A luciferase substrate and methods of making and using the same

Abstract

The application discloses a luciferase substrate, a preparation method and use thereof. The luciferase substrate has the advantages of long emission wavelength, high light intensity and high detection sensitivity after acting on firefly luciferase, and has good performance in live cell and live animal imaging, and can be used as an important analysis tool in the fields of live bio-luminescence imaging and protein quantitative detection.

Description

TECHNICAL FIELD

[0001] The present application belongs to the technical field of luciferase substrate preparation, and particularly relates to a luciferase substrate, a preparation method therefor and use thereof. BACKGROUND

[0002] Optical imaging plays an important role in observing cell structure, real-time monitoring of cell behavior and monitoring various biological events. Biological optical imaging technology can be mainly divided into two categories: fluorescence imaging and luminescence imaging. At present, most fluorescence imaging tools are difficult to achieve in situ real-time dynamic monitoring of in vivo biological processes due to the limitations of biocompatibility and sensitivity.

[0003] Bioluminescence is a phenomenon of converting chemical energy into light energy by using luciferase to catalyze the oxidation of a small molecule substrate, and the monitoring of the released photons can realize the monitoring of proteins. In the specific practice process, researchers can fuse the luciferase gene with the gene of a specific target protein by using molecular biology techniques, and can realize the expression of the corresponding fusion gene in different model organisms by using different technical means, so as to realize the dynamic tracking of the target protein in cells or living animals after the substrate is given. Compared with fluorescence imaging, bioluminescence imaging does not need an external excitation light source, avoids the interference caused by endogenous fluorescence induced by the external excitation light source of the biological tissue, greatly reduces the background signal, and can significantly improve the signal-to-noise ratio of the obtained image. However, in practical application, the bioluminescence imaging technology still faces great challenges in deep tissue imaging. First, the bioluminescence signal is relatively weak; second, the wavelength of the traditional bioluminescence system is 460-620 nm, and substances such as hemoglobin (λ = ~ 415-577 nm) and melanin (λ < 600 nm) in the body can absorb the luminescence signal and hinder the detection of the light signal, thereby reducing the depth and clarity of the imaging. At present, researchers generally believe that the ideal optical window for in vivo imaging is in the wavelength range of 650-900 nm.

[0004] Therefore, in order to improve the biological tissue penetration ability and imaging depth and clarity of the bioluminescence system, the development of a bioluminescence system with high luminous brightness and long emission wavelength has become the focus of research. At present, the ways based on the mutation of luciferase protein and the chemical modification of luciferase substrate have important promoting effect on achieving the above goals. In recent years, several bioluminescence systems (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. Nat Commun. 2018, 9, 1-12; A. Miyawaki.et.al. Science. 2018, 359, 935-939) have been developed, which make the emission wavelength red-shift, but the luminous intensity is still lower than that of natural fluorescein. Therefore, there is still a large space for improvement of the existing bioluminescence system. SUMMARY

[0005] In view of the deficiencies of the existing bioluminescence system, the present application constructs a new luciferase substrate which has the characteristics of red-shifted emission wavelength and high luminous intensity when reacting with firefly luciferase, and has good application prospect in living tissue.

[0006] In order to achieve the above technical content, the present application provides a luciferase substrate, the structure of which is shown in formula (I),

[0007]

[0008] In formula (I),

[0009] R and R1 together and / or R and R2 together form a substituted or unsubstituted alicyclic ring, a substituted or unsubstituted aliphatic heterocyclic ring, a substituted or unsubstituted aromatic ring, or a substituted or unsubstituted aromatic heterocyclic ring;

[0010] Alternatively, when R and R1 or R and R2 do not form a substituted or unsubstituted alicyclic ring, a substituted or unsubstituted aliphatic heterocyclic ring, a substituted or unsubstituted aromatic ring, or a substituted or unsubstituted aromatic heterocyclic ring, R is: a hydroxyl group, or -NR a R b , R a , R b are each independently selected from hydrogen, or an alkyl group, R1, R2 are the same or different, and each independently is -H, a hydroxyl group, an alkyl group, a halogen group;

[0011] The substituents in the "substituted or unsubstituted alicyclo", "substituted or unsubstituted aliphatic heterocyclo", "substituted or unsubstituted aromatic ring", "substituted or unsubstituted aromatic heterocyclo" are the same or different, and each is independently selected from hydroxyl, amino, alkyl, alkylamino, halogen group, cyano:

[0012] n is 0, 1, 2, or 3;

[0013] wherein,

[0014] The alkyl in the "alkyl" or "alkylamino" is C1-C 10 straight chain alkyl or C3-C 10 branched alkyl; preferably, C1-C7straight chain alkyl or C3-C7branched alkyl; preferably, C1-C5straight chain alkyl or C1-C5branched alkyl; preferably, selected from methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, t-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-ethylbutyl, 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;

[0015] The "aliphatic heterocyclo" is a saturated or unsaturated 4-10 membered monocyclic or polycyclic aliphatic heterocyclo; alternatively, the "aliphatic heterocyclo" is a saturated or unsaturated 4, 5, 6, 7, 8, 9, 10 membered monocyclic or polycyclic aliphatic heterocyclo;

[0016] The "aliphatic heterocyclo" is a saturated or unsaturated 4-10 membered monocyclic or polycyclic aliphatic heterocyclo; alternatively, the "aliphatic heterocyclo" is a saturated or unsaturated 4, 5, 6, 7, 8, 9, 10 membered monocyclic or polycyclic aliphatic heterocyclo;

[0017] The "aromatic ring" is a 5-10 membered monocyclic or fused bicyclic aromatic group; alternatively, the "aromatic ring" is a 5, 6, 7, 8, 9, 10 membered monocyclic or fused bicyclic aromatic group; alternatively, the "aromatic ring" is selected from benzene ring, naphthalene ring, fluorene ring, anthracene ring, phenanthrene ring, biphenyl ring;

[0018] 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 in 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 in the ring; alternatively, the aromatic heterocycle is selected from a thiophene ring, a furan ring, a pyrrole ring, an imidazole ring, a thiazole ring, an oxazole ring;

[0019] The "halogen group" is selected from F, Cl, Br, I.

[0020] Alternatively, R and R1 together form a structure in the following formula (I-2-1) to (I-2-6):

[0021] Alternatively, R and R2 together form a structure in the following formula (I-2-7) to (I-2-8):

[0022] Alternatively, R and R1 together and R and R2 together form

[0023] wherein each R' is independently selected from hydrogen, or an alkyl group; and R" is selected from hydrogen, or an alkyl group.

