CN115745987A - Luciferase substrate and preparation method and application thereof - Google Patents

Abstract

After the luciferase substrate and firefly luciferase react, the luciferase substrate has the advantages of long emission wavelength, high luminous intensity, high detection sensitivity and the like, has better performance in live cell and living animal imaging, and can be used as an important analysis tool in the fields of living bioluminescence imaging, protein quantitative detection and the like.

Description

A kind of luciferase substrate and its preparation method and application

technical field

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

Optical imaging plays an important role in observing cell structure, monitoring cell behavior in real time, and monitoring various biological events. Bio-optical imaging technology can be mainly divided into two categories: fluorescence imaging and luminescence imaging. Due to the limitations of biocompatibility and sensitivity, most current fluorescence imaging tools are difficult to realize in situ real-time dynamic monitoring of biological processes in vivo.

Bioluminescence is a phenomenon in which luciferase catalyzes the oxidation of small molecules of the substrate to convert chemical energy into light energy and release it. By monitoring the released photons, the monitoring of proteins can be realized. In the specific practice process, researchers can fuse the luciferase gene with the specific target protein gene through molecular biology techniques, and use different technical means to realize the expression of the corresponding fusion gene in different model organisms. After giving the substrate The dynamic tracking of target proteins in cells or living animals can be realized. Compared with fluorescence imaging, bioluminescence imaging does not require an external excitation light source, avoids the interference caused by the endogenous fluorescence of biological tissues induced by an external excitation light source, greatly reduces the background signal, and can significantly improve the obtained image. signal-to-noise ratio. However, in practical applications, bioluminescent imaging technology still faces great challenges in deep tissue imaging. First, the bioluminescent signal is relatively weak; secondly, the wavelength of the traditional bioluminescent system is 460-620nm, and substances such as hemoglobin (λ=~415-577nm) and melanin (λ<600nm) in the body will absorb the luminescent signal Interference hinders the detection of optical signals and reduces the depth and clarity of imaging. At present, researchers generally believe that the ideal optical window for in vivo imaging is in the wavelength range of 650-900nm.

Therefore, in order to improve the penetrating ability of bioluminescent systems to biological tissues, as well as the imaging depth and clarity, the development of bioluminescent systems with high luminescence brightness and long emission wavelength has become the focus of research. At present, methods based on luciferase protein mutation and luciferase substrate chemical modification play an important role in promoting the above goals. In recent years, several bioluminescent systems have been developed (S.C.Miller.et.al.J.Am.Chem.Soc.2014,136,13277-13282; M.A.Pule.et.al.Angew.Chem.Int.Ed.2014, 53, 13059–13063; L.Mezzanotte.et.al.NatCommun.2018,9,1-12; A.Miyawaki.et. al.Science.2018,359,935–939), which makes the emission wavelength red-shifted, but the luminous intensity Compared with natural luciferin, there is still a gap. Therefore, there is still a lot of room for improvement in the existing bioluminescence system.

Contents of the invention

Aiming at the shortcomings of existing bioluminescent systems, the present invention constructs a new class of luciferase substrates, which react with firefly luciferase and have the characteristics of red-shifted emission wavelength and high luminous intensity, and have good application in living tissues prospect.

In order to achieve the above technical content, the present invention provides a luciferase substrate, the structure of which is shown in formula (I),

In its formula (I),

R together with R and _/ or R and _R together form a substituted or unsubstituted alicyclic ring, a substituted or unsubstituted alicyclic ring, a substituted or unsubstituted aromatic ring, or a substituted or unsubstituted aromatic heterocyclic ring;

Alternatively, when R and _R1 or R and _R2 do not form a substituted or unsubstituted alicyclic ring, a substituted or unsubstituted alicyclic ring, a substituted or unsubstituted aromatic ring, or a substituted or unsubstituted aromatic heterocyclic ring, R is : Hydroxy, or -NR _a R _b , R _a , R _b are each independently selected from hydrogen, or alkyl, R ₁ , R ₂ are the same or different, and each independently is -H, hydroxyl, alkyl, halo group;

The substituents in the "substituted or unsubstituted alicyclic ring", "substituted or unsubstituted aliphatic heterocyclic ring", "substituted or unsubstituted aromatic ring" and "substituted or unsubstituted aromatic heterocyclic ring" are the same or different , each independently selected from hydroxyl, amino, alkyl, alkylamino, halogen, cyano:

n is 0, 1, 2, or 3;

in,

The alkyl in the "alkyl" or "alkylamino" is C ₁ -C ₁₀ straight chain alkyl or C ₃ -C ₁₀ branched chain alkyl; preferably, it is C ₁ -C ₇ straight chain alkyl Or C ₃ -C ₇ branched chain alkyl; preferably, C ₁ -C ₅ straight chain alkyl or C ₁ -C ₅ branched chain alkyl; preferably, selected from methyl, ethyl, n-propyl, Isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, isopentyl, 1 -Ethylpropyl, neopentyl, n-hexyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, isohexyl, 1,1-dimethylbutyl, 2,2 -Dimethylbutyl, 3,3-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,3-dimethylbutyl, 2-ethyl butylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 2,2-dimethylpentyl, 3,3-dimethylpentyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 3-ethylpentyl or 2,2,3-trimethylbutyl;

The "alicyclic ring" is a saturated or unsaturated 4-10 membered monocyclic or polycyclic alicyclic ring; alternatively, the "alicyclic ring" is a saturated or unsaturated 4, 5, 6, 7, 8, 9. 10-membered monocyclic or polycyclic alicyclic rings;

The "aliphatic heterocyclic ring" is a saturated or unsaturated 4-10 membered monocyclic or polycyclic aliphatic heterocyclic ring containing at least one heteroatom selected from N, O, or S on the ring; alternatively, the The "aliphatic heterocyclic ring" is a saturated or unsaturated 4, 5, 6, 7, 8, 9, 10 membered monocyclic or polycyclic ring containing at least one heteroatom selected from N, O, or S. Aliphatic ring;

The "aromatic ring" is a 5-10-membered monocyclic or fused bicyclic aromatic group; optionally, the "aromatic ring" is a 5, 6, 7, 8, 9, 10-membered monocyclic or fused bicyclic Aromatic group; Optionally, the "aromatic ring" is selected from benzene ring, naphthalene ring, fluorene ring, anthracene ring, phenanthrene ring, biphenyl ring;

The "aromatic heterocycle" is a 5-10 membered monocyclic or fused bicyclic heteroaromatic group containing at least one heteroatom selected from N, O, or S on the ring; alternatively, the "aromatic "Heterocycle" is a 5, 6, 7, 8, 9, 10 membered monocyclic or fused bicyclic heteroaromatic group containing at least one heteroatom selected from N, O, or S on the ring; alternatively, The aromatic heterocycle is selected from thiophene ring, furan ring, pyrrole ring, imidazole ring, thiazole ring, oxazole ring;

The "halogen group" is selected from F, Cl, Br, I.

Optionally, the R and _R together form the structures in the following formulas (I-2-1) to (I-2-6):

Alternatively, R and _R together form the structure in the following formulas (I-2-7) to (I-2-8):

Alternatively, R together with _R1 and R together with _R2 form

Wherein, each R' is independently selected from hydrogen or alkyl; R" is selected from hydrogen or alkyl.

或者R 与R₁一起和/或R与R₂一起形成以下式(I-2-1)～(I-2-11)中结构：

R′选自-H或-Me；Optionally, in formula (I), when n is taken from 0, R is

Or R together with _R1 and/or R and _R2 together form the structures in the following formulas (I-2-1) to (I-2-11):

R' is selected from -H or -Me;

时，n可取0,1或2；In formula (I), R is taken from

, n can be 0, 1 or 2;

Optionally, the luciferase substrate is selected from compounds of the following formula:

On the other hand, there is also provided a preparation method for preparing the above-mentioned substrate, including the step of reacting the compound of formula (II) with the compound of formula (III) to obtain the luciferase substrate shown in formula (I),

Wherein, R, R ₁ , R ₂ are as defined in any one of claims 1-3, n=0;

Alternatively, it also includes the product obtained by reacting the compound of formula (IV) with the compound of formula (V), and the product obtained by the condensation reaction of ester bond hydrolysis and formula (VI) removes the sulfhydryl protecting group to obtain the compound of formula (VII), and finally , obtain the luciferase substrate shown in formula (I) through the enzymolysis of ester bond;

Wherein, R, R ₁ , R ₂ are as defined in any one of claims 1-3, n=1, 2, 3, m=0, 1, 2.

