Nucleotide triphosphate with an electroactive label conjugated to the gamma phosphate

A technology of nucleoside triphosphate and nucleoside triphosphate conjugates, applied in the directions of labeling, combinatorial chemistry, organic chemistry, etc. used in chemical analysis, can solve the problems of time-consuming, increased cost, lengthy processing process, etc., and achieve simplified steps Effect

Inactive Publication Date: 2011-04-13
海因茨-伯恩哈德·克拉茨 +2
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, additional modification of the peptide with electroactive or optical labels is required, which adds cost and results in tedious and time-consuming processing

Method used

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  • Nucleotide triphosphate with an electroactive label conjugated to the gamma phosphate
  • Nucleotide triphosphate with an electroactive label conjugated to the gamma phosphate
  • Nucleotide triphosphate with an electroactive label conjugated to the gamma phosphate

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1-F

[0082] The synthesis of embodiment 1-Fc-ATP

[0083] Boc-NH(CH 2 ) 6 Preparation of N(H)COFc (Compound 1): Ferrocenecarboxylic acid (230 mg, 1 mmol) was dissolved in 20 mL of anhydrous DCM. Then, 1.2 equivalents of TEA (0.17 mL) and 1.2 equivalents of HBTU (455 mg) were added sequentially. After 30min, Boc-NH(CH 2 ) 6 NH 2 Add to the solution and continue stirring overnight. After the reaction was complete, the solvent was removed under vacuum, and the residue was purified by flash column chromatography on silica gel (DCM-MeOH, 95:5; R f = 0.25) to afford the desired compound as a yellow solid in 78% yield (334 mg). 1 H-NMR (δ, DMSO): 7.74 (t, 1H, J = 5.2Hz, NH-COFc), 6.78 (t, 1H, J = 5.4Hz, NH-Boc), 4.78 (s, 2H, Cp), 4.32(s, 2H, Cp), 4.14(s, 5H, Cp), 3.15(q, 2H, J=6.4Hz, CH 2 ), 2.90 (q, 2H, J=6.4Hz, CH 2 ), 1.23-1.52(m, 17H). 13 C( 1 H)-NMR (δ, DMSO): 168.57, 155.57, 77.27, 76.94, 69.73, 69.23, 68.06, 39.76, 38.54, 29.50, 29.47, 28.26, 26.17, 26.08. IR: v max...

Embodiment 2

[0088] Example 2 - Electrochemical Detection of Protein Kinase C Phosphorylation

[0089] Cyclic voltammetry (CV) was performed using a CHInstruments 660 system (Austin, TX). DEP-chips with screen-printed gold electrodes (SPEs) were kindly donated by BioDevice Technology Ltd. (Ishikawa, Japan) and prepared as described in Li et al. Anal. Chem. 2005, 77, 5766-5769. The overall length of the SPE is 11mm, while the geometric area of ​​the working electrode is 2.64mm 2 . The reference electrode is an Ag / AgCl paste electrode (Ag / AgCl past electrode), and the counter electrode is a carbon electrode.

[0090] 1 H, 13 C, 31 P NMR experiments were carried out on a Bruker Avance 500MHz spectrometer, and the chemical shifts were compared to residual DMSO (for 1 H is 2.50ppm and for 13 C is 39.52ppm) and H 2 O (4.79ppm). Mass spectrometry was performed using a Perkin Elmer-Sciex API365 instrument.

[0091] Reagents were purchased from Merek unless otherwise noted. All solutio...

Embodiment 3

[0108] Example 3 - Casein Kinase 2 (CK2) and Tyrosine Kinase AbI1 and Detection of HER2 / ErbB2 phosphorylation

[0109] It has previously been demonstrated that the use of nucleoside triphosphate conjugates containing electroactively labeled γ-phosphate groups is suitable for detection of protein kinase C activity using electrochemical biosensing systems. In this example, the utility of nucleoside triphosphate conjugates was to detect another well-described protein serine / threonine kinase, casein kinase-2 (CK2), and two clinically important tyrosine kinases, AbI1 and HER2 / ErbB2 were used to evaluate this method for determining the potency of protein kinase inhibitors.

[0110] First for the kinase-specific peptide RRRDDDDSDDD of serine / threonine kinases 12 Enzyme modification of CK2 was evaluated by mass spectrometry using Fc-ATP as a co-substrate.

[0111] Figure 14 Shown for (A) CK2, (B) AbI1-T315I and (C) HER2 / ErbB2 using Applied Biosystems 4700 Proteomics Analyzer (...

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Abstract

A nucleotide triphosphate (NTP) participates in a phosphorylation reaction, wherein a phosphate group is transferred from the NTP to a substrate by a kinase. Provision in a kinase reaction of a NTP whose gamma phosphate is conjugated to an electroactive label results in the transfer of the gamma phosphate-electroactive label conjugate from the NTP to the substrate. The electroactive label is an organic moiety such as a quinone or a mtrohetercycle, or is a metallocene such as a ferrocene or a cobaltocene. Upon transfer of the gamma phosphate-electroactive label conjugate to an electrode-bound substrate by a kinase, the phosphorylation event is detected electrochemically by cyclic voltammetry. Phosphorylation can also be detected by mass spectrometry of a substrate carrying the electroactive label-conjugated gamma phosphate. NTP comprising the gamma phosphate-electroactive label conjugate is used in methods of detecting the presence of a kinase in a sample, screening candidate compounds that modulate kinase activity, and in methods of diagnosing a disease associated with a kinase.

Description

[0001] prior application [0002] This application claims priority to US Provisional Patent Application 60 / 960,388, filed September 27, 2007. technical field [0003] The present invention relates to a novel electroactive nucleoside triphosphate suitable for monitoring phosphorylation-related events. Background technique [0004] In cellular communication networks, many enzymes and receptors switch between "on" (or up, on) and "off (or down, off"), or in other words, are "phosphorylated" and "dephosphorylated" of ". During phosphorylation, a phosphoryl group from ATP is transferred to a protein-specific serine, threonine or tyrosine residue. Due to these modifications, the function or localization of the protein can change, which in some cases can lead to the formation of tumor proteins 1 . [0005] Aberrant protein phosphorylation is the cause of many diseases, including cancer, diabetes and chronic inflammatory diseases. Assays to quantify protein kinase activity are ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/48C07H19/10C07H19/20C07K16/44C12Q1/00C40B30/08G01N27/403
CPCG01N33/581G01N2458/30C12Q1/485C07H19/20C07H19/10
Inventor 海因茨-伯恩哈德·克拉茨卡甘·克尔曼宋海峰
Owner 海因茨-伯恩哈德·克拉茨
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