[0024] Alternatively, in formula (I), when n is taken as 0, R is Alternatively, R and R1 together and / or R and R2 together form a structure in the following formula (I-2-1) to (I-2-11):

[0025] R' is selected from -H or -Me;

[0026] In formula (I), R is taken as n can be taken as 0, 1 or 2;

[0027] Alternatively, the luciferase substrate is selected from a compound in the following formula:

[0028]

[0029]

[0030] In another aspect, a method for preparing the above-mentioned substrate is also provided, comprising the step of reacting a compound of formula (II) with a compound of formula (III) to obtain a luciferase substrate of formula (I),

[0031]

[0032] Wherein, R, R1, and R2 are defined as described in any one of claims 1-3, and n = 0;

[0033] Alternatively, it may also include the product obtained by reacting compound (IV) with compound (V), the product obtained by hydrolysis of ester bond and condensation reaction of compound (VI) to remove the thiol protecting group, thus obtaining compound (VII), and finally, the luciferase substrate shown in formula (I) is obtained by enzymatic hydrolysis of ester bond.

[0034]

[0035] Wherein, R, R1, and R2 are defined as described in any one of claims 1-3, n = 1, 2, 3, and m = 0, 1, 2.

[0036] On the other hand, the use of the above-mentioned luciferase substrate in the preparation of optical detection products is provided, including but not limited to optical probes and luminescent detection kits.

[0037] On the other hand, a bioluminescent probe is provided, the bioluminescent probe comprising the above-mentioned luciferase substrate.

[0038] On the other hand, a luminescent detection kit is provided, the luminescent detection kit comprising the above-mentioned luciferase substrate or the above-mentioned bioluminescent probe.

[0039] On the other hand, the use of the above-mentioned luciferase substrate, the above-mentioned bioluminescent probe, or the above-mentioned luminescent detection kit in environmental detection is provided.

[0040] On the other hand, the above-mentioned luciferase substrate, the above-mentioned bioluminescent probe, or the above-mentioned luminescent detection kit are used in analytical chemistry.

[0041] On the other hand, the uses of the above-mentioned luciferase substrate, the above-mentioned bioluminescent probe or the above-mentioned luminescent detection kit in bioanalysis and detection;

[0042] Preferably, the bioanalysis and detection include analysis and detection at the cellular, tissue, organ, and individual organism levels.

[0043] Preferably, the organism includes bacteria, mammalian cells, mice, rats, or monkeys.

[0044] The beneficial effects of this invention are:

[0045] The luciferase substrate of this invention, after reacting with firefly luciferase, has the advantages of long emission wavelength, high luminescence intensity, and high detection sensitivity. It has good performance in both live cell and live animal imaging and can be used as an important analytical tool in the fields of live bioluminescence imaging and protein quantification. Attached Figure Description

[0046] Figure 1 . Amino acid sequences of different luciferases and their mutants are shown, all of which are involved in Example 16.

[0047] Figure 2 . Results of protein electrophoresis of purified proteins of different luciferases and their mutants.

[0048] Figure 3 . Relative luminescence intensities of substrate 1, substrate 3, substrate 5 and substrate 19 reacted with different luciferases.

[0049] Figure 4 . Time-dependent curves of relative luminescence intensities of substrate 1, substrate 3, substrate 5 and substrate 19 reacted with firefly luciferase within 30 min.

[0050] Figure 5 . Luminescence spectra of substrate 1, substrate 3, substrate 5, substrate 16 and substrate 19 reacted with firefly luciferase, in which the spectra of mutants x5g and x5r are those of reaction with luciferin.

[0051] Figure 6 . Live cell imaging results of substrate 1, substrate 3, substrate 5 and substrate 19 in HeLa cell lines expressing firefly luciferase.

[0052] Figure 7 . Test results of substrate 1, substrate 3, substrate 5 and substrate 19 at different concentrations in HEK293T cell lines.

[0053] 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 of substrate 3 and substrate 5 in mouse legs (3 biological replicates). DETAILED DESCRIPTION

[0054] The following detailed description of the embodiments of the present application is given on the premise of the technical solutions of the present application, and detailed implementation manners and specific operation processes are given, but the protection scope of the present application is not limited to the following examples. The following examples involve conventional molecular biology cloning methods and cell culture and mouse test methods, which are well known to those skilled in the art, and those skilled in the art can easily modify or change the embodiments according to the specific circumstances to implement the present application.

[0055] The pEGFP-C1 plasmid vector used in the examples was purchased from Invitrogen Corporation, and the pCDFDuet-1 plasmid vector was purchased from Novagen Corporation. All primers used for PCR were synthesized, purified and identified correctly by mass spectrometry by Shanghai Jerui Biological Engineering Technology Co., Ltd. The expression plasmids constructed in the examples were all sequenced, and the sequencing was completed by Jilei Sequencing Company. The PrimeSTAR DNA polymerase used in the PCR reaction in each example was purchased from TaKaRa Corporation. The restriction endonucleases such as NheI, BamHI, NotI, and Acc65I were purchased from Fermentas Corporation. Inorganic salt chemicals were purchased from Shanghai Chemical Reagent Company of National Pharmaceutical Group. Kanamycin and Streptomycin were purchased from Ameresco Corporation. D-luciferin was purchased from Sigma Corporation. The 384-well and 96-well white plates were purchased from Grenier Corporation.

[0056] The BL21(DE3) strain used in the examples was purchased from Invitrogen Corporation. HeLa cells and HEK293T cells were purchased from the Chinese Academy of Sciences Typical Culture Collection Cell Bank. ICR mice were purchased from Shanghai Jiesijie Experimental Animal Co., Ltd. The endotoxin-free plasmid maxi kit used in the examples was purchased from Tian Gen Biochemical Technology (Beijing) Co., Ltd. The main instruments used in the examples were Synergy Neo2 multifunctional microplate reader (Bio-Tek Corporation, USA), X-15R high-speed refrigerated centrifuge (Beckman Corporation, USA), PCR amplifier (Biometra Corporation, Germany), IVIS Spectrum CT in vivo imaging system (PerkinElmer Corporation, USA), and nucleic acid electrophoresis instrument (Shen Gong Bocai Company).

[0057] The abbreviations have the following meanings: “h” refers to hours, “min” refers to minutes, “s” refers to seconds, “d” refers to days, “μL” refers to microliters, “mL” refers to milliliters, “L” refers to liters, “mM” refers to millimoles, and “μM” refers to micromoles.

