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Novel chemiluminescent substrates for factor xa

A chemiluminescence, factor technology, applied in the fields of organic chemistry, bulk chemical production, biochemical equipment and methods, etc., can solve problems such as discomfort measurement, continuous measurement of discomfort, reduction of physiological conditions, etc.

Pending Publication Date: 2021-07-30
ENZYRE BV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Chromogenic tests are flawed: because their method depends on the measurement of optical density, they cannot be performed in mixtures that have become cloudy due to clot formation, and the chromogenic yellow interferes with the inherent yellow color of plasma
[0009] Fluorescent substrates are flawed: Commercial platforms for coagulation analysis often do not support fluorescent assays, thus requiring additional instrumentation
Cosby et al. (―Customenzyme substrates for luciferase-based assays", Cell Notes, No. 18, pp. 9-11, 2007) deal with luminescent substrates, however they are not suitable for measurements in aqueous solutions (such as plasma), nor Not suitable for continuous measurement
Poor solubility often requires organic co-solvents, which degrade the physiological conditions of the assay, or require larger sample volumes or addition of larger volumes of reagents

Method used

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  • Novel chemiluminescent substrates for factor xa
  • Novel chemiluminescent substrates for factor xa
  • Novel chemiluminescent substrates for factor xa

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0180] Example 1 - Providing a substrate of the invention

[0181] Such as Figure 1A As shown, the substrate is synthesized through the key intermediate A1. The synthesis of this intermediate started with the conversion of benzothiazole 1 to nitrile 2 in 91% yield. Reduction afforded pure aniline in 54% yield after direct and reverse phase flash column chromatography. In pyridine, with PCl 3 Coupling with Fmoc-Orn(Boc)-OH(4). After Fmoc-deprotection, compound 6 was successfully obtained. In these two steps, peptide coupling of 6 with Fmoc-Gly-OH 7 to 8 and Boc deprotection gave 9 in 84% yield. After purification by flash column chromatography, reaction with 1,3-bis(tert-butoxycarbonyl)-2-(trifluoromethanesulfonyl)guanidine afforded guanidine 10 in good yield and purity. To provide compounds of general formula 1-3 or 1-4 in which other amino acids are present, the above scheme is repeated with said other amino acids, or compound 8 is not converted to compound 10.

[018...

Embodiment 2

[0184] Example 2 - Kinetic Behavior of Substrates of the Invention

[0185] The Michaelis-Menten kinetics of the enzyme FXa and its substrate MePEG2-IEGR were determined (TFA salt was used in these examples). Prepare the following compositions in microtubes on ice:

[0186] Composition 1:

[0187] 160 μL Tris-buffered saline (TBS) 50 mM Tris-HCl, 150 mM NaCl (pH 7.4)

[0188] 4 μL ATP (final concentration 333 μM)

[0189] 4 μL MgCl 2 (final concentration 8.3mM)

[0190] 12 μL luciferase (final concentration 0.9 mg / ml)

[0191] Substrate compositions 2a-g:

[0192] Prepare microtiter tubes with 30 µL of substrate concentrations resulting in final concentrations of 2000, 1333, 1000, 666, 333, 167, and 66 µM.

[0193] Enzyme composition 3:

[0194] Prepare FXa (Coachrom, 16nM) compositions in microtubes on ice

[0195] Subsequently, the following compositions were added together to a white 384-well plate:

[0196] 24 μL composition 1

[0197] 3 μL of compositions 2a-g ...

Embodiment 3

[0201] Example 3 - Cross-reactivity

[0202] To determine cross-reactivity, assays were performed using a series of coagulases at concentrations representative of those found in human plasma. The selected enzyme concentrations were comparable to those expected in vivo after activation. For tissue plasminogen activator (tPA), this is the usual concentration to promote fibrinolysis.

[0203] FXa at a concentration of 80 nm produced a signal of 1.000.000 RLU. The other thrombins produced the following signals: 52 nM thrombin -30,000 RLU, 8 nM plasmin -8,000 RLU, 193 IU / mL tPA -6,000 RLU, 145 nMFXIIa -500 RLU (Figure 3). These data indicate that the substrates of the invention are preferentially cleaved by FXa.

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Abstract

The present invention relates to chemiluminescent substrates of formulas (I-3) and (I-4) for blood clotting enzyme Factor Xa. The substrates are particularly useful for assaying coagulation factors and for quantifying an anticoagulant in a sample.

Description

technical field [0001] The present invention relates to chemiluminescent substrate molecules suitable for monitoring thrombin Factor Xa. The substrate is particularly useful for the determination of coagulation factors in samples and the quantification of anticoagulants in samples. Background technique [0002] Coagulation of blood (also known as clotting) is the transition of blood from a liquid to a gel, resulting in clots. Coagulation is part of the process of stopping bleeding and ultimately preventing excessive blood loss. Coagulation begins shortly after damage to the endothelium lining of blood vessels. Exposure of the subendothelial space results in the binding of plasma factor VII (FVII) to tissue factor, which ultimately leads to the formation of fibrin. In so-called primary hemostasis, platelets immediately form a plug at the site of injury. So-called secondary hemostasis occurs simultaneously: coagulation factors respond in a complex cascade to form fibrin ch...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07D277/68C12Q1/66G01N33/86
CPCG01N33/86G01N2333/96463C07D277/68C12Q1/66C07K5/101G01N21/6408
Inventor W·L·范海尔德M·范格芬D·斯蒂格斯
Owner ENZYRE BV
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