In another aspect, the use of the luciferase substrate above in the preparation of optical detection products is provided, and the optical detection products include but not limited to optical probes and luminescent detection kits.

In another aspect, there is provided a bioluminescent probe comprising the luciferase substrate described above.

In another aspect, a luminescence detection kit is provided, the luminescence detection kit comprises the above-mentioned luciferase substrate or the above-mentioned bioluminescence probe.

In another aspect, the application of the above-mentioned luciferase substrate, the above-mentioned bioluminescence probe or the above-mentioned luminescence detection kit in environmental detection is provided.

In another aspect, the use of the above-mentioned luciferase substrate, the above-mentioned bioluminescence probe or the above-mentioned luminescence detection kit in analytical chemistry.

In another aspect, the use of the above-mentioned luciferase substrate, the above-mentioned bioluminescence probe or the above-mentioned luminescence detection kit in biological analysis and detection;

Preferably, the biological analysis and detection include the analysis and detection of the biological cell level, tissue level, organ level and biological individual level;

Preferably, said organisms include bacteria, mammalian cells, mice, rats or monkeys.

Beneficial effects of the present invention:

After the luciferase substrate of the present invention reacts with firefly luciferase, it has the advantages of long emission wavelength, high luminous intensity, and high detection sensitivity. It has good performance in imaging of living cells and living animals, and can be used as An important analytical tool in the fields of in vivo bioluminescent imaging, protein quantitative detection, etc.

Description of drawings

Figure 1 shows the amino acid sequences of luciferases from different sources and their mutants, all of which are luciferases involved in Example 16.

Figure 2. Electrophoresis results of purified proteins from different sources of luciferase and its mutants.

Figure 3. The relative luminescence intensities measured by the reactions of substrate 1, substrate 3, substrate 5 and substrate 19 with different luciferases.

Fig. 4. Curves of the relative luminous intensity versus time within 30 minutes obtained from the reaction of substrate 1, substrate 3, substrate 5 and substrate 19 with firefly luciferase.

Figure 5. The luminescence spectra corresponding to the reaction of substrate 1, substrate 3, substrate 5, substrate 16 and substrate 19 with firefly luciferase, wherein the corresponding spectra of firefly luciferase mutants x5g and x5r are the same as those of firefly luciferase Obtained by photoreaction.

Figure 6. Live cell imaging results of Substrate 1, Substrate 3, Substrate 5, and Substrate 19 in a HeLa cell line expressing firefly luciferase.

Figure 7. Test results of different concentrations of substrate 1, substrate 3, substrate 5 and substrate 19 in HEK293T cell line.

Figure 8. Test results of substrate 3 and substrate 5 in mice. (A) Luminescence images of substrate 3 and substrate 5 in ICR mice; (B) Quantitative results of luminescence signals in the legs of substrate 3 and substrate 5 mice (3 biological replicates).

Detailed ways

The embodiments of the present invention are described in detail below. These embodiments are implemented under the premise of the technical solution of the present invention, and detailed implementation methods and specific operating procedures are provided, but the protection scope of the present invention is not limited to the following implementation example. The following examples relate to conventional molecular biology cloning methods and cell culture and mouse testing methods. These methods are well known to those of ordinary skill in the art. Those of ordinary skill in the art are not difficult to make according to the specific circumstances according to the following examples. Modifications or transformations successfully carry out the invention.

The pEGFP-C1 plasmid vector used in the examples was purchased from Invitrogen, and the pCDFDuet-1 plasmid vector was purchased from Novagen. All primers used for PCR were synthesized, purified and identified by mass spectrometry by Shanghai Jereh Bioengineering Technology Co., Ltd. The expression plasmids constructed in the examples have all been sequenced, and the sequence determination was completed by Jerry Sequencing Company. The PrimeSTAR DNA polymerase used in the PCR reaction in each example was purchased from TaKaRa Company. NheI, BamHI, NotI, Acc65I and other restriction endonucleases were purchased from Fermentas Company. Inorganic salt chemical reagents were purchased from Sinopharm Shanghai Chemical Reagent Company. Both Kanamycin and Streptomycin were purchased from Ameresco. D-luciferin was purchased from Sigma Company. 384-well and 96-well white plates were purchased from Grenier.

The BL21(DE3) strain used in the examples was purchased from Invitrogen. HeLa cells and HEK293T cells were purchased from the Cell Bank of the Type Culture Collection Committee of the Chinese Academy of Sciences. ICR mice were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd. The endotoxin-free plasmid large-scale extraction kit used in the examples was purchased from Tiangen Biochemical Technology (Beijing) Co., Ltd. The main instrument used in the embodiment: Synergy Neo2 multifunctional microplate reader (U.S. Bio-Tek company), X-15R high-speed refrigerated centrifuge (U.S. Beckman company), PCR amplification instrument (Germany Biometra company), IVIS Spectrum CT In vivo imaging system (PerkinElmer, USA), nucleic acid electrophoresis instrument (Shenergy Gaming Company).

The meanings of the abbreviations are as follows: "h" means hour, "min" means minute, "s" means second, "d" means day, "μL" means microliter, "mL" means milliliter, "L" means liter, "mM " means millimole, and "μM" means micromole.

Example 1

Synthesis of Substrate 1:

Compound 2: Dissolve compound 1 (10 g, 80 mmol) in 200 ml of ethanol, add methyl isopropyl ketone (8.3 g, 96 mmol), then add 3 ml of concentrated sulfuric acid, heat to reflux, and stir overnight. The next day, the reaction was returned to room temperature, added water, and extracted 3 times with ethyl acetate (100ml), the organic phases were combined, dried over anhydrous sodium sulfate, the organic phase was removed under reduced pressure, and the compound 2 (13.0g, yield 86%). ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.70(d,J=8.7,1H),6.72(t,J=16.7,1H),6.67(s,1H),3.80(s,3H),2.29 (s,3H),1.46(s,6H).

Compound 3: Dissolve compound 2 (10g, 53mmol) in 100ml 1,4-dioxane, add selenium dioxide (11.7g, 106mmol), heat the reaction system to 80°C, and stir for 2h. The reaction was returned to room temperature, selenium dioxide was removed by filtration, the organic solvent was removed under reduced pressure, and compound 3 (8.0 g, yield 75%) was obtained by column chromatography. ¹ H-NMR (400MHz, CDCl ₃ ): δ=9.90(s,1H),7.72 (d,J=8.6,1H),6.70(t,J=16.6,1H),6.68(s,1H),3.80 (s,3H),1.46(s,6H).

Compound 4: Dissolve compound 3 (5.0 g, 24.6 mmol) in 15 ml of tetrahydrofuran, add 25 ml of ammonia water, then add iodine (9.4 g, 36.9 mmol), and stir at room temperature for 6 h. Add 30ml of saturated sodium thiosulfate to the reaction system, extract with ethyl acetate, combine the organic phases, dry over anhydrous sodium sulfate, remove the organic phase under reduced pressure, and separate by column chromatography to obtain compound 4 (2.2g, yield 46%) . ¹ H-NMR (400MHz, CDCl3): 7.72 (d, J = 8.9, 1H), 6.70 (t, J = 16.8, 1H), 6.68 (s, 1H), 3.80 (s, 3H), 1.46 (s, 6H).

Compound 5: Dissolve compound 4 (3.0g, 15mmol) in 50ml of dry tetrahydrofuran, place the reaction system at 0°C, add BBr ₃ (1.7ml, 18mmol) dropwise to the reaction system, stir for half an hour, then add water Quenched. DCM was extracted 3 times, the organic phases were combined, dried over anhydrous sodium sulfate, the organic phase was removed under reduced pressure, and compound 5 (2.57 g, yield 92%) was obtained by column chromatography. ¹ H-NMR (400MHz, CDCl ₃ ): δ=9.30(s, 1H), 7.70(d, J=8.6, 1H), 6.70(t, J=16.6, 1H), 6.68(s, 1H), 1.46 (s,6H).