[0058] Example 1

[0059] Synthesis of Substrate 1:

[0060]

[0061] Compound 2: Compound 1 (10 g, 80 mmol) was dissolved in 200 ml of ethanol, methyl isopropyl ketone (8.3 g, 96 mmol) was added, followed by 3 ml of concentrated sulfuric acid, and heated to reflux and stirred overnight. The next day, the reaction was brought to room temperature, water was added, and the reaction was extracted three times with ethyl acetate (100 ml), the organic phases were combined, dried over anhydrous sodium sulfate, and the organic phase was removed under reduced pressure, and compound 2 (13.0 g, 86% yield) was obtained by column chromatography. 1 H-NMR (400 MHz, CDC13): 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).

[0062] Compound 3: Compound 2 (10 g, 53 mmol) was dissolved in 100 ml of 1,4-dioxane, selenium dioxide (11.7 g, 106 mmol) was added, and the reaction was heated to 80°C and stirred for 2 h. The reaction was brought to room temperature, the selenium dioxide was filtered off, the organic solvent was removed under reduced pressure, and compound 3 (8.0 g, 75% yield) was obtained by column chromatography. 1 H-NMR (400 MHz, CDC13): 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).

[0063] Compound 4: Compound 3 (5.0 g, 24.6 mmol) was dissolved in 15 ml of tetrahydrofuran, 25 ml of aqueous ammonia was added, followed by iodine (9.4 g, 36.9 mmol), and stirred at room temperature for 6 h. To the reaction, 30 ml of saturated sodium thiosulfate was added, extracted with ethyl acetate, the organic phases were combined, dried over anhydrous sodium sulfate, the organic phase was removed under reduced pressure, and compound 4 (2.2 g, 46% yield) was obtained by column chromatography. 1 H-NMR (400 MHz, CDC13): 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).

[0064] Compound 5: Compound 4 (3.0 g, 15 mmol) was dissolved in 50 ml of dry tetrahydrofuran, the reaction was placed at 0°C, BBr3(1.7 ml, 18 mmol) was added dropwise to the reaction, and after stirring for half an hour, it was quenched with water. It was extracted three times with DCM, the organic phases were combined, dried over anhydrous sodium sulfate, the organic phase was removed under reduced pressure, and compound 5 (2.57 g, 92% yield) was obtained by column chromatography. 1H-NMR (400 MHz, CDC13): 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).

[0065] 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 and added to the reaction system, stirred at room temperature for 1 h. After the reaction was completed, it was extracted with DCM for 3 times, the organic phase was combined, dried over anhydrous sodium sulfate, and the organic phase was removed under reduced pressure. Compound 1 (0.98 g, yield 63%) was obtained by column chromatography. 1 H NMR (600 MHz, D20): 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).

[0066] Example 2

[0067] Synthesis of Substrate 2:

[0068]

[0069] Compound 7: Refer to the synthesis of compound 2, yield 85%. 1 H-NMR (400 MHz, CDC13): 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).

[0070] Compound 8: Refer to the synthesis of compound 3, yield 71%. 1 H-NMR (400 MHz, CDC13): 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).

[0071] Compound 9: Refer to the synthesis of compound 4, yield 43%. 1H-NMR (400 MHz, CDC13): δ = 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).

[0072] Compound 10: Compound 9 (0.3 g, 0.7 mmol) was dissolved in 10 ml of DCM, 2.5 ml of trifluoroacetic acid was added, and stirred at room temperature for 1 h. After the reaction was completed, the solvent was removed under reduced pressure, and DCM was added several times to remove the trifluoroacetic acid, to obtain compound 10 (0.13 g, yield 96%). 1 H-NMR (400 MHz, CDC13): δ = 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).

[0073] Substrate 2: Reference the synthesis of Substrate 1, yield 59%. 1 H-NMR (400 MHz, D20): 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).

[0074] Example 3

[0075] Synthesis of Substrate 3:

[0076]

[0077] Compound 12: Reference the synthesis of Compound 2, yield 87%. 1 H-NMR (400 MHz, CDC13): 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).

[0078] Compound 13: Compound 12 (1.0 g, 4.2 mmol), potassium phosphate (1.8 g, 8.4 mmol) were weighed into a pressure bottle, then 7 ml of dimethylamine aqueous solution and 3 ml of dimethylaminoethanol were added, as well as a catalytic amount of copper powder and cuprous iodide. Heat to 80°C, stir overnight. The next day, cool to room temperature, add water, extract with ethyl acetate 3 times, combine the organic phase, dry with anhydrous sodium sulfate, remove the solvent under reduced pressure, and separate by column chromatography to obtain compound 13 (0.53 g, yield 63%). 1H-NMR (400 MHz, CDC13): 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).

[0079] Compound 14: Refer to the synthesis of compound 3, yield 69%. 1 H-NMR (400 MHz, CDC13): 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).

[0080] Compound 15: Refer to the synthesis of compound 4, yield 42%. 1 H-NMR (400 MHz, CDC13): 1 H NMR (400 MHz, CDC13): δ = 7.62 (d, J = 8.7, 1H), 6.83 - 6.74 (m, 2H), 3.08 (s, 6H), 1.44 (s, 6H).

[0081] Substrate 3: Refer to the synthesis of substrate 1, yield 59%. 1 H-NMR (400 MHz, CDC13): 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).

[0082] Example 4

[0083] Synthesis of substrates 4, 5:

[0084]

[0085] Compound 17: Refer to the literature disclosed method S. C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 56%.

[0086] Compound 18: Compound 17 (3.0 g, 13 mmol) was dissolved in 250 ml of concentrated hydrochloric acid in an ice bath; sodium nitrite (1.2 g, 17 mmol) was dissolved in 12 ml of water and added to the reaction system, then SnCl2H2O (7.6 g, 34 mmol) was dissolved in 9 ml of concentrated hydrochloric acid and added to the reaction system, and the reaction was stirred at room temperature for 1 h to obtain compound 18 (2.4 g, yield 76%). 1 H NMR (400 MHz, 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).

[0087] Compound 19: Refer to the synthesis of compound 2, yield 79%. 1 H-NMR (400 MHz, CDCl3): 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).

[0088] Compound 20: Refer to the synthesis of compound 3, yield 69%. 1 H-NMR (400 MHz, CDCl3): 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).

[0089] Compound 21: Refer to the synthesis of compound 4, yield 42%. 1 H-NMR (400 MHz, CDCl3): 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).

[0090] Compound 22: Refer to the literature method (S. C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282), yield 86%. 1 H-NMR (400 MHz, CDCl3): 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).

