A class of light-activated covalent probes for trans-thyretin protein and methods of synthesis thereof

Abstract

The present application relates to a kind of photocontrol activation covalent probe for transthyretin and its synthesis method.The probe has the structure shown in formula I, raw material is low in price, method is simple and easy to operate, and easy to purify;This kind of probe can target transthyretin, and under the irradiation of ultraviolet light or white light, it is activated to produce carbene, and then covalently labeled protein;It can be applied to the field such as quantitative analysis of transthyretin.

Description

Technical Field

[0001] This invention relates to the field of probes, and more specifically, to a class of photoactivated covalent probes for transthyretin and their synthesis methods. Background Technology

[0002] Transthyretin (TTR) is a plasma tetramer protein composed of 127 amino acids, primarily responsible for the transport of thyroid hormones and vitamin A. Under the influence of factors such as gene mutations, environmental stress, and natural aging, TTR tetramers can abnormally dissociate and misfold, forming fibrous amyloid deposits. These deposits may lead to transthyretin amyloid cardiomyopathy (ATTR-CM) and transthyretin amyloid polyneuropathy (ATTR-PN).

[0003] Currently, researchers have developed TTR tetramer covalent probes based on the lysine activation mode of pKa perturbation, which are labeled using chemical reactions such as nucleophilic substitution and affinity addition. Reactive groups include 1,4-Michael acceptors, sulfonyl fluorides, and active esters. However, there are no reports on TTR labeling using photoactivated covalent probes. Compared with traditional covalent probes, photoactivated probes have the advantage of spatiotemporal controllability, allowing for precise regulation of the reaction at specific times and spaces.

[0004] Therefore, developing photoactivated TTR covalently labeled probes will provide new research tools for the development of TTR tetramer stabilizers and the quantitative analysis of TTR concentration. This has significant scientific and clinical value for a deeper understanding of the mechanisms of transthyretin amyloidosis and for promoting its clinical diagnosis and treatment. Summary of the Invention

[0005] According to one aspect of this application, a compound I is provided, which is a photosensitive activated covalent probe capable of covalently labeling transthyretin (TTR) protein. The aforementioned properties of this fluorescent molecule can be used for the development of TTR tetramer stabilizers and for quantitative analysis of TTR concentrations.

[0006] According to one aspect of this application, a compound I is provided, having the chemical formula shown in Formula I:

[0007]

[0008] In Formula I:

[0009] R1 is selected from one of C1-C5 alkyl groups, substituted C1-C5 alkyl groups, and C3-C8 cycloalkyl groups;

[0010] R2 may be the same or different, and each is independently selected from one of hydrogen, halogen, C1-C5 alkyl, C1-C5 alkoxy, substituted C1-C5 alkyl, and hydroxyl.

[0011] R3 is selected from one of hydrogen, hydroxyl, amino, -OR4, -NHR4, C1-C5 alkyl, and substituted C1-C5 alkyl;

[0012] R4 is selected from one of the following: C1-C5 alkyl, substituted C1-C5 alkyl, C2-C6 alkenyl, substituted C2-C6 alkenyl, C2-C6 alkynyl, and substituted C2-C6 alkynyl.

[0013] In one implementation, "substituted" means mono- or poly-substituted with deuterium, halogen, or hydroxyl.

[0014] In one embodiment, the substituted C1-C5 alkyl group includes deuterated C1-C5 alkyl group and halogenated C1-C5 alkyl group; the substituted C2-C6 alkenyl group includes deuterated C2-C6 alkenyl group and halogenated C2-C6 alkenyl group; and the substituted C2-C6 alkynyl group includes deuterated C2-C6 alkynyl group and halogenated C2-C6 alkynyl group.

[0015] In one embodiment, R1 is selected from methyl, deuterated methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, trifluoromethyl, chloromethyl, bromomethyl, iodomethyl, cyclopropane, cyclobutane, cyclohexane, cycloheptane, and cyclooctane.