Substrate 1: Compound 5 (1 g, 5 mmol) was dissolved in 10 ml of methanol, D-cysteine (0.65 g, 5 mmol) was dissolved in 10 ml of pH=8 buffer, added to the reaction system, and stirred at room temperature for 1 h. After the reaction was complete, DCM was extracted three times, the organic phases were combined, dried over anhydrous sodium sulfate, the organic phase was removed under reduced pressure, and the substrate 1 (0.98 g, yield 63%) was obtained by column chromatography. ¹ H NMR (600MHz, D ₂ O): δ = 7.50 (d, J = 8.7, 1H), 6.97 (d, J = 2.4, 1H), 6.87 (dd, J = 8.7, 2.4, 1H), 5.23 ( dd,J=9.7,7.9,1H),3.57(dd,J=10.9,10.0,1H),3.39(dd,J=11.1,7.8,1H),1.45(s,3H),1.39(s,3H) .

Example 2

Synthesis of Substrate 2:

Compound 7: the synthesis of reference compound 2, the yield is 85%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.80(d, J=8.7,1H), 6.76(t, J=16.7,1H), 6.72(s,1H), 1.49(s,9H), 2.28 (s,3H),1.46(s,6H).

Compound 8: the synthesis of reference compound 3, the yield is 71%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=9.90(s,1H), 7.78(d,J=8.6,1H), 6.72(t,J=16.6,1H), 6.68(s,1H), 1.50 (s,9H),1.47(s,6H).

Compound 9: the synthesis of reference compound 4, the yield is 43%. ¹ H-NMR (400MHz, CDCl ₃ ): 7.78 (d, J = 8.8, 1H), 6.70 (t, J = 16.3, 1H), 6.68 (s, 1H), 1.51 (s, 9H), 1.46 (s ,6H).

Compound 10: Compound 9 (0.3 g, 0.7 mmol) was dissolved in 10 ml of DCM, added to 2.5 ml of trifluoroacetic acid, and stirred at room temperature for 1 h. After the reaction was complete, the solvent was removed under reduced pressure, and DCM was added several times to remove trifluoroacetic acid to obtain compound 10 (0.13 g, yield 96%). ¹ H-NMR (400 MHz, CDCl ₃ ): 7.78(d, J=8.8,1H), 6.70(s,1H), 6.68(s,1H), 1.45(s,6H).

Substrate 2: Refer to the synthesis of substrate 1 with a yield of 59%. ¹ H-NMR (400MHz, D ₂ O): δ = 7.51 (d, J = 8.6, 1H), 6.96 (d, J = 2.3, 1H), 6.86 (dd, J = 8.6, 2.4, 1H), 5.21 (dd,J=9.6,7.8,1H),3.56(m,1H),3.39(m,1H),1.46(s,3H),1.38(s,3H).

Example 3

Synthesis of Substrate 3:

Compound 12: the synthesis of reference compound 2, the yield is 87%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.71(d, J=8.7, 1H), 6.75(d, J=8.0, 1H), 6.65(s, 1H), 2.28(s, 3H), 1.46 (s,6H).

Compound 13: Weigh compound 12 (1.0g, 4.2mmol) and potassium phosphate (1.8g, 8.4 mmol) respectively in a pressure bottle, then add 7ml of dimethylamine aqueous solution and 3ml of dimethylaminoethanol and a catalytic amount of copper powder and cuprous iodide. Heat to 80°C and stir overnight. The next day, cooled to room temperature, added water, extracted three times with ethyl acetate, combined the organic phases, dried over anhydrous sodium sulfate, removed the solvent under reduced pressure, and separated by column chromatography to obtain compound 13 (0.53 g, yield 63%). ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.72(d,J=8.7,1H),6.77(d,J=8.0,1H),6.65(s,1H),3.08(s,6H),2.30 (s,3H), 1.46(s,6H).

Compound 14: the synthesis of reference compound 3, the yield is 69%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=9.91(s, 1H), 7.70(d, J=8.7, 1H), 6.75(d, J=8.0, 1H), 6.65(s, 1H), 3.08 (s,6H),1.46(s,6H).

Compound 15: the synthesis of reference compound 4, the yield is 42%. ¹ H-NMR (400MHz, CDCl ₃ ): ¹ H NMR (400MHz, CDCl ₃ ) δ=7.62(d,J=8.7,1H),6.83–6.74(m,2H), 3.08(s,6H),1.44 (s,6H).

Substrate 3: refer to the synthesis of substrate 1, with a yield of 59%. ¹ H-NMR (400MHz, CDCl ₃ ): δ = 7.50 (d, J = 8.7, 1H), 6.97 (d, J = 2.4, 1H), 6.87 (dd, J = 8.7, 2.4, 1H), 5.23 ( dd,J=9.7,7.9,1H),3.57(dd,J=10.9,10.0,1H),3.39(dd,J=11.1,7.8,1H),2.88(s,6H),1.45(s,3H) ,1.39(s,3H).

Example 4

Synthesis of substrates 4 and 5:

Compound 17: The method disclosed in the literature S.C.Miller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 56%.

Compound 18: Weigh compound 17 (3.0g, 13mmol) and dissolve it in 250ml eggplant with 30ml of concentrated hydrochloric acid, and place it in an ice bath; dissolve sodium nitrite (1.2g, 17mmol) with 12ml of water and add to the reaction system , and then SnCl·2H ₂ O (7.6g, 34mmol) was dissolved in 9ml concentrated hydrochloric acid, added to the reaction system, returned to room temperature and stirred for 1h, and filtered to obtain compound 18 (2.4g, yield 76%). ¹ H NMR (400MHz, DMSO): δ = 7.75 (d, J = 8.6, 1H), 6.53 (s, 1H), 6.42 (d, J = 8.5, 1H), 4.18 (t, J = 7.9, 2H) ,3.10(t,J=8.0,2H).

Compound 19: the synthesis of reference compound 2, the yield is 79%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.76(d, J=8.6, 1H), 7.15(s, 1H), 4.20(t, J=7.9, 2H), 3.12(t, J=8.0, 2H), 2.30(s,3H), 1.44(s,6H).

Compound 20: the synthesis of reference compound 3, the yield is 69%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=9.6(s, 1H), 7.76(d, J=8.6, 1H), 7.16(s, 1H), 4.22(t, J=7.9, 2H), 3.12 (t,J=8.1,2H),1.39(s,6H).

Compound 21: the synthesis of reference compound 4, the yield is 42%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.76(d, J=8.6, 1H), 7.16(s, 1H), 4.20(t, J=7.6, 2H), 3.12(t, J=8.1, 2H), 1.44(s, 6H).

Compound 22: The method disclosed in the reference (SC Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282), the yield was 86%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.66(d, J=8.6, 1H), 7.20(s, 1H), 3.60(t, J=7.6, 2H), 3.12(t, J=8.0, 2H), 1.44(s, 6H).

Substrate 4: refer to the synthesis of substrate 1, with a yield of 59%. ¹ H-NMR (400MHz, D ₂ O): δ = 7.32 (s, 1H), 6.75 (s, 1H), 5.18 (dd, J = 9.7, 7.9, 1H), 3.53 (dd, J = 13.9, 7.0 ,1H), 3.41– 3.31(m,3H), 2.91(t,J=8.3,2H), 1.34(s,3H), 1.26(s,3H).

Compound 23: The method published in references SCMiller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 69%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.66(d, J=8.6, 1H), 7.20(s, 1H), 3.60(t, J=7.6, 2H), 3.12(t, J=8.2, 2H), 2.75(s,3H), 1.44(s,6H).

Substrate 5: refer to the synthesis of substrate 1, with a yield of 61%. ¹ H-NMR (400MHz, CDCl3): δ=7.32(s,1H),6.75(s,1H),5.20(dd,J=9.7,7.9,1H),3.53(dd,J=13.9,7.0,1H ),3.42-3.32(m,3H),2.91(t,J＝8.3,2H),2.75(s,3H),1.34(s,3H),1.26(s,3H).