[0091] Substrate 4: Refer to the synthesis of substrate 1, yield 59%. 1 H-NMR (400 MHz, D20): δ = 7.32 (s, 1 H), 6.75 (s, 1 H), 5.18 (dd, J = 9.7, 7.9, 1 H), 3.53 (dd, J = 13.9, 7.0, 1 H), 3.41 - 3.31 (m, 3 H), 2.91 (t, J = 8.3, 2 H), 1.34 (s, 3 H), 1.26 (s, 3 H).

[0092] Compound 23: Refer to the literature published method S.C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 69%. 1 H-NMR (400 MHz, CDC13): δ = 7.66 (d, J = 8.6, 1 H), 7.20 (s, 1 H), 3.60 (t, J = 7.6, 2 H), 3.12 (t, J = 8.2, 2 H), 2.75 (s, 3 H), 1.44 (s, 6 H).

[0093] Substrate 5: Refer to the synthesis of substrate 1, yield 61%. 1 H-NMR (400 MHz, CDC13): δ = 7.32 (s, 1 H), 6.75 (s, 1 H), 5.20 (dd, J = 9.7, 7.9, 1 H), 3.53 (dd, J = 13.9, 7.0, 1 H), 3.42 - 3.32 (m, 3 H), 2.91 (t, J = 8.3, 2 H), 2.75 (s, 3 H), 1.34 (s, 3 H), 1.26 (s, 3 H).

[0094] Example 5

[0095] Synthesis of substrates 6, 7:

[0096]

[0097] Compound 25: Refer to the literature published method S.C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 56%.

[0098] Compound 26: Refer to the synthesis of compound 18, yield 70%. 1 H-NMR (400 MHz, CDC13): δ = 7.76 (d, J = 8.7, 1 H), 6.53 (s, 1 H), 6.40 (d, J = 8.6, 1 H), 4.09 (t, J = 7.9, 2 H), 2.90 (t, J = 8.0, 2 H), 1.60 (m, 2 H).

[0099] Compound 27: Reference to the synthesis of compound 2, yield 76%. 1 H-NMR (400 MHz, CDC13): 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).

[0100] Compound 28: Reference to the synthesis of compound 3, yield 69%. 1 H-NMR (400 MHz, CDC13): 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).

[0101] Compound 29: Reference to the synthesis of compound 4, yield 41%. 1 H-NMR (400 MHz, CDC13): 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).

[0102] Compound 30: Reference to the published method S.C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 86%. 1 H-NMR (400 MHz, CDC13): 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).

[0103] Substrate 6: Reference to the synthesis of substrate 1, yield 60%. 1 H-NMR (400 MHz, D20): 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).

[0104] Compound 31 : reference literature disclosed method S.C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 69%. 1 H-NMR (400 MHz, CDC13): 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).

[0105] Substrate 7: reference synthesis of substrate 1, yield 59%. 1 H-NMR (400 MHz, D20): 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).

[0106] Example 6

[0107] Synthesis of substrates 8, 9:

[0108]

[0109] Compound 33: reference literature disclosed method S.C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 53%.

[0110] Compound 34: reference synthesis of compound 18, yield 69%. 1 H-NMR (400 MHz, CDC13): 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).

[0111] Compound 35: reference synthesis of compound 2, yield 76%. 1 H-NMR (400 MHz, CDC13): 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).

[0112] Compound 36: reference synthesis of compound 3, yield 72%.1 H-NMR (400 MHz, CDC13): 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).

[0113] Compound 37: Refer to the synthesis of compound 4, yield 43%. 1 H-NMR (400 MHz, CDC13): 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).

[0114] Compound 38: Refer to the literature published method S.C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 87%. 1 H-NMR (400 MHz, CDC13): 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).

[0115] Substrate 8: Refer to the synthesis of substrate 1, yield 59%. 1 H-NMR (400 MHz, D20): 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).

[0116] Compound 39: Refer to the literature published method S.C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 72%. 1 H-NMR (400 MHz, CDC13): 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).

[0117] Substrate 9: Refer to the synthesis of substrate 1, yield 57%. 1H-NMR (400 MHz, D20): δ = 7.30 (s, 1 H), 6.77 (s, 1 H), 5.23 (dd, J = 9.8, 8.0, 1 H), 4.32 (t, J = 4.8, 2 H), 3.56 (dd, J = 13.9, 7.0, 1 H), 3.46 (m, 3 H), 2.94 (s, 3 H), 1.36 (s, 3 H), 1.29 (s, 3 H).

[0118] Example 7

[0119] Synthesis of substrates 10, 11:

[0120]

[0121] Compound 41 : Reference literature disclosed method S.C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 52%.

[0122] Compound 42: Reference compound 18 synthesis, yield 72%. 1 H-NMR (400 MHz, CDC13): δ = 7.68 (d, J = 8.0, 1 H), 7.28 (d, J = 8.2, 1 H), 6.40 (d, J = 3.4, 1 H), 3.86 (t, J = 4.8, 2 H), 3.02 (t, J = 4.8, 2 H).

[0123] Compound 43: Reference compound 2 synthesis, yield 73%. 1 H-NMR (400 MHz, CDC13): δ = 7.66 (s, 1 H), 7.18 (s, 1 H), 3.80 (t, J = 5.0, 2 H), 3.02 (t, J = 5.0, 2 H), 2.30 (s, 3 H), 1.44 (s, 6 H).

[0124] Compound 44: Reference compound 3 synthesis, yield 70%. 1 H-NMR (400 MHz, CDC13): δ = 9.69 (s, 1 H), 7.78 (s, 1 H), 7.18 (s, 1 H), 3.82 (t, J = 4.8, 2 H), 3.03 (t, J = 4.8, 2 H), 1.40 (s, 6 H).

[0125] Compound 45: Reference compound 4 synthesis, yield 42%. 1 H-NMR (400 MHz, CDC13): δ = 7.79 (s, 1 H), 7.18 (s, 1 H), 3.80 (t, J = 7.9, 2 H), 3.06 (t, J = 8.1, 2 H), 1.44 (s, 6 H).

[0126] Compound 46: Reference literature disclosed method S. C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 86%. 1 H-NMR (400 MHz, CDC13): 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).

[0127] Substrate 10: Reference synthesis of substrate 1, yield 57%. 1 H-NMR (400 MHz, D20): 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).

[0128] Compound 47: Reference literature disclosed method S. C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 73%. 1 H-NMR (400 MHz, CDC13): 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).

[0129] Substrate 11: Reference synthesis of substrate 1, yield 59%. 1 H-NMR (400 MHz, D20): 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).

[0130] Example 8

[0131] Synthesis of substrates 12, 13:

[0132]

[0133] Compound 49: Reference literature disclosed method S. C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 56%.