[0016] In one embodiment, the R2 may be the same or different, each independently selected from hydrogen, fluorine, chlorine, bromine, iodine, methyl, deuterated methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, trifluoromethyl, chloromethyl, bromomethyl, iodomethyl, methoxy, ethoxy, and propoxy.

[0017] In one embodiment, R3 is selected from -OR4 and -NHR4; R4 is selected from methyl, deuterated methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, pentyl, etc. Where m and n are independently selected from 0, 1, 2, 3 or 4.

[0018] In one embodiment, the compound is selected from:

[0019]

[0020] According to another aspect of this application, a method for preparing the above-mentioned compound I is also provided, comprising the following steps:

[0021]

[0022] In one implementation, step (1) includes:

[0023] The alkaline substance was dissolved in solvent I and cooled to -5 to 5°C. Compound II was added under inert gas protection and stirred at -5 to 5°C for 5 to 60 minutes. Then, compound III was added and the temperature was raised to 20 to 40°C and stirred. After the reaction was completed, acid was added to adjust to acidity, solvent I was removed, and organic solvent and water were added for extraction. Organic phase column chromatography was used for separation to obtain intermediate product I.

[0024] Preferably, the alkaline substance is selected from at least one of sodium tert-butoxide, potassium tert-butoxide, sodium methoxide, sodium ethoxide, potassium hydroxide, sodium hydroxide, and sodium hydride; the molar ratio of the alkaline substance to compound II is 2 to 6:1.

[0025] Preferably, the reaction conditions for step (1) are: temperature of 20-30℃ and time of 2-12h.

[0026] Preferably, the molar ratio of compound II to compound III is 1:1 to 2.

[0027] Preferably, solvent I is selected from at least one of methanol, ethanol, tetrahydrofuran, 1,4-dioxane, and toluene.

[0028] Preferably, the inert gas is selected from at least one of nitrogen and argon.

[0029] Preferably, the acid is selected from at least one of acetic acid, hydrochloric acid, and sulfuric acid.

[0030] Preferably, the organic solvent used in the extraction is selected from at least one of ethyl acetate, dichloromethane, chloroform, and toluene.

[0031] In one implementation, step (2) includes:

[0032] Intermediate product I, compound IV, and 4-dimethylaminopyridine were dissolved in solvent II and cooled to -5 to 5°C. Then, dicyclohexylcarbodiimide was added and the temperature was raised to 20 to 40°C with stirring. After the reaction was completed, organic solvent and water were added for extraction, and the mixture was separated by organic phase column chromatography to obtain intermediate product II.

[0033] Preferably, the reaction conditions for step (2) are: temperature of 20-30℃ and time of 12-24h.

[0034] Preferably, the molar ratio of intermediate product I to compound IV is 1:1.0 to 3.0.

[0035] Preferably, solvent II is selected from at least one of dichloromethane, chloroform, carbon tetrachloride, and N,N-dimethylformamide.

[0036] Preferably, the organic solvent used in the extraction is selected from at least one of ethyl acetate, dichloromethane, chloroform, and toluene.

[0037] In one implementation, step (3) includes:

[0038] Intermediate II and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) were dissolved in solvent III and cooled to -5 to 5°C. Compound V was slowly added to the solvent and the temperature was raised to 20 to 40°C with stirring. After the reaction was completed, solvent III was removed, and organic solvent and water were added for extraction. Organic phase column chromatography was used to separate the extract to obtain compound I.

[0039] Preferably, the reaction conditions for step (3) are: temperature of 20-30℃ and time of 2-24h.

[0040] Preferably, the molar ratio of intermediate product II to DBU is 1:2.0 to 6.0.

[0041] Preferably, the molar ratio of intermediate product II to compound V is 1:1.0 to 3.0.

[0042] Preferably, solvent III is selected from at least one of acetonitrile, tetrahydrofuran, dichloromethane, chloroform, and N,N-dimethylformamide.

[0043] Preferably, the organic solvent used in the extraction is selected from at least one of ethyl acetate, dichloromethane, chloroform, and toluene.

[0044] The probe of this invention has the characteristics of targeting transthyretin (TTR) protein and activating it under ultraviolet light to achieve covalent labeling, and can be used in the fields of TTR protein quantitative analysis, stabilizer development, and related disease research.