Example 5

Synthesis of substrates 6 and 7:

Compound 25: The method disclosed in the literature S.C.Miller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 56%.

Compound 26: Synthesized with reference to Compound 18, with a yield of 70%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.76(d, J=8.7,1H),6.53(s,1H),6.40(d,J=8.6,1H),4.09(t,J=7.9, 2H), 2.90(t, J=8.0, 2H), 1.60(m, 2H).

Compound 27: the synthesis of reference compound 2, the yield is 76%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.66(s,1H),7.15(s,1H),4.10(t,J=7.8,2H),3.10(t,J=8.2,2H),2.19 (s,3H),1.56(m,2H),1.46(s,6H).

Compound 28: the synthesis of reference compound 3, the yield is 69%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=10.00(s,1H),7.60(s,1H),7.16(s,1H),4.10(t,J=7.8,2H),3.12(t,J ＝8.2,2H),1.51(m,2H),1.40(s,6H).

Compound 29: the synthesis of reference compound 4, the yield is 41%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.60(s,1H),7.16(s,1H),4.12(t,J=7.9,2H),3.12(t,J=8.2,2H),1.50 (m,2H),1.41(s,6H).

Compound 30: The method published in references SCMiller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 86%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.40(s,1H),6.70(s,1H),3.42(t,J=7.9,2H),2.79(t,J=8.2,2H),1.96 (m,2H),1.41(s,6H).

Substrate 6: refer to the synthesis of substrate 1, with a yield of 60%. ¹ H-NMR (400MHz, D ₂ O): δ = 7.50 (s, 1H), 6.90 (s, 1H), 5.20 (dd, J = 9.7, 7.9, 1H), 3.56 (dd, J = 10.9, 10.0 ,1H),3.39 (dd,J=11.1,7.8,1H),3.22(t,J=7.9,2H),2.80(t,J=8.2,2H),1.96(m,2H),1.45(s, 3H), 1.39(s, 3H).

Compound 31: The method published in references SCMiller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 69%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.20(s,1H),6.72(s,1H),3.40(t,J=7.8,2H),2.79(t,J=8.0,2H),2.75 (s,3H),2.00(m,2H),1.44(s,6H).

Substrate 7: the synthesis of the reference substrate 1, the yield is 59%. ¹ H-NMR (400MHz, D ₂ O): δ = 7.56 (s, 1H), 6.96 (s, 1H), 5.22 (dd, J = 9.7, 7.9, 1H), 3.56 (dd, J = 10.9, 10.0 ,1H),3.40 (dd,J=10.1,7.9,1H),3.20(t,J=7.9,2H),2.82(m,5H),1.98(m,2H),1.45(s,3H), 1.39 (s,3H).

Example 6

Synthesis of substrates 8 and 9:

Compound 33: The method disclosed in the literature S.C.Miller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 53%.

Compound 34: Synthesized with reference to Compound 18, with a yield of 69%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.68(d, J=8.2,1H), 7.28(d, J=8.2,1H), 6.32(d, J=2.4,1H), 4.30(t, J=4.8,2H),3.82(t,J=4.8,2H).

Compound 35: the synthesis of reference compound 2, the yield is 76%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.66(s,1H),7.16(s,1H),4.30(t,J=4.8,2H),3.86(t,J=4.8,2H),2.29 (s,3H),1.44(s,6H).

Compound 36: the synthesis of reference compound 3, the yield is 72%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=9.6(s, 1H), 7.76(d, J=8.6, 1H), 7.16(s, 1H), 4.32(t, J=4.8, 2H), 3.82 (t,J=4.8,2H),1.42(s,6H).

Compound 37: the synthesis of reference compound 4, the yield was 43%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.78(d, J=8.8, 1H), 7.18(s, 1H), 4.30(t, J=7.9, 2H), 3.80(t, J=8.1, 2H), 1.44(s, 6H).

Compound 38: The method published in references SCMiller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 87%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.30(s,1H),6.83(s,1H),4.30(t,J=4.8,2H),3.46(t,J=4.2,2H),1.44 (s,6H).

Substrate 8: refer to the synthesis of substrate 1, with a yield of 59%. ¹ H-NMR (400MHz, D ₂ O): δ=7.30(s,1H),6.75(s,1H),5.20(dd,J=9.7,7.9,1H),4.26(t,J=4.6,2H ),3.58(dd, J＝10.8,10.0,1H),3.44(m,3H),1.34(s,3H),1.30(s,3H).

Compound 39: The method published in references SCMiller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 72%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.29(s,1H),6.80 (s,1H),4.27(t,J=4.8,2H),3.44(t,J=4.0,2H),2.96 (s,3H),1.44(s,6H).

Substrate 9: refer to the synthesis of substrate 1, with a yield of 57%. ¹ H-NMR (400MHz, D ₂ O): δ=7.30(s,1H),6.77(s,1H),5.23(dd,J=9.8,8.0,1H),4.32(t,J=4.8,2H ), 3.56(dd, J=13.9,7.0,1H), 3.46(m,3H), 2.94(s,3H), 1.36(s,3H), 1.29(s,3H).

Example 7

Synthesis of substrates 10, 11:

Compound 41: The method disclosed in the reference S.C.Miller, J.Am.Chem.Soc.2014, 136, 13277-13282, the yield was 52%.

Compound 42: Synthesized with reference to Compound 18, with a yield of 72%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.68(d, J=8.0,1H),7.28(d,J=8.2,1H),6.40(d,J=3.4,1H),3.86(t, J=4.8,2H),3.02(t,J=4.8,2H).

Compound 43: the synthesis of reference compound 2, the yield was 73%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.66(s,1H),7.18(s,1H),3.80(t,J=5.0,2H),3.02(t,J=5.0,2H),2.30 (s,3H),1.44(s,6H).

Compound 44: the synthesis of reference compound 3, the yield is 70%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=9.69(s,1H),7.78(s,1H),7.18(s,1H),3.82(t,J=4.8,2H),3.03(t,J =4.8,2H),1.40(s,6H).

Compound 45: the synthesis of reference compound 4, the yield is 42%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.79(s,1H),7.18(s,1H),3.80(t,J=7.9,2H),3.06(t,J=8.1,2H),1.44 (s,6H).

Compound 46: The method published in references SCMiller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 86%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.30(s,1H),6.68(s,1H),3.60(t,J=5.8,2H),3.08(t,J=5.2,2H),1.42 (s,6H).

Substrate 10: Refer to the synthesis of substrate 1 with a yield of 57%. ¹ H-NMR (400MHz, D ₂ O): δ=7.30(s,1H),6.76(s,1H),5.22(dd,J=9.9,7.9,1H),3.80(t,J=4.6,2H ),3.58(dd, J＝10.8,10.2,1H),3.40(m,3H),1.34(s,3H),1.26(s,3H).

Compound 47: The method published in references SCMiller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 73%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.29(s,1H),6.80(s,1H),3.80(t,J=4.8,2H),3.04(t,J=4.4,2H),3.00 (s,3H),1.46(s,6H).

Substrate 11: the synthesis of the reference substrate 1, the yield is 59%. ¹ H-NMR (400MHz, D ₂ O): δ=7.30(s,1H),6.77(s,1H),5.23(dd,J=9.8,8.0,1H),3.80(t,J=4.8,2H ),3.56(dd, J＝13.7,9.0,1H),3.39(m,3H),2.96(s,3H),1.36(s,3H),1.29(s,3H).

Example 8

Synthesis of substrates 12, 13:

Compound 49: The method disclosed in the literature S.C.Miller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 56%.

Compound 50: Synthesized with reference to Compound 18, with a yield of 72%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.30(s,1H),7.12(s,1H),6.42(s,1H),3.53(t,J=6.9,2H),3.15(t,J =6.8,2H).

Compound 51: the synthesis of reference compound 2, the yield is 73%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.86(s,1H),7.26(s,1H),3.56(t,J=6.0,2H),3.18(t,J=6.0,2H),2.30 (s,3H),1.46(s,6H).