[0134] Compound 50: Synthesized by reference to compound 18, yield 72%. 1 H-NMR (400 MHz, CDC13): 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).

[0135] Compound 51: Synthesized by reference to compound 2, yield 73%. 1 H-NMR (400 MHz, CDC13): 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).

[0136] Compound 52: Synthesized by reference to compound 3, yield 70%. 1 H-NMR (400 MHz, CDC13): 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).

[0137] Compound 53: Synthesized by reference to compound 4, yield 41%. 1 H-NMR (400 MHz, CDC13): 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).

[0138] Compound 54: Synthesized by reference to literature method S.C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 86%. 1 H-NMR (400 MHz, CDC13): 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).

[0139] Substrate 12: Synthesized by reference to substrate 1, yield 61%. 1 H-NMR (400 MHz, D20): 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).

[0140] Compound 55: Reference literature disclosed method S. C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 73%. 1 H-NMR (400 MHz, CDC13): 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).

[0141] Substrate 13: Reference the synthesis of substrate 1, yield 56%. 1 H-NMR (400 MHz, D20): 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).

[0142] Example 9

[0143] Synthesis of substrate 14:

[0144]

[0145] Compound 57: Reference literature disclosed method S. C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 51%.

[0146] Compound 58: Reference the synthesis of compound 18, yield 72%. 1 H-NMR (400 MHz, CDC13): δ = 7.30 (s, 2H), 3.30 (t, J = 6.9, 4H), 2.79 (t, J = 6.9, 4H), 1.99 (m, 4H).

[0147] Compound 59: Reference the synthesis of compound 2, yield 73%. 1 H-NMR (400 MHz, CDC13): δ = 7.06 (s, 1H), 3.25 (t, J = 3.6, 4H), 2.86 (t, J = 6.5 Hz, 2H), 2.80 (t, J = 6.5 Hz, 2H), 2.30 (s, 3H), 1.96 (m, 4H), 1.44 (s, 6H).

[0148] Compound 60: Reference the synthesis of compound 3, yield 70%. 1H-NMR (400 MHz, CDC13): δ = 9.69 (s, 1H), 7.09 (s, 1H), 3.26 (t, J = 6.8, 4H), 2.89 (t, J = 6.6 Hz, 2H), 2.82 (t, J = 6.5 Hz, 2H), 1.96 (m, 4H), 1.39 (s, 6H).

[0149] Compound 61 : Reference to the synthesis of compound 4, yield 41 %. 1 H-NMR (400 MHz, CDC13): δ = 9.69 (s, 1H), 7.09 (s, 1H), 3.26 (t, J = 6.8, 4H), 2.89 (t, J = 6.6 Hz, 2H), 2.82 (t, J = 6.5 Hz, 2H), 1.96 (m, 4H), 1.39 (s, 6H).

[0150] Substrate 14: Reference to the synthesis of substrate 1, yield 59%. 1 H-NMR (400 MHz, CDC13): δ = 9.69 (s, 1H), 7.09 (s, 1H), 3.26 (t, J = 6.8, 4H), 2.89 (t, J = 6.6 Hz, 2H), 2.82 (t, J = 6.5 Hz, 2H), 1.96 (m, 4H), 1.39 (s, 6H).

[0151] Example 10

[0152] Synthesis of substrates 15, 16:

[0153]

[0154] Compound 62: Reference to the published method S.C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 53%.

[0155] Compound 63: Reference to the synthesis of compound 2, yield 73%. 1 H-NMR (400 MHz, CDC13): δ = 9.69 (s, 1H), 7.09 (s, 1H), 3.26 (t, J = 6.8, 4H), 2.89 (t, J = 6.6 Hz, 2H), 2.82 (t, J = 6.5 Hz, 2H), 1.96 (m, 4H), 1.39 (s, 6H).

[0156] Compound 64: Reference to the synthesis of compound 3, yield 70%. 1H-NMR (400 MHz, CDC13): 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).

[0157] Compound 65: Refer to the synthesis of compound 4, yield 43%. 1 H-NMR (400 MHz, CDC13): 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).

[0158] Compound 66: Refer to the literature published method S.C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 86%. 1 H-NMR (400 MHz, CDC13): 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).

[0159] Substrate 15: Refer to the synthesis of substrate 1, yield 55%. 1 H-NMR (400 MHz, D20): 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).

[0160] Compound 67: Refer to the literature published method S.C. Miller, J. Am. Chem. Soc. 2014, 136, 13277-13282, yield 73%. 1 H-NMR (400 MHz, CDC13): 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).

[0161] Substrate 16: Refer to the synthesis of substrate 1, yield 56%. 1H-NMR (400 MHz, D20): 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).

[0162] Example 11

[0163] Synthesis of substrate 17:

[0164]

[0165] Compound 69: Synthesized according to compound 2 with a yield of 86%. 1 H-NMR (400 MHz, CDC13): 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).

[0166] Compound 70: Synthesized according to compound 3 with a yield of 72%. 1 H-NMR (400 MHz, CDC13): 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).

[0167] Compound 71: Synthesized according to compound 4 with a yield of 42%. 1 H-NMR (400 MHz, CDC13): 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).

[0168] Compound 72: Synthesized according to compound 5 with a yield of 93%. 1 H-NMR (400 MHz, CDC13): 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).

[0169] Substrate 17: Reference to the synthesis of substrate 1, yield 60%. 1 H-NMR (400 MHz, D20): δ = 7.86 (d, J = 9.2, 1 H), 7.56 (m, 2 H), 7.22 (d, J = 8.3, 1 H), 7.06 (s, 1 H), 5.20 (t, J = 8.9, 1 H), 3.60 (t, J = 10.0, 1 H), 3.37 (t, J = 10.6, 1 H), 1.46 (s, 3 H), 1.26 (s, 3 H).

[0170] Example 12

[0171] Synthesis of substrate 18:

[0172]

[0173] Compound 74: Reference to the synthesis of compound 2, yield 89%. 1 H-NMR (400 MHz, CDC13): δ = 8.19 (d, J = 2.0, 1 H), 8.08 (d, J = 9.0, 1 H), 7.89 (d, J = 8.6, 1 H), 7.76 (d, J = 8.6, 1 H), 7.60 (dd, J = 9.2, 2.2, 1 H), 2.30 (s, 3 H), 1.51 (s, 9 H), 1.44 (s, 6 H).

