

P¹ and P² are each members independently selected from the group consisting of a hydrogen atom, an activating group and a protecting group;

X¹ and X² are each independently selected from the group consisting of a bond, —

O—

, —

NH—

, —

NR— and

—

CO₂—

, wherein R is a lower alkyl group having one to four carbon atoms;

W¹ and W² are each independently selected from the group consisting of methylene, oxyethylene and oxypropylene; and

n and m are each independently integers of from 2 to 12 with the proviso that n and m are not the same when W¹ and W² are the same;

to produce a derivatized solid support having attached unsymmetrical disulfide linking groups suitably protected with protecting groups;

(b) optionally removing said protecting groups from said derivatized solid support to provide a derivatized solid support having unsymmetrical disulfide linking groups with synthesis initiation sites; and

(c) coupling said small ligand molecules to said synthesis initiation sites on said derivatized solid support to produce a solid support having attached small ligand molecules which are removable therefrom upon application of said disulfide cleaving reagent.

11. A compound of the formula:

12. A compound in accordance with claim 11, wherein P¹¹ and P¹² are both hydrogen.

13. A compound in accordance with claim 11, wherein P¹¹ is a protecting group and P¹² is a phosphoramidite.

14. A compound in accordance with claim 11, wherein P¹¹ is DMT and P¹² is a phosphoramidite.

15. A substrate for the solid phase synthesis of oligonucleotides, said substrate having the formula:

A¹¹—

B¹¹—

L¹¹—

Fl

wherein A¹¹ is a solid support, B¹¹ is a bond or a derivatizing group, L¹¹ is a linking group, and Fl is a fluorescent moiety having the formula;

16. A substrate bound, fluorescently labeled oligonucleotide having the formula:

A¹¹—

B¹¹—

L¹¹—

Nu—

Fl

wherein A¹¹ is a solid support, B¹¹ is a bond or a derivatizing group, L¹¹ is a linking group, Nu is an oligonucleotide and Fl is a fluorescent moiety having the formula;

17. A substrate bound, fluorescently labeled oligonucleotide having the formula:

A¹¹—

B¹¹—

L¹¹—

Fl—

Nu

wherein A¹¹ is a solid support, B¹¹ is a bond or a derivatizing group, L¹¹ is a linking group, Fl is a fluorescent moiety having the formula;

18. A selectively cleavable linkage molecule useful in solid phase compound synthesis, said linkage molecule having the formula:

19. A selectively cleavable linkage molecule in accordance with claim 18, wherein X²¹ is an amino alkoxy group, Y is —

C(═

O)R, Z is —

C(O)— and

Q is a phosphoramidite.

20. A selectively cleavable linkage molecule in accordance with claim 18, wherein P²¹ is removable under photolytic conditions, P²² is removable under acidic conditions, X²¹ is an amino alkoxy group, Y is —

C(═

O)R, Z is —

C(O)— and

Q is a phosphoramidite.

21. A selectively cleavable linkage molecule in accordance with claim 18, wherein P²¹ is MeNPOC, P²² is DMT, X²¹ is —

NH—

CH₂CH(CH₃)—

O—

, Y is —

C(═

O)R, Z is —

C(O)— and

Q is a phosphoramidite.

22. A modified substrate for use in solid phase chemical synthesis, said substrate having the formula:

L²¹—

B²¹—

A²¹

wherein A²¹ is a solid support, B²¹ is a bond or a derivatizing group, and L²¹ is a linking group having the formula;