Optical pure small peptide chemiluminescent probe and application thereof in identification of Mycobacterium tuberculosis

Abstract

The invention belongs to the technical field of organic synthesis and medicine, and particularly relates to an optical pure small peptide chemiluminescent probe. By changing the absolute configuration of amino acid on a peptide chain, the length of the peptide chain, the variety of the amino acid and the end-capping group of the peptide, the invention discloses the influence of the small peptide chemiluminescence probe on the recognition performance of mycobacterium tuberculosis based on chiral control and structural transformation, the chemiluminescence intensity and chiral structure-activity relationship, and the application of the small peptide chemiluminescence probe to the mycobacterium tuberculosis. The capability of distinguishing and specifically recognizing clinical sample mycobacteria is achieved. Compared with existing reports, the small peptide chemiluminescent probe disclosed by the invention creates and innovates a chiral environment of a small peptide terminal recognition fragment of the probe from a molecular level, so that a brand-new three-dimensional structure of the small peptide recognition fragment is constructed; further, an electronic effect, a steric hindrance effect, a pi-pi stacking effect, an interaction mode between a subject and an object and the like of a recognition center are changed, and the chemiluminescence probe which is higher in luminous intensity and excellent in recognition performance compared with an existing small peptide type mycobacterium tuberculosis probe is prepared and screened.

Description

Technical Field

[0001] The invention belongs to the technical field of organic synthesis and medicine, and specifically relates to an optically pure small peptide chemiluminescent probe and application thereof in the identification of Mycobacterium tuberculosis. Background Art

[0002] Chirality plays an extremely important role in the field of medicine, and often has specific interactions with biological targets. Many targets in organisms, such as enzymes and receptors, have chiral characteristics. Different enantiomers of chiral drugs (different absolute configurations) interact with these biological targets, and only specific chiral configurations can perfectly match the targets and produce the expected pharmacological effects. For example, the antimalarial drug quinine has a specific chiral structure, and only one of its enantiomers can effectively bind to the relevant targets of malarial parasites, thereby exerting an antimalarial effect. In addition, different enantiomers of chiral drugs may have significant differences in pharmacological activity. One enantiomer may have strong therapeutic activity, while the other enantiomer may be less active or even inactive. For example: Taking the β-receptor blocker propranolol as an example, the activity of its S-enantiomer in blocking β-receptors is about 100 times that of the R-enantiomer, so S-propranolol plays a major pharmacological role in the treatment of cardiovascular diseases. Chiral drugs of different configurations have different pharmacokinetic properties. Different enantiomers of chiral drugs may have different pharmacokinetic processes such as absorption, distribution, metabolism and excretion in the body. This is because the enzymes and transporters involved in these processes are usually also chiral selective. For example, the non-steroidal anti-inflammatory drug naproxen has a higher plasma protein binding rate for its S-enantiomer and is eliminated slower in the body than the R-enantiomer, resulting in a longer duration of action in the body and a more stable drug concentration. Chiral drugs also have great differences in toxicity and side effects. One enantiomer may produce serious toxic side effects, while the other enantiomer is relatively safe. If chirality is not controlled, it may bring unnecessary risks to patients. For example, thalidomide, its R-enantiomer has the effect of inhibiting pregnancy reactions, while the S-enantiomer has a strong teratogenic effect, which can cause serious limb deformities in the fetus.

[0003] Therefore, in the process of drug development, the study and control of the chirality of chiral drugs is a crucial link. It is necessary to ensure that drugs with specific chiral configurations are obtained and to conduct strict quality control. Chirality largely determines the safety, efficacy and quality of drugs, and is crucial in the development, production and clinical application of chiral drugs.