[0045] Therefore, the present invention also provides the use of said compound I in the preparation of products for detecting transthyretin.

[0046] The present invention also provides the use of compound I in the preparation of diagnostic transthyretin amyloidosis.

[0047] Preferably, the transthyretin amyloidosis includes transthyretin amyloid cardiomyopathy (ATTR-CM) and transthyretin amyloid polyneuropathy (ATTR-PN).

[0048] The beneficial effects of this application include, but are not limited to:

[0049] (1) The synthetic raw materials provided by this invention are inexpensive, the method is simple and easy to operate, and easy to purify;

[0050] (2) This type of probe can be activated to generate an active carbene intermediate under ultraviolet or white light irradiation. In methanol, the absorbance decreases with the extension of light irradiation time.

[0051] (3) This type of probe can target transthyretin protein and activate the production of carbene under ultraviolet or white light irradiation, thereby covalently labeling the protein; it can be applied to fields such as quantitative analysis of transthyretin protein. Attached Figure Description

[0052] Figure 1 This is a synthetic route diagram for this type of probe.

[0053] Figure 2 The 1H NMR spectrum of probe P1 prepared in Example 3.

[0054] Figure 3 The carbon NMR spectrum of probe P1 prepared in Example 3.

[0055] Figure 4 The graph shows the change in the absorption spectrum of probe P1 after UV irradiation in methanol. The horizontal axis represents wavelength, and the vertical axis represents absorbance. The probe concentration is 50 μM.

[0056] Figure 5 The fluorescence spectra of probe P1 and transthyretin protein before and after ultraviolet light irradiation are shown. The probe concentration is 50 μM and the protein concentration is 14.4 μM.

[0057] Figure 6 The fluorescence spectra of probe P1 and transthyretin protein before and after white light irradiation are shown. The probe concentration is 50 μM and the protein concentration is 14.4 μM.

[0058] Figure 7 This is a gel electrophoresis image of probe P1 covalently labeled with transthyretin under ultraviolet light irradiation.

[0059] Figure 8 This is a gel electrophoresis image of probe P1 covalently labeled with transthyretin under white light irradiation.

[0060] Figure 9 This is the mass spectrum of probe P1 covalently labeled with transthyretin protein. Detailed Implementation

[0061] The present invention will be further illustrated below with reference to specific embodiments, but the embodiments do not limit the present invention in any way. Simple modifications or substitutions made to the methods, steps or conditions of the present invention without departing from the spirit and substance of the present invention are all within the scope of the present invention; unless otherwise specified, the technical means used in the embodiments are conventional means well known to those skilled in the art.

[0062] Unless otherwise specified, all raw materials and reagents used in this application are commercially purchased and used directly without processing. The instruments and equipment used adopt the manufacturer's recommended scheme and parameters.

[0063] The NMR data in this embodiment were obtained using a Bruker AVANCE III 700MHz NMR spectrometer.

[0064] The matrix-assisted ionization mass spectrometer used in the embodiments of this application is a Bruker Ultra Flex III MALDI-TOF-TOF.

[0065] Example 1

[0066] Potassium tert-butoxide (491 mg, 5.0 mmol) was added to tetrahydrofuran (10 mL) and cooled to 0 °C. Compound II (500 mg, 1.7 mmol) was added under nitrogen protection and stirred at 0 °C for 10 minutes. Then, compound III (345 mg, 1.8 mmol) was added. After stirring at room temperature overnight, acetic acid was added to adjust to acidity. Most of the tetrahydrofuran was removed by rotary evaporation. Extraction was performed with appropriate amounts of ethyl acetate and water. The organic phase was collected and concentrated. Finally, it was purified by column chromatography (elution with a petroleum ether / ethyl acetate gradient of 100:0-50:50, v / v) to give intermediate I (257 mg, yield: 48%).