[0174] Compound 75: Reference to the synthesis of compound 3, yield 69%. 1 H-NMR (400 MHz, CDC13): δ = 9.96 (s, 1 H), 8.09 (d, J = 2.3, 1 H), 7.93 (d, J = 9.3, 1 H), 7.79 (d, J = 8.6, 1 H), 7.66 (d, J = 8.6, 1 H), 7.30 (d, J = 9.2, 1 H), 1.56 (s, 6 H), 1.50 (s, 9 H).

[0175] Compound 76: Reference to the synthesis of compound 4, yield 43%. 1 H-NMR (400 MHz, CDC13): δ = 7.89 (d, J = 9.0, 1 H), 7.76 (m, 2 H), 7.30 (d, J = 9.2, 1 H), 7.06 (s, 1 H), 1.59 (s, 6 H), 1.50 (s, 9 H).

[0176] Compound 77: Reference to the synthesis of compound 10, yield 96%. 1H-NMR (400 MHz, CDC13): δ = 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).

[0177] Substrate 18: Reference synthesis of Substrate 1, yield 60%. 1 H-NMR (400 MHz, D20): 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).

[0178] Example 13

[0179] Synthesis of Substrate 19:

[0180]

[0181] Compound 79: Reference synthesis of Compound 2, yield 83%. 1 H-NMR (400 MHz, CDC13): 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).

[0182] Compound 80: Reference synthesis of Compound 13, yield 43%. 1 H-NMR (400 MHz, CDC13): 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).

[0183] Compound 81: Reference synthesis of Compound 3, yield 66%. 1 H-NMR (400 MHz, CDC13): 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).

[0184] Compound 82: Refer to the synthesis of compound 4, yield 49%. 1 H-NMR (400 MHz, CDC13): 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).

[0185] Substrate 19: Refer to the synthesis of substrate 1, yield 56%. 1 H-NMR (400 MHz, D20): 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).

[0186] Synthesis of substrate 20:

[0187]

[0188] Compound 83: NaH (0.13 g, 3.2 mmol) was weighed into a dry three-necked flask under argon protection, which was dissolved in 20 ml THF. Phosphorous acid triethyl ester (0.73 ml, 3.6 mmol) was dissolved in 5 ml THF under ice bath conditions, and added dropwise into the reaction flask, stirred at 0°C for 30 min, then compound 14 (0.4 g, 1.8 mmol) was dissolved in 10 ml THF, and added dropwise into the reaction flask. The reaction was gradually restored to room temperature, and after the reaction was completed, the reaction was quenched with water, extracted with EA (100 ml x 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 (400 MHz, CDC13): 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).

[0189] Compound 84: Compound 83 (1.0 g, 3.5 mmol) was dissolved in 30 ml of isopropyl alcohol, and 10 ml of NaOH solution (1M) was added, and stirred at room temperature for 1 h. After the reaction was completed, the pH was adjusted to 7, the solvent was removed under reduced pressure, extracted with DCM (100 ml x 3), the organic phases were combined and dried over anhydrous sodium sulfate, the solvent was removed under reduced pressure, and compound 84 (0.87 g, yield 96%) was obtained by column chromatography.1 H NMR (400 MHz, CDC13): δ = 7.56 (m, 2H), 6.63 (m, 3H), 3.02 (s, 6H), 1.39 (s, 6H).

[0190] Compound 85: Compound 84 (0.15 g, 0.58 mmol) and S-(trityl)-D-cysteine methyl ester (0.24 g, 0.64 mmol) were dissolved in 20 ml DMF, EDC (0.36 g, 1.9 mmol) and DMAP (0.18 g, 1.5 mmol) were added respectively, and the reaction was carried out under argon protection for 3 h. After the reaction was completed, 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, CDC13): δ = 7.56 (m, 2H), 6.63 (m, 3H), 3.02 (s, 6H), 1.39 (s, 6H).

[0191] Compound 86: Compound 85 (0.13 g, 0.21 mmol) was dissolved in 10 ml DCM, Ph3PO (0.12 g, 0.42 mmol) and Tf20 (360 ul, 2.1 mmol) were added under ice bath condition, and the reaction was carried out for 1 h. After the reaction was completed, the organic solvent was removed under reduced pressure, and compound 85 (46 mg, 61%) was obtained by column chromatography. 1 H-NMR (400 MHz, CDC13): 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).

[0192] Substrate 20: 50 mg of compound 86 was dissolved in 2 ml of ethanol, 6 ml of 10 mM NH4HCO3 solution was added, 25 mg of esterase was added, and the reaction was carried out at 37°C for 19 h. After the reaction was completed, the solvent was removed under reduced pressure, and the substrate (46 mg, 96%) was obtained by column chromatography. 1H-NMR (400MHz, D2O): 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).

[0193] Example 15

[0194] Synthesis of substrate 21:

[0195]

[0196] Substrate 21: Refer to the synthetic route of substrate 20, except that the starting material triethyl phosphonoacetate is replaced with trans-ethyl-4-(diethylphosphono)crotonate, and the other procedures and starting materials are the same. 1 H-NMR (400MHz, D2O): 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).

[0197] Example 16

[0198] Expression and purification of different luciferase proteins

[0199] To determine the efficacy of the synthesized novel luciferase substrates, the amino acid sequences encoding different luciferases (such as...) were compared. Figure 1 As shown, the gene sequences of Fluc, x5g, x5r, Eluc, and CBR2 were cloned into the pCDFDuet1 vector via enzyme digestion and ligation. The selected restriction enzyme sites were BamHI and Acc65I. The plasmids of the constructed different luciferase expression vectors, after being correctly sequenced, were transformed into BL21(DE3) for protein expression. Colonies obtained from the transformation were picked and cultured overnight in test tubes. 1 mL of bacterial culture was inoculated into 100 mL of LB medium at a 1% ratio and cultured at 37°C and 220 rpm on a shaker. When the bacterial culture OD... 600When the value reaches 0.6, IPTG (final concentration of 1 mM) is added to induce protein expression, and the induction is carried out at 18°C for 24-48 h. After the expression is completed, the bacterial cells are collected by centrifugation, resuspended in an appropriate amount of His Buffer A, and the bacterial cells are lysed by ultrasonic waves. The protein supernatant is collected by centrifugation at 12,000 rpm at 4°C for 20 min, and then added to an affinity column in batches. A 5-fold column volume of a washing buffer containing 50 mM imidazole is added to remove most of the impurities. Then, the target protein is eluted with an elution buffer containing 300 mM imidazole. The target protein is collected according to the Bradford colorimetric process, labeled, and placed on ice for standby. The concentration of the purified protein is determined by the BCA method. After the concentration of the purified protein is determined, the size and purity of the purified protein are verified by SDS-PAGE. Figure 2 For the electrophoretogram of the purified different luciferase proteins, the electrophoresis results show that there is a protein slightly smaller than 66.2 kDa, which is consistent with the size of 62 kDa of luciferase, which is consistent with the expectation. On the other hand, Figure 2 The different luciferases show ideal purity, and there is basically no impurity.