[0004] As a class of chiral bioactive molecules with unique structure and function, small peptides have shown many advantages in the field of chiral drug research and development. Small peptides are short-chain molecules formed by connecting chiral amino acids through peptide bonds. As the basic building blocks of small peptides, the chiral configuration of amino acids has a decisive influence on the overall structure and function of small peptides. Naturally occurring amino acids are mainly in the L-configuration. However, through artificial synthesis or modification, the introduction of D -configuration amino acids, small peptides containing different chiral amino acid configurations can be constructed. The introduction of different chiral amino acid configurations will change the spatial conformation of the small peptide, thereby affecting the interaction mode and affinity of the small peptide with the biological target. For example, in some studies on antimicrobial peptides, it was found that after replacing some L-amino acids with D-amino acids, the antibacterial activity and stability of the small peptide were significantly improved. This is because the changed chiral configuration enables the small peptide to better bind to specific targets on the bacterial cell membrane, while enhancing its resistance to protease degradation.

[0005] Since small peptides composed of amino acids with different configurations have huge differences in pharmacological activity, in-depth research on small peptides based on different chiral amino acid configurations is expected to screen out small peptide drugs with higher activity and efficacy. By systematically changing the chiral configuration of amino acids in small peptides and evaluating their activity, we can accurately find the small peptide structure that binds most closely to biological targets and can produce the strongest pharmacological effects. This will provide more effective drug options for the treatment of various difficult diseases, significantly improve the treatment effect of diseases, and bring new hope to patients. At the same time, studying small peptides composed of different chiral amino acid configurations can clarify the configuration factors that cause toxic and side effects, so as to avoid or reduce configurations with potential toxic and side effects by rationally designing small peptide structures, and improve the safety of small peptide drugs. In addition, small peptides containing different chiral amino acid configurations often have different interaction patterns with biological targets, thereby revealing new drug action mechanisms, which is helpful for in-depth exploration of physiological and pathological processes in organisms and provides an opportunity for the development of drugs with new mechanisms of action.

[0006] At present, there are few studies on the design and research of small peptide-based Mycobacterium tuberculosis recognition probes, and the small peptide amino acid fragments reported in the literature all use L-configuration amino acids ( ACS Cent. Sci . 2021, 7 , 803-814). The absolute configuration of amino acids has a crucial influence on the performance of small peptide probes. However, the design of small peptide probes for Mycobacterium tuberculosis based on the absolute configuration of amino acids and the influence of the absolute configuration on the luminescence intensity and recognition ability of small peptide probes have not been reported. In addition, the influence of small peptide capping groups on the luminescence intensity and recognition ability of probes has not been reported. Summary of the invention

[0007] Based on the previous research and development of small peptide probes, the present invention designed and synthesized a series of novel small peptide probes, targeting the defects of the reported small peptide chemiluminescent probes, such as the single structure and the failure to consider the influence, contribution, and chiral structure-activity relationship of the absolute configuration of amino acids in the small peptide fragments on the reflection intensity and the recognition of Mycobacterium tuberculosis. The main strategies are as follows: (1) the absolute configuration of amino acids in the small peptide, (2) the peptide chain length, (3) the type of amino acids, and (4) the capping group of the small peptide. A series of optically pure small peptide chemiluminescent probes were synthesized, which innovatively revealed for the first time the influence of factors such as the absolute chiral configuration of amino acids in the small peptide chemiluminescent probes, the peptide chain length, the type of amino acids, and the capping group of the small peptide on the recognition performance of Mycobacterium tuberculosis, the intensity of chemiluminescence and the chiral structure-activity relationship, as well as the ability to distinguish and specifically recognize mycobacteria in clinical samples. Compared with the single configuration small peptide probe reported by predecessors, the small peptide chemiluminescent probe developed by the present invention creates and innovates the chiral environment of the probe small peptide end recognition fragment at the molecular level by changing the absolute configuration of amino acids, introducing amino acids of different absolute configurations, changing the peptide chain length, amino acid type, and the end-capping group of small peptides, thereby constructing a new three-dimensional structure of the small peptide recognition fragment, and then changing the electronic effect, steric hindrance effect, π-π stacking effect, weak interaction and pattern between host and guest of the recognition center, etc., to construct the specific recognition, chemiluminescence intensity and chiral structure-activity relationship of small peptide chemiluminescent probes for Mycobacterium tuberculosis, prepare and screen out chemiluminescent probes with high luminescence intensity and excellent recognition performance, and provide feasible solutions, experimental basis and theoretical guidance for the development of optically pure small peptide chemiluminescent probes and chiral structure-activity relationship research. This method has been successfully applied to the specific recognition of mycobacteria in clinical samples.