[0067] Intermediate I was tested using nuclear magnetic resonance spectroscopy, and the results are as follows: 1 H-NMR (400MHz, CDCl3) δ7.46(d,J=7.9Hz,2H),7.28(s,2H),7.17(s,2H),6.99(s,2H),4.52(m,2H),3.66(s,2H),2.51(m,1H),2.34(s,6H).

[0068] The synthesis route in this embodiment is as follows:

[0069]

[0070] Example 2

[0071] Intermediate I (83 mg, 0.26 mmol), compound IV (9.6 mg, 0.3 mmol), and 4-dimethylaminopyridine (173 mg, 0.75 mmol) were dissolved in dichloromethane (5 mL) and cooled to 0 °C. Then, dicyclohexylcarbodiimide (155 mg, 0.75 mmol) was added, and the mixture was stirred overnight at room temperature. After the reaction was complete, water and dichloromethane were added for extraction. The organic phase was collected and concentrated, and finally purified by column chromatography (elution with a petroleum ether / ethyl acetate gradient of 100:0-50:50, v / v) to give intermediate II (32 mg, yield: 37%).

[0072] Intermediate product II was tested using nuclear magnetic resonance spectroscopy, and the results are as follows: 1 H-NMR (400MHz, CDCl3) δ7.45 (d, J = 7.9Hz, 2H), 7.26 (m, 2H), 7.17 (s, 2H), 6. 99(s,2H),4.52(m,2H),3.70(s,3H),3.63(s,2H),2.52(s,1H),2.34(s,6H).

[0073] The synthesis route in this embodiment is as follows:

[0074]

[0075] Example 3

[0076] Intermediate II (67 mg, 0.2 mmol) and 1,8-diazabicyclo[5.4.0]undec-7-ene (91.3 mg, 0.6 mmol) were dissolved in acetonitrile and cooled to 0 °C. Then, compound V (52.9 mg, 0.22 mmol) was slowly added, the mixture was brought to room temperature, and stirred overnight. After the reaction was complete, most of the acetonitrile was removed by rotary evaporation. Then, water and ethyl acetate were added for extraction, the organic phase was collected and concentrated, and finally purified by column chromatography (elution with a petroleum ether / ethyl acetate gradient of 100:0-50:50, v / v) to give the yellow product P1 (62 mg, yield: 65%).

[0077] P1 was tested using a nuclear magnetic resonance spectrometer, and the results are as follows: 1 H-NMR (400MHz, CDCl3) δ7.55-7.43(m,4H),7.18(d,J=1.9Hz,2H),6.98(d,J=2.2Hz,2H),4.52(s,2H),3.88(d,J=2.0Hz,3H),2.52(m,1H),2.35(m,6H). 13C-NMR (100MHz, CDCl3) δ135.3,133.5,131.6,128.2,127.2,127.1,127.1,124.2,79.4,75.2,60.0,52.2,16.8.

[0078] The synthesis route in this embodiment is as follows:

[0079]

[0080] Test Example 1: Absorption Spectroscopy Measurement of P1 Probe in Methanol After Irradiation

[0081] The P1 prepared in the examples was dissolved in methanol (50 μM) and irradiated with 360-375 nm ultraviolet light. The absorption spectra were measured using a microplate reader at 0, 5, 20, 40, and 60 minutes. The results are as follows: Figure 4 As shown, the absorption value of P1 gradually decreases with the extension of illumination time, indicating that ultraviolet light can activate P1.

[0082] Test Example 2: Fluorescence spectrum of P1 covalently labeled transthyretin protein

[0083] The probe P1 (50 μM) was incubated with TTR (14.4 μM) at room temperature for 120 minutes, followed by irradiation with 360-375 nm ultraviolet light or 400-760 nm white light for 30 minutes, and fluorescence spectroscopy was performed. The excitation wavelength was 341 nm. The results are as follows: Figure 5 and Figure 6 As shown, the fluorescence intensity is enhanced after P1 is covalently labeled with transthyretin.