Compound 52: the synthesis of reference compound 3, the yield is 70%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=9.60(s,1H),7.79(s,1H),7.22(s,1H),3.56(t,J=6.8,2H),3.20(t,J =6.8,2H),1.39(s,6H).

Compound 53: the synthesis of reference compound 4, the yield is 41%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.76(s,1H),7.18(s,1H),3.56(t,J=6.9,2H),3.18(t,J=6.1,2H),1.40 (s,6H).

Compound 54: The method published in references SCMiller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 86%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.20(s,1H),6.69(s,1H),3.36(t,J=5.8,2H),3.08(t,J=6.2,2H),1.44 (s,6H).

Substrate 12: Refer to the synthesis of substrate 1, with a yield of 61%. ¹ H-NMR (400MHz, D ₂ O): δ=7.20(s,1H),6.78(s,1H),5.20(dd,J=9.7,7.7,1H),3.58(dd,J=10.8,10.2 ,1H), 3.30(m,5H),1.32(s,3H),1.24(s,3H).

Compound 55: The method published in references SCMiller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 73%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.29(s,1H),6.68(s,1H),3.59(t,J=5.8,2H),3.02(t,J=5.6,2H),3.02 (s,6H),1.46(s,6H).

Substrate 13: refer to the synthesis of substrate 1, with a yield of 56%. ¹ H-NMR (400MHz, D ₂ O): δ = 7.29 (s, 1H), 6.77 (s, 1H), 5.33 (dd, J = 9.8, 8.0, 1H), 3.56 (m, 5H), 3.36 ( dd,J=11.0, 7.9,1H), 2.96(s,6H), 1.39(s,3H), 1.31(s,3H).

Example 9

Synthesis of Substrate 14:

Compound 57: The method disclosed in the literature S.C.Miller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 51%.

Compound 58: Synthesized with reference to Compound 18, with a yield of 72%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.30(s,2H), 3.30(t,J=6.9,4H), 2.79(t,J=6.9,4H), 1.99(m,4H).

Compound 59: the synthesis of reference compound 2, the yield was 73%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.06(s, 1H), 3.25(t, J=3.6, 4H), 2.86(t, J=6.5Hz, 2H), 2.80(t, J= 6.5 Hz,2H),2.30(s,3H),1.96(m,4H),1.44(s,6H).

Compound 60: the synthesis of reference compound 3, the yield is 70%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=9.69(s,1H),7.09(s,1H),3.26(t,J=6.8,4H),2.89(t,J=6.6Hz,2H), 2.82(t, J=6.5Hz, 2H), 1.96(m, 4H), 1.39(s, 6H).

Compound 61: the synthesis of reference compound 4, the yield was 41%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.06(s, 1H), 3.30(t, J=6.9, 4H), 2.86(t, J=6.3Hz, 2H), 2.80(t, J= 5.5 Hz,2H),1.96(m,4H),1.44(s,6H).

Substrate 14: the synthesis of the reference substrate 1, the yield is 59%. ¹ H-NMR (400MHz, D ₂ O): δ=6.90(s, 1H), 5.22(dd, J=9.6, 7.6, 1H), 3.60(dd, J=9.8, 9.2, 1H), 3.30(m ,5H),2.83 (t,J=6.6Hz,2H),2.80(t,J=5.6Hz,2H),1.99(m,4H),1.32(s,3H),1.24(s,3H).

Example 10

Synthesis of substrates 15, 16:

Compound 62: The method disclosed in the literature S.C.Miller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 53%.

Compound 63: the synthesis of reference compound 2, the yield is 73%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.80(s,1H),7.16(s,1H),5.66(s,1H),2.30(s,3H),2.13(s,3H),1.54( s,6H),1.44(s,6H).

Compound 64: the synthesis of reference compound 3, the yield is 70%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=9.66(s,1H),7.79(s,1H),7.20(s,1H),5.60(s,1H),2.03(s,3H),1.50( s,6H),1.39(s,6H).

Compound 65: the synthesis of reference compound 4, the yield is 43%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.76(s,1H), 7.18(s,1H), 5.63(s,1H), 2.06(s,3H), 1.52(s,6H), 1.44 ( s,6H).

Compound 66: The method published in references SCMiller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 86%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.56(s,1H),6.78(s,1H),5.53(s.1H),2.03(s,3H),1.42(s,6H),1.30( s,6H).

Substrate 15: refer to the synthesis of substrate 1, with a yield of 55%. ¹ H-NMR (400MHz, D ₂ O): δ=7.78(s, 1H), 6.86(s, 1H), 5.47(s, 1H), 5.26(dd, J=8.7, 6.7, 1H), 3.58( dd,J=10.8,10.2,1H),3.36(dd,J=10.1,7.8,1H),2.05(s,3H),1.36(s,3H),1.30(s,6H),1.24(s,3H ).

Compound 67: The method published in references SCMiller, J.Am.Chem.Soc.2014, 136, 13277-13282, yield 73%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.76(s,1H),6.79(s,1H),5.50(s.1H),3.01(s,3H),2.06(s,3H),1.44( s,6H),1.39(s,6H).

Substrate 16: refer to the synthesis of substrate 1, with a yield of 56%. ¹ H-NMR (400MHz, D ₂ O): δ=7.76(s, 1H), 6.80(s, 1H), 5.46(s, 1H), 5.36(dd, J=8.6, 6.6, 1H), 3.56( dd,J=9.8,9.2,1H),3.36(dd,J=10.0,6.9,1H),3.02(s,3H),2.06(s,3H),1.39(m,9H),1.31(s,3H ).

Example 11

Synthesis of Substrate 17:

Compound 69: the synthesis of reference compound 2, the yield is 86%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=8.30(d, J=2.0,1H), 8.09(d, J=9.1,1H), 7.92(d, J=8.6,1H), 7.76(d, J=8.6,1H),7.66(s,1H),3.81(s,3H),2.31(s,3H),1.49(s,6H).

Compound 70: the synthesis of reference compound 3, the yield was 72%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=9.90(s, 1H), 8.00(d, J=9.3, 1H), 7.86(d, J=8.6, 1H), 7.76(d, J=8.7, 1H), 7.30(d, J=6.8, 1H), 7.09(s, 1H), 3.80(s, 3H), 1.63(s, 6H).

Compound 71: the synthesis of reference compound 4, the yield is 42%. ¹ H-NMR (400MHz, CDCl ₃ ): 7.90(d, J=9.3,1H), 7.76(m,2H), 7.30(d,J=9.3,1H), 7.07(s,1H), 3.81 (s ,3H),1.59(s,6H).

Compound 72: the synthesis of reference compound 5, the yield is 93%. ¹ H-NMR (400MHz, CDCl ₃ ): 7.96 (d, J = 9.3, 1H), 7.77 (m, 2H), 7.32 (d, J = 9.4, 1H), 7.07 (s, 1H), 1.60 (s ,6H).

Substrate 17: refer to the synthesis of substrate 1, with a yield of 60%. ¹ H-NMR (400MHz, D ₂ O): δ=7.86(d, J=9.2,1H), 7.56(m,2H), 7.22(d,J=8.3,1H), 7.06(s,1H), 5.20(t, J=8.9, 1H), 3.60(t, J=10.0, 1H), 3.37(t, J=10.6, 1H), 1.46(s, 3H), 1.26(s, 3H).

Example 12

Synthesis of Substrate 18:

Compound 74: the synthesis of reference compound 2, the yield is 89%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=8.19(d, J=2.0,1H), 8.08(d, J=9.0,1H), 7.89(d, J=8.6,1H), 7.76(d, J=8.6,1H),7.60(dd,J=9.2,2.2,1H),2.30(s,3H),1.51(s,9H),1.44(s,6H).

Compound 75: the synthesis of reference compound 3, the yield was 69%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=9.96(s, 1H), 8.09(d, J=2.3, 1H), 7.93(d, J=9.3, 1H), 7.79(d, J=8.6, 1H), 7.66(d, J=8.6, 1H), 7.30(d, J=9.2, 1H), 1.56(s, 6H), 1.50(s, 9H).