[0008] In order to achieve the above-mentioned object of the invention, the present invention provides the following technical solutions: An optically pure small peptide chemiluminescent probe, the structural formula of which is shown in the following formula (I), (II) or (III): ; ; ; Wherein, the symbol * represents a chiral carbon atom, whose configuration is R or S, and 1* represents chiral carbon atom No. 1; Among them, the chiral configurations from 1* to 4* can be the same or different; In formula I, Y represents or CH2SCH2Ph ​​or other alpha-substituent groups of alpha-amino acids.

[0009] In formula II, X represents an -NH2 protecting group, and the -NH2 protecting group is benzyloxycarbonyl, tert-butyloxycarbonyl or fluorenylmethoxycarbonyl; benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (BOC), fluorenylmethoxycarbonyl (Fmoc) are the preferred α-NH2 protecting groups in peptide synthesis.

[0010] In formula III, X1 represents acetyl, benzyloxycarbonyl, tert-butyloxycarbonyl or fluorenylmethoxycarbonyl; Y1 represents hydrogen or a C1-C3 saturated alkyl group.

[0011] Preferably, X represents fluorenylmethoxycarbonyl.

[0012] Preferably, in formula III, X1 represents acetyl or fluorenylmethoxycarbonyl; and Y1 represents hydrogen or methyl.

[0013] Preferably, when Y in Formula I represents When the chiral configuration of 1* to 4* is one of the following chiral configurations: SRRR, SSRR, SSSR, SRRS, SSRS, SRSR, SRSS, RSSS, RSRR, RSSR, RRRR, RSRS, RRSR, RRRS, RRSS.

[0014] More preferably, the chiral configuration of 1* to 4* in Formula I is one of the following chiral configurations: SSSR, SSRS, SRSS, SSRR, SRSR.

[0015] Preferably, when Y in formula I represents -CH2SCH2Ph, the chiral configurations of 1* to 4* are one of the following chiral configurations: SSSS, SRRR, SSRR, SSSR, SRRS, SSRS, SRSR, SRSS, RSSS, RSRR, RSSR, RRRR, RSRS, RRSR, RRRS, RRSS.

[0016] Preferably, the chiral configurations of 1* to 3* in Formula II are one of the following chiral configurations in sequence: SSS, SSR, SRR, SRS, RSS, RSR, RRR, RRS.

[0017] Compared with the prior art, the present invention has the following beneficial effects: Compared with existing reports, the present invention creates and innovates the chiral environment of the recognition fragment of the small peptide end of the probe at the molecular level, thereby constructing a new three-dimensional structure of the small peptide recognition fragment, and then changing the electronic effect, steric hindrance effect, π-π stacking effect, weak interaction and pattern between the host and the guest, etc. of the recognition center, and prepares and screens a chemiluminescent probe with higher luminescence intensity and better recognition performance than the existing reported small peptide probe (SSSS-A7). BRIEF DESCRIPTION OF THE DRAWINGS

[0018] Figure 1 A7 series tetrapeptide probes were used to study the recognition and luminescence intensity of Mycobacterium tuberculosis; Figure 2 A8 series tetrapeptide probes were used to study the recognition and luminescence intensity of Mycobacterium tuberculosis; Figure 3 The figure shows the study of the recognition and luminescence intensity of Mycobacterium tuberculosis by tripeptide Fmoc-A5-O2 series probes and tripeptide Ac-A5-O2 series probes; Figure 4 Figure 2 shows the ability to differentiate and identify low concentration clinical isolates of tuberculosis. DETAILED DESCRIPTION

[0019] The technical scheme of the present invention will be clearly and completely described below in combination with the embodiments of the present invention and the comparative examples. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0020] Unless otherwise specified, the experimental methods used in the following examples are all conventional methods; the materials and reagents used are reagents and materials that can be obtained from commercial channels unless otherwise specified.

