Luciferase substrates, and preparation method and application thereof

A technology of luciferase and luciferase, which is applied in the field of luciferase substrate and its preparation, can solve the problems that the kinetic isotope effect is still less, and achieve good bioluminescence advantages, long bioluminescence time, Effect of strong bioluminescence intensity

Active Publication Date: 2020-09-18
SHANDONG UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

At present, bioluminescent imaging technology has been widely used, mainly in food, tumor, heav

Method used

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  • Luciferase substrates, and preparation method and application thereof
  • Luciferase substrates, and preparation method and application thereof
  • Luciferase substrates, and preparation method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0134] Embodiment 1: preparation compound cycluc

[0135]

[0136] 2,2,2-trifluoro-1-(5-nitroindolin-1-yl)ethan-1-one (Intermediate 1)

[0137] Add dichloromethane (20mL) to 5-nitroindoline (4g, 24.3mmol) and triethylamine (2.6g, 26.8mmol) and stir, while adding trifluoroacetic anhydride (5.6g, 26.8mmol) dropwise . The mixture was stirred for 45 minutes, then water (50 mL) was added, after stirring for 10 minutes, the mixture was acidified with 5M HCl, the organic layer was washed with brine, dried and concentrated to give Intermediate 1 as a yellow solid 6 g, 94% yield , mp: 136-138°C. 1 H NMR (400MHz, DMSO-d 6 )δ8.23(d, J=10.9Hz, 3H), 4.40(t, J=8.3Hz, 2H), 3.36(d, J=8.2Hz, 2H).

[0138] 1-(5-aminoindolin-1-yl)-2,2,2-trifluoroethan-1-one (Intermediate 2)

[0139] SnCl 2 2H 2 O (14.2 g, 63.1 mmol) was added to a solution of Intermediate 1 (5.5 g, 21.5 mmol) in ethanol (20 mL), and the reaction mixture was heated to reflux at 60° C. for 4 h. After cooling to room t...

Embodiment 2

[0150] Embodiment 2: preparation compound d 2 -cycluc:

[0151]

[0152] indoline-2,3-d 2 (Intermediate 8)

[0153] 1H-indole (20.0g, 170.7mmol) was added into a round-bottomed flask, dissolved with deuterated methanol, added palladium carbon (2.0g, 18.7mmol), and deuterium was bubbled into the reaction under reflux at 40 ° C, and used A pressure of eight atmospheres was applied to the autoclave, monitored by TLC, and the reaction was still incomplete for about 72 hours. Filter to remove palladium carbon, spin dry the reaction solution, add water and extract with ethyl acetate to obtain 11 g of yellow liquid with a yield of 50%, spin dry and directly throw it into the next step.

[0154] 1-(indolin-1-yl-2,3-d 2 )ethan-1-one (Intermediate 9)

[0155] Intermediate 8 (5g, 41.3mmol) was dissolved in glacial acetic acid, and acetyl chloride (19.5mL, 247.6mmol) was added dropwise to the solution at room temperature, and then the reaction solution was moved to 90°C for heat...

Embodiment 3

[0170] Embodiment 3: luciferase substrate cycluc, d 2 Experimental study on metabolism of -cycluc in vitro

[0171] Add compounds cycluc and d in all black 96-well plates 2 -cycluc (20 μM, 50 μL), then add 50 μL of luciferase solution containing 2 mM ATP, start shooting immediately, every 5 min, record the number of photons until the end of 120 min, and use a multifunctional fluorescent microplate reader ( ) to measure the intensity of bioluminescence, each experiment was repeated three times, and statistical calculations were performed with Graphpad.

[0172] Depend on Figure 9 It can be known that firefly luciferase substrates cycluc and d 2 - The bioluminescent intensity of cycluc in vitro decreases with time, and is determined by Figure 9 A-D known d 2 The bioluminescent intensity of -cycluc is much greater than that of cycluc. Within 120min, d 2 - The ratio of bioluminescent intensity between cycluc and cycluc reaches 10.4 times at the minimum and 158.1 times ...

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Abstract

The invention provides luciferase substrates, and a preparation method and application thereof, belonging to the technical field of luciferase substrate preparation. The luciferase substrates have a structural formula as described in the specification. In the structural formula, R is hydrogen or deuterium. The luciferase substrates provided by the invention have the advantages of good selectivity,high sensitivity, low detection line, good biocompatibility and the like; and further studies prove that d2-cycluc is higher in bioluminescence intensity and longer in bioluminescence time in vitro,in cells and in vivo compared with cycluc, and the two luciferase substrates have good concentration dependence. The preparation method for the luciferase substrates in the inventions simple in process, high in operability and relatively low in cost, and thus has a good practical application value.

Description

technical field [0001] The invention belongs to the technical field of luciferase substrate preparation, and in particular relates to a luciferase substrate and its preparation method and application. Background technique [0002] The information disclosed in this background section is only intended to increase the understanding of the general background of the present invention, and is not necessarily taken as an acknowledgment or any form of suggestion that the information constitutes the prior art already known to those skilled in the art. [0003] Many drugs are carbon-based, and carbon-hydrogen bonds are particularly relevant for understanding important properties of drug molecules. Deuteration refers to the process of selectively replacing the position of protium hydrogen isotope in small molecule drugs to realize deuterium hydrogen isotope drug. On the one hand, deuteration of a drug is most likely to affect pharmacokinetic properties, such as metabolism, rather than...

Claims

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Application Information

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IPC IPC(8): C07D513/04C07B59/00C09K11/06G01N21/64G01N21/76
CPCC07D513/04C07B59/002C09K11/06G01N21/6428G01N21/6452G01N21/6458G01N21/763C07B2200/05C09K2211/1037C09K2211/1051G01N2021/6439
Inventor 李敏勇杜吕佩陈新新
Owner SHANDONG UNIV
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