[0200] Example 17

[0201] Reaction of substrate 1, substrate 3, substrate 5, and substrate 19 with different luciferases

[0202] The different luciferase proteins are diluted to 0.2 mM. In a black 96-well plate, different luciferases (0.2 mM, 40 mL) are added, followed by ATP (8 mM, 10 mL) and MgSO4(32 mM, 10 mL). Finally, different substrates with a final concentration of 100 μM are added immediately for testing by using a BioTek Neo2 enzyme marker. The relative luminescence intensity is determined. The test results are shown in Table 1. Figure 3 The test results show that substrate 1, substrate 3, substrate 5, and substrate 19 react with firefly luciferase (Fluc) to obtain a higher luminescence intensity.

[0203] Example 18

[0204] Changes of substrate 1, substrate 3, substrate 5, and substrate 19 with Fluc over time within 30 min

[0205] The purified Fluc protein is diluted to 0.5 mM. In a white 96-well plate, Fluc (0.5 mM, 40 mL) is added, followed by ATP (8 mM, 10 mL) and MgSO4(32 mM, 10 mL). Finally, different substrates with a final concentration of 100 μM are added immediately for luminescence intensity testing by using a BioTek Neo2 enzyme marker. The testing is performed once every 20 seconds, and the testing is continuously performed for 30 min. The test results are shown in Table 2. Figure 4As shown: Under the same test conditions, the order of luminescence intensity from strongest to weakest is substrate 3 > substrate 5 > substrate 1 > substrate 19 (without considering the difference in quantum yield of different bands of PMT detector); after substrate 3 is added to the reaction system, the luminescence intensity decreases slightly in the first minute, and then remains basically stable; the reaction process of substrate 5 is basically the same as that of substrate 3; the luminescence intensity of substrate 1 reacting with Fluc initially decreases slightly, and then remains basically stable; the luminescence intensity of substrate 19 reacting with Fluc remains basically stable.

[0206] Example 19

[0207] The emission spectra of substrates 1, 3, 5, 7, 16, 19, and 20 reacting with Fluc were as follows.

[0208] The purified Fluc protein was diluted to 2 mM, and Fluc (2 mM, 40 mL) was added to a white 96-well plate. Then ATP (8 mM, 10 mL) and MgSO4 (32 mM, 10 mL) were added. Finally, different substrates with a final concentration of 100 μM were added, and emission spectroscopy was performed. The test was conducted using a BioTek Neo2 microplate reader.

[0209] Test results are as follows Figure 5 As shown, the obtained emission spectra indicate that the maximum emission peak value of the reaction between substrate 1 and Fluc corresponds to a wavelength of 650 nm; the maximum emission peak value of the reaction between substrate 3 and Fluc corresponds to a wavelength of 660 nm; the maximum emission peak value of the reaction between substrate 5 and Fluc corresponds to a wavelength of 655 nm; the maximum emission peak value of the reaction between substrate 7 and Fluc corresponds to a wavelength of 670 nm; the maximum emission peak value of the reaction between substrate 16 and Fluc corresponds to a wavelength of 680 nm; the maximum emission peak value of the reaction between substrate 19 and Fluc corresponds to a wavelength of 750 nm; and the maximum emission peak value of the reaction between substrate 20 and Fluc corresponds to a wavelength of 745 nm.

[0210] Under the same conditions described above, fluorescein (Fluorescence) was tested. The emission peaks of the reaction between fluorescein and x5g and x5r (purchased from Bid Pharmaceutical) were measured. The test results showed that the maximum emission peak of fluorescein reacting with x5g corresponds to a wavelength of 560 nm, and the maximum emission peak of fluorescein reacting with x5r corresponds to a wavelength of 610 nm, which is consistent with the results reported in the literature (Branchini, Ablamsky et al. 2007).

[0211] In summary, the novel luciferase substrates of this invention have wavelengths reaching the far-infrared or near-infrared bands, which have strong tissue penetration capabilities and are of great application value in in vivo imaging.

[0212] Example 20

[0213] Microscopic imaging assays of substrates 1, 3, 5 and 19 in HeLa cell lines

[0214] The Fluc gene sequence was cloned into the pEGFP-C1 vector using restriction enzyme digestion at NheI and NotI sites. The constructed plasmid pEGFP-Fluc, after DNA sequencing confirmation, was transformed into DH5α. Plasmid extraction was performed using an endotoxin-free plasmid extraction kit (Tiangen Biotech Co., Ltd.). The concentration of the obtained plasmid was determined using a microplate reader (BioTek Neo2), and the plasmids were stored at -20°C for later use. HeLa cell lines (purchased from the Chinese Academy of Sciences Type Culture Collection) were passaged into 96-well glass-bottom plates and cultured in a CO2 incubator using high-glucose medium (DMEM) containing 10% fetal bovine serum (FBS), streptomycin, and penicillin. When the cells reached 50-60% confluence, plasmid transfection was performed using HieffTrans. TM (Purchased from Yensen) The procedure was performed according to standard operating procedures. Microscopic imaging was performed 48 hours after transfection: the microscope used was a Nikon Ti2, the lens was a Nikon 100x oil immersion lens (NA=1.40), and the camera was a Prime 95Bs CMOS camera (TELEDYNE PHOTOMETRICS). At the start of the test, different substrates were dissolved to 200 μM in HBSS buffer, and the DMEM medium was replaced with 60 μL of HBSS buffer (containing 10 mM glucose). After the test started, a suitable field of view was found and images were taken using bright field imaging. Then, 60 μL of the prepared substrate solution was added, and the luminescence signal was collected immediately. The exposure time for samples corresponding to substrates 3 and 5 was 1 min, the exposure time for samples corresponding to substrate 1 was 2 min, and the exposure time for samples corresponding to substrate 19 was 5 min. The results were obtained at this point. Figure 6 As shown in the image, it is important to note that the collection of the luminescent signal must be carried out in a strictly dark environment.

[0215] Figure 6 The results showed that the HeLa cell line exhibited high levels of Flux expression 48 hours after transfection with the pEGFP-Fluc plasmid. The luminescence signal intensities of substrates 1, 3, 5, and 19 in live cells (ignoring the low quantum yield of the camera in this wavelength band) were consistent with the protein level test results. In summary, the different substrates of this invention all possess good cell permeability and can be used for testing in live cells.

