Reagents for labeling biomolecules having aldehyde or ketone moieties

• US 6,967,251 B2
• Filed: 10/02/2001
• Issued: 11/22/2005
• Est. Priority Date: 10/02/2000
• Status: Expired due to Term

• Current Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Original Assignee: Molecular Probes Incorporated (Thermo Fisher Scientific Incorporated)
• Inventors: Diwu, Zhenjun, Patton, Wayne F., Steinberg, Thomas H., Haugland, Richard P.
• Primary Examiner(s): Berch, Mark L.
• Application Number:
  US09/970,215
• Publication Number:
  US 20020137068A1
• Time in Patent Office: 1,512 Days
• Field of Search: 544/289, 544/287, 544/249, 544/283, 435/4, 435/23, 435/28, 525/54.1, 530/300, 546/33, 546/109, 546/153, 252/62.51
• US Class Current: 544/289
• CPC Class Codes: C07D 239/91 with aryl or aralkyl radicals attached in position 2 or 3
• CPC Class Codes: C07D 265/22 Oxygen atoms
• CPC Class Codes: Y10T 436/143333 Saccharide [e.g., DNA, etc.]
• CPC Class Codes: Y10T 436/200833 Carbonyl, ether, aldehyde or ketone containing

Abstract

Novel fluorescent derivitization reagents are described that are suitable for coupling to biomolecules that contain aldehyde or ketone functional groups. In one embodiment is provided reagents that have the following formula:
wherein Q is carbonyl, thiocarbonyl, or sulfonyl, and R⁵ is -L-Z; L is arylene, or a C_1-6 perfluoroalkylene, or a single covalent bond; Z is a carbonyl hydrazide, hydrazide, sulfonyl hydrazide, or a thiocarbonyl hydrazide; R¹¹-R¹⁴ are independently H, C_1-6 alkyl, C_1-6 alkoxy, C_1-6 perfluoroalkyl, C_1-6 alklyamino, di(C_2-12alkyl)amino, amino, carboxy, cyano, halogen, hydroxy, nitro, phenyl, or sulfo; and R²¹-R²⁴ are independently H, C_1-6 alkyl, C_1-6 alkoxy, C_1-6 perfluoroalkyl, C_1-6 alklyamino, di(C_2-12-alkyl)amino, amino, carboxy, cyano, halogen, hydroxy, nitro, phenyl, sulfo, or -L-Z. The method of treating a sample with the derivativization reagents is described. The reagents are particularly useful for labeling glycoproteins or glycopeptides, nucleic acids, and lipopolysaccharides in electrophoresis gels.

Claims

1. A compound of the formula:

wherein Q is carbonyl, thiocarbonyl, or sulfonyl, and R⁶ is -L-Z;

L is arylene, or a C_1-6 perfluoroalkylene; and

Z is a (C═

0)NR⁸NH₂ (carbonyl hydrazide), —

NR⁶—

NH₂ (hydrazide), —

(SO₂)NR⁶NH₂ (sulfonyl hydrazide) or —

(C═

S)NR⁶NH₂ (thiocarbonyl hydrazide);

R¹¹-R¹⁴ are independently H, C_1-6 alkyl, C_1-6 alkoxy, C_1-6 perfluoroalkyl, C_1-6 alklyamino, di(C_2-12-alkyl)amino, amino, carboxy, cyano, halogen, hydroxy, nitro, phenyl, or sulfo;

R²¹-R²⁴ are independently H, C_1-6 alkyl, C_1-6 alkoxy, C_1-6 perfluoroalkyl, C_1-6 alklyamino, di(C_2-12alkyl)amino, amino, carboxy, cyano, halogen, hydroxy, nitro, phenyl, sulfo, or -L-Z;

R6 is H or alkyl having 1-6 carbons.

2. The compound, as claimed in claim 1 wherein Q is carbonyl.

3. The compound, as claimed in claim 1, wherein L is arylene.

4. The compound, as claimed in claim 1, wherein R¹¹-R¹⁴ are each H.

5. The compound according to claim 1, wherein said compound is selected from the group consisting of

6. The compound according to claim 1, wherein said Z is sulfonyl hydrazide or carbonyl hydrazide.

7. The compound according to claim 1, wherein L is C_1-6 perfluoroalkylene.

8. The compound according to claim 1, wherein R²¹-R²⁴ are each hydrogen.

9. A compound of the formula:

wherein Q is carbonyl, thiocarbonyl, or sulfonyl, and R⁶ is -L-Z;

L is a single covalent bond;

Z is —

NR⁶—

NH₂ (hydrazide);

R¹¹-R¹⁴ are independently H, C_1-6 alkyl, C_1-6 alkoxy, C_1-6 perfluoroalkyl, C_1-6 alklyamino, di(C_2-12-alkyl)amino, amino, carboxy, cyano, halogen, hydroxy, nitro, phenyl, or sulfo; and

R²¹-R²⁴ are independently H, C_1-6 alkyl, C_1-6 alkoxy, C_1-6 perfluoroalkyl, C_1-6 alklyamino, di(C_2-12-alkyl)amino, amino, carboxy, cyano, halogen, hydroxy, nitro, phenyl, sulfo, or -L-Z;

R6 is H or alkyl having 1-6 carbons.