Compound 76: the synthesis of reference compound 4, the yield is 43%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.89(d, J=9.0,1H), 7.76(m,2H), 7.30(d,J=9.2,1H), 7.06(s,1H), 1.59 (s,6H),1.50(s,9H).

Compound 77: the synthesis of reference compound 10, the yield was 96%. ¹ H-NMR (400 MHz, CDCl ₃ ): 7.86 (d, J=9.2,1H), 7.76 (m, 2H), 7.30 (d, J=9.2, 1H), 7.06 (s, 1H), 1.60 (s ,6H).

Substrate 18: refer to the synthesis of substrate 1, with a yield of 60%. ¹ H-NMR (400MHz, D ₂ O): δ=7.83(d, J=9.2,1H), 7.56(m,2H), 7.22(d,J=8.0,1H), 7.06(s,1H), 5.20(t, J=8.9, 1H), 3.56(t, J=9.2, 1H), 3.36(t, J=10.2, 1H), 1.50(s, 3H), 1.29(s, 3H).

Example 13

Synthesis of Substrate 19:

Compound 79: the synthesis of reference compound 2, the yield is 83%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=8.29(d, J=1.9,1H), 8.08(d, J=9.0,1H), 7.91(d, J=8.5,1H), 7.75(d, J=8.5,1H),7.66(dd,J=9.0,2.0,1H),2.30(s,3H),1.44(s,6H).

Compound 80: the synthesis of reference compound 13, the yield was 43%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.90(d,J=9.3,1H),7.69(m,2H),7.28(m,1H),7.09(s,1H),3.06(s,6H ),2.42(s,3H),1.54(s,6H).

Compound 81: the synthesis of reference compound 3, the yield is 66%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=10.00(s, 1H), 7.98(d, J=9.3, 1H), 7.84(d, J=8.6, 1H), 7.72(d, J=8.7, 1H), 7.27(d, J=6.8, 1H), 7.06(s, 1H), 3.11(s, 6H), 1.69(s, 6H).

Compound 82: the synthesis of reference compound 4, the yield was 49%. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.88(d, J=9.3,1H), 7.74(m,2H), 7.29(d,J=9.2,1H), 7.05(s,1H), 3.11 (s,6H),1.65(s,6H).

Substrate 19: refer to the synthesis of substrate 1, with a yield of 56%. ¹ H-NMR (400MHz, D ₂ O): δ=7.83(d, J=9.0,1H), 7.53(m,2H), 7.19(d,J=8.2,1H), 7.04(s,1H), 5.23(t, J=8.8, 1H), 3.57(t, J=10.2, 1H), 3.39(t, J=10.4, 1H), 2.78(s, 6H), 1.49(s, 3H), 1.26(s ,3H). Example 14

Synthesis of Substrate 20:

Compound 83: under argon protection, NaH (0.13 g, 3.2 mmol) was weighed into a dry three-necked flask, and dissolved in 20 ml of THF. Under ice bath conditions, triethyl phosphonoacetate (0.73ml, 3.6mmol) was dissolved in 5ml THF, added dropwise to the reaction flask, stirred at 0°C for 30min, and compound 14 (0.4g, 1.8mmol) Dissolve in 10ml THF and add dropwise to the reaction flask. The reaction gradually returned to room temperature. After the reaction was complete, the reaction was quenched by adding water, extracted with EA (100ml×3), and the organic phases were combined and dried over anhydrous sodium sulfate. The solvent was removed under reduced pressure, and compound 83 (0.46 g, yield 90%) was obtained by column chromatography. ¹ H NMR (400MHz, CDCl ₃ ): δ=7.57(m,2H),6.68(m,3H),4.28(q, J=7.1,2H),3.04(s,6H),1.41(s,6H) ,1.34(t,J=7.1,3H).

Compound 84: Dissolve compound 83 (1.0 g, 3.5 mmol) in 30 ml of isopropanol, add 10 ml of NaOH solution (1 M), and stir at room temperature for 1 h. After the reaction was complete, adjust the pH to 7, remove the solvent under reduced pressure, extract with DCM (100ml×3), combine the organic phases, dry over anhydrous sodium sulfate, remove the solvent under reduced pressure, and separate by column chromatography to obtain compound 84 (0.87g, yield The rate is 96%). ¹ H NMR (400MHz, CDCl ₃ ): δ=7.56(m,2H), 6.63(m,3H), 3.02(s,6H), 1.39(s,6H).

Compound 85: Dissolve compound 84 (0.15g, 0.58mmol) and S-(trityl)-D-cysteine methyl ester (0.24g, 0.64mmol) in 20ml DMF, add EDC (0.36g , 1.9mmol) and DMAP (0.18g, 1.5mmol), under the protection of argon, reacted for 3h. After the reaction was complete, the organic solvent was removed under reduced pressure, and compound 85 (0.32 g, 89%) was obtained by column chromatography. ¹ H NMR (400 MHz, CDCl ₃ ): δ=7.53(d, J=8.5,1H),7.44(d,J=15.6,1H),7.39(m,6H),7.28(m,8H),7.21 (m,4H),6.80(d,J=15.6,1H),6.67(m,2H),6.22(d,J=7.8,1H),4.72 (m,1H),3.73(s,3H),3.03 (s,6H),1.39(d,J=4.3,6H).

Compound 86: Dissolve compound 85 (0.13g, 0.21mmol) in 10ml of DCM, add Ph ₃ PO (0.12g, 0.42mmol) and Tf ₂ O (360ul, 2.1mmol) respectively under ice-bath conditions, and stir for 1h , the organic solvent was removed under reduced pressure, and compound 85 (46 mg, 61%) was obtained by column chromatography. ¹ H-NMR (400MHz, CDCl ₃ ): δ=7.52(d, J=8.7,1H), 7.39(d, J=13.9,1H), 7.30(d, J=13.9,1H), 6.96(d, J＝2.4,1H),6.87(dd,J＝8.7,2.4,1H),5.20(dd,J＝9.6,7.7,1H), 3.70(s,3H),3.56(dd,J＝9.9,9.0, 1H), 3.36(dd, J=11.0, 7.8, 1H), 2.98(s, 6H), 1.45(s, 3H), 1.39(s, 3H).

Substrate 20: Dissolve 50mg of compound 86 in 2ml of ethanol, add 6ml of 10mM NH ₄ HCO ₃ solution, add 25mg of esterase, stir at 37°C for 19h, after the reaction is complete, remove the solvent under reduced pressure, and separate by column chromatography to obtain the substrate ( 46 mg, 96%). ¹ H-NMR (400MHz, D ₂ O): δ=7.50(d, J=8.6,1H), 7.36(d, J=11.9,1H), 7.29(d, J=11.9,1H), 6.93(d ,J=2.6,1H),6.85(dd, J=8.6,2.2,1H),5.22(dd,J=9.2,7.6,1H),3.56(dd,J=10.9,10.0,1H),3.36(dd , J＝10.9,8.8,1H),3.02(s,6H),1.46(s,3H),1.40(s,3H).

Example 15

Synthesis of Substrate 21:

Substrate 21: Refer to the synthetic route of substrate 20, wherein only the raw material triethyl phosphonoacetate is replaced by trans-ethyl-4-(diethylphosphono)crotonate, and other operating methods are the same as the raw materials . ¹ H-NMR (400 MHz, D ₂ O): δ = 7.60 (d, J = 9.6, 1H), 7.33 (d, J = 10.9, 1H), 7.29 (m, 1H), 7.12 (m, 2H) ,6.93(d,J=8.6,1H),6.85(d,J=8.6,1H),5.26(d,J=7.6,1H),3.66(dd,J=8.8,8.0,1H),3.39(dd ,J=9.6,8.6,1H), 3.03(s,6H),1.44(s,3H),1.39(s,3H).