[0084] Test Example 3: P1 covalently labeled fluorescent gel containing transthyretin

[0085] The probe P1 (50 μM) was incubated with TTR (14.4 μM) at room temperature for 120 min, then irradiated with 360-375 nm UV light or 400-760 nm white light for 60 min. Following this, a copper-catalyzed electrochemical reaction (50 mM CuSO4, 50 mM TMR-N3, 50 mM MTCEP, 1.7 mM TBTA) was performed at room temperature for 1 h. Acetone precipitation was carried out overnight, centrifuged, and the supernatant was discarded. The sample was denatured at 95 °C with 4% SDS solution and 5x loading buffer, and analyzed by gel electrophoresis after cooling. The experimental results are as follows: Figure 7 and Figure 8 As shown, transthyretin protein was covalently labeled under ultraviolet irradiation.

[0086] Test Example 4: Mass spectrum of P1 covalently labeled transthyretin protein

[0087] The probe P1 (300 μM) was incubated with TTR (14.4 μM) at room temperature for 120 minutes, then irradiated with 360-375 nm UV light for 120 minutes. After dilution with deionized water, matrix-assisted laser desorption / ionization analysis was performed. The experimental results are as follows: Figure 9 As shown, after transthyretin was covalently labeled with P1, a peak with a molecular weight increase of 320.2 was observed.

[0088] The above description is only a preferred embodiment of the present invention. It should be noted that those skilled in the art can make several improvements and additions without departing from the method of the present invention, and these improvements and additions should also be considered within the scope of protection of the present invention.

Claims

1. A compound I having the chemical formula shown in Formula I: Equation I In Formula I: R1 is selected from one of C1-C5 alkyl groups and substituted C1-C5 alkyl groups; R2 may be the same or different, and each is independently selected from one of C1 to C5 alkyl groups or substituted C1 to C5 alkyl groups; R3 is selected from -OR4; R4 is selected from ,in, m is selected from 1, 2, 3, or 4; The term "substituted" indicates that it is substituted by deuterium in one or more ways.

2. Compound I according to claim 1, characterized in that, R1 is selected from methyl, deuterated methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, and pentyl.

3. Compound I according to claim 1, characterized in that, The R2 may be the same or different, and each is independently selected from methyl, deuterated methyl, ethyl, n-propyl, isopropyl, n-butyl, sec-butyl, isobutyl, tert-butyl, and pentyl.

4. Compound I according to claim 1, characterized in that, m is selected from 1 or 2.

5. Compound I according to claim 1, characterized in that, The compound is selected from: 。 6. A method for preparing compound I according to any one of claims 1-5, comprising the following steps: 。 7. The preparation method according to claim 6, characterized in that, Step (1) includes: dissolving the alkaline substance in solvent I and cooling it to -5 to 5°C. o C, under inert gas protection, add compound II and apply at -5 to 5°C. o Stir at C for 5-60 minutes, then add compound III and raise the temperature to 20-40°C and stir. After the reaction is complete, add acid to adjust to acidity, remove solvent I, add organic solvent and water for extraction, and separate by organic phase column chromatography to obtain intermediate product I. Step (2) includes: dissolving intermediate I, compound IV, and 4-dimethylaminopyridine in solvent II and cooling to -5 to 5°C. o C, then dicyclohexylcarbodiimide was added and the temperature was raised to 20-40°C and stirred. After the reaction was completed, organic solvent and water were added for extraction, and the mixture was separated by organic phase column chromatography to obtain intermediate product II. Step (3) includes: dissolving intermediate II and 1,8-diazabicyclo[5.4.0]undec-7-ene (DBU) in solvent III and cooling to -5 to 5°C. o C. Compound V was slowly added to the mixture and the temperature was raised to 20-40°C with stirring. After the reaction was completed, solvent III was removed, and organic solvent and water were added for extraction. The mixture was then separated by organic phase column chromatography to obtain compound I.

8. The use of compound I according to any one of claims 1-5 in the preparation of a product for detecting transthyretin.

9. The use of compound I according to any one of claims 1-5 in the preparation of a product for diagnosing transthyretin amyloidosis.

10. The application according to claim 9, characterized in that, The transthyretin amyloidosis is selected from transthyretin amyloid cardiomyopathy and transthyretin amyloid polyneuropathy.

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