[0216] Example 21

[0217] Results of testing different concentrations of substrate 1, substrate 3, substrate 5 and substrate 19 in HEK293T cell line

[0218] The cell culture method in Reference Example 19 was used to test different concentrations of substrates in HEK293T cell line. HEK293T cells were passaged into 24-well plates, and when the cells reached 50-60% confluence, plasmid transfection was performed. The test was performed 36 h after transfection. At the beginning of the test, the culture medium was discarded, the cells were washed once with PBS, and then cell lysis solution (70 μL of lysis solution per cm2of cells) was added to lyse the cells on ice for 5 min (during which time the cells were gently shaken). The supernatant was collected by centrifugation at 12000 rpm for 5 min at 4°C, and then tested. 10 μL of the lysis supernatant and 15 μL of the activity assay buffer were mixed in each well of a 384-well white luminescence plate. The luminescence intensity was tested immediately after 15 μL of substrate solution was added to each well. The test results are shown in Table 2. Figure 7 .

[0219] The test results show that the luminescence signal intensity of substrate 1, substrate 3, substrate 5, and substrate 19 in HEK293T cells is consistent with the test results of the protein level. The test results of different concentrations of substrates show that the luminescence signal can also be detected when the substrate concentration is less than 1 μM, that is, the different substrates of the present application have good application prospects in cells.

[0220] The components of the different solutions involved in this example are as follows:

[0221] Activity assay buffer: 15 mM potassium phosphate (pH 7.8), 25 mM dipeptide, 15 mM MgSO4, 4 mM EGTA, 2 mM ATP;

[0222] Cell lysis solution: 25 mM dipeptide (pH 7.8), 15 mM MgSO4, 4 mM EGTA, 1% Triton X-100;

[0223] Substrate solution: 25 mM dipeptide (pH 7.8), 15 mM MgSO4, 4 mM EGTA, 0.1 mM substrate X.

[0224] Example 22

[0225] Test results of substrate X in mice

[0226] The endotoxin-free plasmid pEGFP-Fluc obtained in Example 20 was delivered to the legs of ICR mice by muscle electroporation, 12 μg of plasmid was electroporated to the outer side of each leg, and the electroporation process was performed using a living gene introduction instrument (Shanghai Teresa Health Technology Co., Ltd.) according to the standard operation procedure. After the mice woke up from the anesthesia, they were sent back to the animal house for feeding. Two days later, the IVIS Spectrum CT live imaging system was used for testing. The testing process was as follows: the mice were anesthetized with sodium pentobarbital, and 100 μL of 10 mM different substrates (dissolved in PBS, filtered through a 0.22 μm filter) was injected intraperitoneally after the mice were anesthetized. The live imaging instrument was used for testing after 5 min. The test results are shown in Figure 8 Figure B: The luminescence intensity of different samples after injection of substrate 3 or 5 was counted. The results showed that the mice injected with substrate 3 detected strong luminescence signals in both legs, and the mice injected with substrate 5 also detected luminescence signals. The luminescence intensity of the mice injected with substrate 3 was higher than that of the mice injected with substrate 5.

[0227] In summary, the different substrates introduced in the present application have good tissue penetration, and have good application prospects in live small animal imaging.

Claims

1. A luciferase substrate having the structure shown in formula (I): In its formula (I), R is: hydroxyl, or -NR a R b , R a , R b are each independently selected from the group consisting of hydrogen, or alkyl, R1, R2 are -H; Alternatively, R and R1 together form a structure of the following formula (I-2-1) to (I-2-6): Alternatively, R and R2 together form the structure of the following formulae (I-2-7) ~ (I-2-8): wherein, Each R′ is independently selected from hydrogen or alkyl; R″ is selected from hydrogen or alkyl. n can be 0, 1, 2, or 3; in, said "alkyl" is a C1-C 10 straight-chain alkyl or C3-C 10 branched-chain alkyl.

2. The luciferase substrate according to claim 1, characterized in that, The "alkyl" is a C1-C7 straight-chain alkyl or a C3-C7 branched alkyl.

3. The luciferase substrate according to claim 1, characterized in that, The "alkyl" is a C1-C5 straight-chain alkyl or a C3-C5 branched alkyl.

4. The luciferase substrate of claim 1, wherein, The "alkyl" is 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-ethylbutyl, 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.

5. The luciferase substrate of claim 1, wherein, The luciferase substrate is selected from compounds of the following formula:

6. A method of preparing a luciferase substrate according to any one of claims 1 to 5, characterised in that: The steps include reacting a compound of formula (II) with a compound of formula (III) to obtain the luciferase substrate shown in formula (I). Wherein, R, R1, and R2 are defined as described in any one of claims 1-5, and n = 0; Alternatively, the product obtained by reacting compound (IV) with compound (V) is subjected to ester bond hydrolysis and condensation reaction with compound (VI) to remove the thiol protecting group, thereby obtaining compound (VII). Finally, the luciferase substrate shown in formula (I) is obtained by enzymatic hydrolysis of the ester bond. Wherein, R, R1, and R2 are defined as described in any one of claims 1-5, n = 1, 2, 3, and m = 0, 1, 2.

7. Use of the luciferase substrate according to any one of claims 1-5 in the preparation of optical detection products.

8. Use according to claim 7, characterized in that, The optical detection product is an optical probe or a luminescence detection kit.

9. A bioluminescent probe, characterized by The bioluminescent probe comprises the luciferase substrate according to any one of claims 1-5.

10. A luminescent detection kit, characterized by The luminescent detection kit comprises the luciferase substrate of any one of claims 1-5 or the bioluminescent probe of claim 9.

11. Use of the luciferase substrate according to any one of claims 1-5, the bioluminescent probe according to claim 9, or the luminescent detection kit according to claim 10 in the preparation of environmental detection reagents.

12. Use of the luciferase substrate according to any one of claims 1-5, the bioluminescent probe according to claim 9, or the luminescent detection kit according to claim 10 in the preparation of analytical chemical detection reagents.

13. Use of the luciferase substrate of any one of claims 1-5, the bioluminescent probe of claim 9, or the luminescent detection kit of claim 10 in the preparation of bioanalytical and detection reagents.

14. Use according to claim 13, characterized in that, The biological analysis and detection include analysis and detection at the cellular level, tissue level, organ level, and individual organism level of the organism.

15. Use according to claim 13 or 14, characterized in that, The organism is a bacterium, a mammalian cell, a mouse, a rat, or a monkey.

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