Example 16

Expression and purification of different luciferase proteins

In order to determine the effectiveness of the new luciferase substrates synthesized, the gene sequences encoding different luciferases (the amino acid sequence is shown in Figure 1, followed by: Fluc, x5g, x5r, Eluc and CBR2) were connected by enzyme digestion Cloned into the pCDFDuet1 vector by the method, and the selected enzyme cutting sites were BamHI and Acc65I. The constructed different luciferase expression vectors were transformed into BL21(DE3) with correct DNA sequencing plasmids for protein expression: Pick the transformed colonies and culture them overnight in test tubes, and add 1 mL of bacterial liquid to 100 mL of LB at a ratio of 1%. The culture medium was cultured on a shaker at 37°C and 220 rpm. When the OD ₆₀₀ value of the bacterial solution reached 0.6, IPTG (final concentration: 1 mM) was added to induce protein expression, and induced at 18°C for 24-48 hours. After the expression, the bacteria were collected by centrifugation, resuspended to an appropriate amount of His Buffer A, ultrasonically lysed, centrifuged at 12000rpm at 4°C for 20min, and the protein supernatant was added to the affinity column in batches; add 5 times the column volume containing 50mM imidazole wash buffer to remove most of the impurity proteins; then the target protein was eluted with 300mM imidazole elution buffer, and the target protein was collected according to the Bradford chromogenic process, labeled and placed on ice for later use; determined by BCA method Purify the resulting protein concentration for use. After the concentration of the purified protein was determined, the size and purity of the purified protein were verified by SDS-PAGE. Figure 2 is the electrophoresis diagram of different purified luciferase proteins. The electrophoresis results show that there is a protein slightly smaller than 66.2kDa, which is consistent with the size of luciferase 62kDa, as expected; on the other hand, Figure 2 shows that different luciferases The purity of the enzyme is ideal, and there is basically no foreign protein.

Example 17

Reaction of substrate 1, substrate 3, substrate 5 and substrate 19 with different luciferases

Dilute different luciferase proteins to 0.2mM, add different luciferases (0.2mM, 40mL) to black 96-well plates, then add ATP (8mM, 10mL) and MgSO ₄ (32mM, 10mL), and finally After adding the substrate with a final concentration of 100 μM, it was immediately tested using a BioTek Neo2 microplate reader to determine the relative luminescence intensity. The test results are shown in FIG. 3 , and the test results show that: Substrate 1, Substrate 3, Substrate 5, and Substrate 19 react with firefly luciferase (Fluc), all of which obtain higher luminous intensities.

Example 18

Substrate 1, Substrate 3, Substrate 5, and Substrate 19 reacted with Fluc in 30 min over time

Dilute the purified Fluc protein to 0.5mM, add Fluc (0.5mM, 40mL) to a white 96-well plate, then add ATP (8mM, 10mL) and MgSO ₄ (32mM, 10mL), and finally use the BioTek Neo2 microplate reader to After adding different substrates with a final concentration of 100 μM to the perfusion system, the luminous intensity test was carried out immediately, and the measurement was performed at intervals of 20 seconds, and the test was continued for 30 minutes. Substance 3 > Substrate 5 > Substrate 1 > Substrate 19 (without considering the difference in quantum yield of different bands of PMT detectors); after substrate 3 was added to the reaction system, the luminous intensity decreased slightly in the first minute, and then remained basically stable ; The reaction process of substrate 5 is basically the same as that of substrate 3; the luminescence intensity of the reaction between substrate 1 and Fluc decreased slightly at first, and then basically remained stable; the luminescence intensity of substrate 19 and Fluc remained basically stable.

Example 19

Substrate 1, Substrate 3, Substrate 5, Substrate 7, Substrate 16, Substrate 19 and Substrate 20 respectively react with Fluc emission spectra

Dilute the purified Fluc protein to 2mM, add Fluc (2mM, 40 mL) to a white 96-well plate, then add ATP (8mM, 10mL) and MgSO ₄ (32mM, 10mL), and finally add different bases with a final concentration of 100μM The substances were scanned by emission spectrum, and the test process was carried out with BioTek Neo2 microplate reader.

The test results are shown in Figure 5, and the obtained emission spectrum shows: the wavelength corresponding to the maximum emission peak of the reaction between substrate 1 and Fluc is 650nm; the maximum wavelength of the emission peak corresponding to the reaction between substrate 3 and Fluc is 660nm; The wavelength corresponding to the peak maximum value is 655nm; the corresponding wavelength of the maximum emission peak of the reaction between substrate 7 and Fluc is 670nm; the corresponding wavelength of the maximum emission peak of the reaction between substrate 16 and Fluc is 680 nm; the maximum emission peak of the reaction between substrate 19 and Fluc corresponds to The wavelength is 750nm; the wavelength corresponding to the maximum emission peak of the reaction between substrate 20 and Fluc is 745nm.

购自于毕得医药) 与x5g和x5r反应的发射峰，测试结果显示萤光素与x5g反应发射峰最大值对应波长为560nm，萤光素与x5r反应发射峰最大值对应波长为610nm，与文献 (Branchini,Ablamsky et al.2007)报道结果一致。Under the same conditions as above, luciferin (

(purchased from Beide Pharmaceuticals) and x5g and x5r reaction emission peaks, the test results show that the maximum emission peak corresponding to the wavelength of 560nm for the reaction between luciferin and x5g, and the corresponding wavelength for the maximum emission peak of the reaction between luciferin and x5r is 610nm, and The literature (Branchini, Ablamsky et al.2007) reported the same results.

To sum up, the wavelength of a series of novel luciferase substrates of the present invention reaches the far-red or near-infrared band, which has strong photon tissue penetration ability and has great application value in in vivo imaging.

Example 20

Microscopic imaging test of substrate 1, substrate 3, substrate 5 and substrate 19 in HeLa cell line

The Fluc gene sequence was cloned into the pEGFP-C1 vector by restriction digestion, and the restriction restriction sites selected were NheI and NotI. The constructed plasmid pEGFP-Fluc was transformed into DH5a after the correct DNA sequencing, and the plasmid was extracted using the endotoxin-free plasmid extraction kit (Tiangen Biochemical Technology Co., Ltd.). ) to determine the concentration, and store in a -20°C refrigerator for later use. Passage the HeLa cell line (purchased from the cell bank of the Type Culture Collection Committee of the Chinese Academy of Sciences) to a glass-bottomed 96-well plate in a CO2 incubator with a high-glucose medium containing 10% fetal bovine serum (FBS) and streptomycin and penicillin (DMEM) culture, plasmid transfection was carried out when the growth reached 50-60% confluence, transfection was carried out using Hieff Trans ^TM (purchased from Yensen), and the operation process was performed according to standard operating procedures. Microscope imaging was carried out 48 hours after transfection: the microscope used was Nikon Ti2, the lens was Nikon 100 times oil lens (NA=1.40), and the camera used Prime95B sCMOS camera (TELEDYNE PHOTOMETRICS). At the beginning of the test, dissolve the different substrates to 200 μM with HBSS buffer, and replace the DMEM medium with 60 μL of HBSS buffer (containing 10 mM glucose). A good substrate solution, then began to collect luminescent signals. The exposure time for the samples corresponding to substrate 3 and substrate 5 is 1 min, the exposure time for the sample corresponding to substrate 1 is 2 min, and the exposure time for the sample corresponding to substrate 19 is 5 min. At this point, the image shown in Figure 6 can be obtained. It should be noted that , the collection of luminescent signals must be carried out in a strict light-proof environment.

Figure 6 shows that the HeLa cell line will have a higher level of Fluc expression 48 hours after transfection with the pEGFP-Fluc plasmid, and the luminescent signal intensity of substrate 1, substrate 3, substrate 5 and substrate 19 in living cells (regardless of the low quantum yield of the camera in this band) is consistent with the test results at the protein level. In summary, the different substrates of the present invention have good cell permeability and can be used for testing in living cells.

Example 21

Test results of different concentrations of substrate 1, substrate 3, substrate 5 and substrate 19 in HEK293T cell line

Referring to the cell culture method in Example 19, different concentrations of substrates were tested in the HEK293T cell line. Passage HEK293T cells to a 24-well plate, transfect the plasmid when the cells grow to a confluence of 50-60%, and test 36 hours after transfection, discard the medium at the beginning of the test, wash the cells once with PBS, and then Add cell lysate (70 μL lysate/cm2 cells) and place on ice to lyse for 5 min (during this period, gently tap and shake), centrifuge at 12,000 rpm at 4°C for 5 min to take the supernatant for testing. Add 10 μL of lysed supernatant and 15 μL of activity assay buffer to each well of a 384-well white luminescent plate and mix well. After adding 15 μL of substrate solution to each well, test the luminescence intensity immediately. The test results are shown in Figure 7.

The test results show that: the luminescent signal intensity of substrate 1, substrate 3, substrate 5, and substrate 19 in HEK293T cells is consistent with the test results at the protein level; the test results of different concentrations of substrates show that when the substrate concentration is less than Luminescent signals can also be detected at 1 μM, that is, the different substrates of the present invention all have good application prospects in cells.

The corresponding components of different solutions involved in this embodiment are:

Activity assay buffer: 15mM potassium phosphate (pH 7.8), 25mM glycylglycine, 15mM MgSO ₄ , 4mM EGTA, 2mM ATP;

Cell lysate: 25mM glycylglycine (pH 7.8), 15mM MgSO ₄ , 4mM EGTA, 1% Triton X-100;

Substrate solution: 25 mM glycylglycine (pH 7.8), 15 mM MgSO ₄ , 4 mM EGTA, 0.1 mM substrate X.

Example 22

Substrate X test results in mice

The endotoxin-free plasmid pEGFP-Fluc obtained in Example 20 was delivered to the legs of ICR mice by means of muscle electroporation, and 12 μg of the plasmid was electroporated on the outer side of each leg. The electroporation process used a living gene transfer instrument (Shanghai Tarisa Health Technology Co., Ltd. Company) followed the standard operating procedures. After the electroporation was completed, the mice were recovered from anesthesia and returned to the animal room for feeding. Two days later, the IVIS Spectrum CT in vivo imaging system was used for testing. The test process is as follows: the mice were anesthetized with pentobarbital sodium, and after the mice were anesthetized, 100 μL of 10 mM different substrates (dissolved in PBS, filtered with a 0.22 μm filter membrane) were injected intraperitoneally, and the in vivo imager was used for testing after 5 minutes of injection. The test results are shown in Figure 8: in Figure B, the luminescence intensities of different samples were counted after injection of substrate 3 or 5, and the results showed that strong luminescence signals were detected in both legs of mice injected with substrate 3, and the injected bottom The luminescent signal was also detected in the mice injected with Substance 5, and the luminous intensity of the legs of the mice injected with Substrate 3 was higher than that of the mice injected with Substrate 5.

In summary, the different substrates introduced in the present invention have good tissue permeability and have good application prospects in live small animal imaging.

Claims (10)

1. A luciferase substrate having a structure represented by the following formula (I):

in the formula,

r and R ₁ Together and/or R and R ₂ Together form a substituted or unsubstituted alicyclic ring, a substituted or unsubstituted alicyclic heterocyclic ring, a substituted or unsubstituted aromatic ring, or a substituted or unsubstituted aromatic heterocyclic ring;

or R and R ₁ Or R and R ₂ When a substituted or unsubstituted alicyclic ring, a substituted or unsubstituted alicyclic heterocyclic ring, a substituted or unsubstituted aromatic ring, or a substituted or unsubstituted aromatic heterocyclic ring is not formed, R is: hydroxy, or-NR _a R _b ，R _a 、R _b Each independently selected from hydrogen, or alkyl, R ₁ 、R ₂ Are the same or different and are each independently-H, hydroxy, alkyl, halo;

the substituents in the "substituted or unsubstituted alicyclic ring", "substituted or unsubstituted aromatic ring" are the same or different and are each independently selected from the group consisting of a hydroxyl group, an amino group, an alkyl group, an alkylamino group, a halogen group, a cyano group:

n is 0,1, 2, or 3;

wherein,

the alkyl group in the "alkyl group" or "alkylamino group" is C ₁ -C ₁₀ Straight chain alkyl or C ₃ -C ₁₀ A branched alkyl group; preferably, is C ₁ -C ₇ Straight chain alkyl or C ₃ -C ₇ A branched alkyl group; preferably, is C ₁ -C ₅ Straight chain alkyl or C ₁ -C ₅ A branched alkyl group; preferably selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, tert-butyl, sec-butyl, n-pentyl, 1-methylbutyl,2-methylbutyl, 3-methylbutyl, isopentyl, 1-ethylpropyl, neopentyl, n-hexyl, 1-methylpentyl, 2-methylpentyl 3-methylpentyl, isohexyl, 1-dimethylbutyl, 2-dimethylbutyl, 3-dimethylbutyl, 1, 2-dimethylbutyl 1, 3-dimethylbutyl, 2-ethylbutyl, n-heptyl, 2-methylhexyl, 3-methylhexyl, 2-dimethylpentyl, 3-dimethylpentyl, 2, 4-dimethylpentyl, 3-ethylpentyl or 2, 3-trimethylbutyl;

the alicyclic ring is a saturated or unsaturated 4-to 10-membered monocyclic or polycyclic alicyclic ring;

the "aliphatic heterocyclic ring" is a saturated or unsaturated 4-to 10-membered (optionally 4-, 5-or 6-membered) monocyclic or polycyclic aliphatic heterocyclic ring containing at least one heteroatom selected from N, O or S on the ring;

the aromatic ring is a 5-to 10-membered monocyclic or fused bicyclic aromatic group; alternatively, the "aromatic ring" is selected from a benzene ring, a naphthalene ring, a fluorene ring, an anthracene ring, a phenanthrene ring, a biphenyl ring;

the aromatic heterocyclic ring is a 5-to 10-membered monocyclic or fused bicyclic heteroaromatic group containing at least one heteroatom selected from N, O or S on the ring; optionally, the aromatic heterocycle is selected from thiophene ring, furan ring, pyrrole ring, imidazole ring, thiazole ring, oxazole ring;

the halogen group is selected from F, cl, br and I.

2. The luciferase substrate according to claim 1, which is characterized in that;

r and R ₁ Together form structures of the following formulae (I-2-1) to (I-2-6):

or R and R ₂ Together form structures of the following formulae (I-2-7) to (I-2-8):

or, R and R ₁ Together with R and R ₂ Are formed together

Wherein each R' is independently selected from hydrogen, or alkyl; r' is selected from hydrogen, or alkyl.

3. The luciferase substrate of claim 1 or 2, wherein the luciferase substrate is selected from a compound of the formula:

4. a method of producing a luciferase substrate as claimed in any one of claims 1 to 3, wherein: comprising the step of reacting a compound of formula (II) with a compound of formula (III) to obtain a luciferase substrate of formula (I),

wherein R and R ₁ 、R ₂ As defined in any one of claims 1 to 3, n =0;

or, the luciferase substrate comprises a product obtained by the reaction of the compound of the formula (IV) and the compound of the formula (V), and the product obtained by the hydrolysis of an ester bond and the condensation reaction of the formula (VI) is subjected to thiol protecting group removal to obtain a compound of the formula (VII), and finally, the luciferase substrate shown in the formula (I) is obtained by the enzymolysis of the ester bond;

wherein R and R ₁ 、R ₂ Defined as in any one of claims 1 to 3, n =1, 2,3,m =0, 1, 2.

5. Use of the luciferase substrate as claimed in any one of claims 1 to 3 in the manufacture of an optical detection product including, but not limited to, optical probes and luminescent detection kits.

6. A bioluminescent probe comprising the luciferase substrate of any one of claims 1 to 3.

7. A luminescent detection kit comprising a luciferase substrate as claimed in any one of claims 1 to 3 or a bioluminescent probe as claimed in claim 6.

8. Use of the luciferase substrate of claim 1, the bioluminescent probe of claim 6 or the luminescent detection kit of claim 7 in an environmental assay.

9. Use of the luciferase substrate of claim 1, the bioluminescent probe of claim 6 or the luminescence detection kit of claim 7 in analytical chemistry.

10. Use of the luciferase substrate of claim 1, the bioluminescent probe of claim 6 or the luminescence detection kit of claim 7 in bioassays and assays;

preferably, the biological analysis and detection comprises the analysis and detection of cell level, tissue level, organ level and individual level of organism;

preferably, the organism comprises a bacterium, a mammalian cell, a mouse, a rat or a monkey.

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