[0021] Example 1: Synthesis of optically pure tetrapeptide probe A7 series:

[0022] (1) Compound S -A1 and R -Synthesis of A1:

[0023] General synthesis method: Under the protection of inert gas, N 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (934 mg, 3.80 mmol) was added to a tetrahydrofuran solution (20 mL) of -fluorenylmethoxycarbonyl-L (or D)-leucine (S-A0 or R-A0, 1020 mg, 2.88 mmol) and 4-aminobenzyl alcohol (400 mg, 3.24 mmol), and the mixture was reacted at room temperature and monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was dissolved in ethyl acetate (100 mL), washed with saturated brine (50 ml), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 60:40) to obtain the corresponding S -A1 and S -A1, as a white solid.

[0024]

[0025] Using a general synthetic method, N -Fluorenylmethoxycarbonyl- L -Leucine ( S -A0) Preparation S -A1, yield 95%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.01 (s, 1H), 7.89 (d, J = 7.6 Hz, 2H), 7.76 (t, J =6.3 Hz, 2H), 7.67 (d, J = 8.2 Hz, 1H), 7.60 (d, J = 8.1 Hz, 2H), 7.42 (t, J =7.6 Hz, 2H), 7.36 – 7.23 (m, 4H), 5.14 (t, J = 5.6 Hz, 1H), 4.47 (d, J = 5.5Hz, 2H), 4.31 (t, J = 6.5 Hz, 2H), 4.25 (d, J = 7.2 Hz, 2H), 1.78 – 1.59 (m,2H), 1.50 (ddd, J = 13.8, 8.8, 5.1 Hz, 1H), 0.93 (t, J = 7.1 Hz, 6H). 13 C NMR(101 MHz, DMSO) δ 171.87, 156.53, 144.37, 144.22, 141.18, 138.09, 137.92,128.09, 127.36, 125.79, 120.55, 119.51, 66.08, 63.10, 54.31, 47.18, 41.64,24.80, 23.51, 21.96.

[0026]

[0027] Using a general synthetic method, N -Fluorenylmethoxycarbonyl- D -Leucine ( R -A0) Preparation R -A1, yield 98%.

[0028] 1H NMR (400 MHz, CDCl3) δ 8.50 (s, 1H), 7.66 (d, J = 7.5 Hz, 2H), 7.46 (t, J = 7.6 Hz, 2H), 7.37 – 7.25 (m, 4H), 7.22 – 7.14 (m, 2H), 7.11 (d, J =8.0 Hz, 2H), 5.52 (d, J = 8.4 Hz, 1H), 4.50 (s, 2H), 4.31 (dd, J = 13.3, 7.7Hz, 2H), 4.12 – 4.06 (m, 1H), 1.73 – 1.48 (m, 4H), 0.87 (td, J = 7.4, 6.9,3.0 Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ 170.26, 155.87, 142.69, 142.49,140.20, 136.08, 126.56, 126.07, 126.04, 123.97, 123.95, 119.03, 118.94,114.12, 76.33, 76.01, 75.69, 66.27, 63.62, 53.32, 45.94, 40.20, 23.73, 21.91,20.93.

[0029] (2) Compounds S -A2 and R -Synthesis of A2:

[0030] General synthesis method: Under inert gas protection, the intermediate S -A1 or R -A1 (912 mg, 2.0 mmol) and sodium iodide (900 mg, 6.0 mmol) were dissolved in acetonitrile (30 mL) solution. Trimethylsilyl chloride (1.5 mL) was added at 0°C, and after reacting at 0°C for five minutes, the mixture was moved to room temperature and protected from light for 1 h. The reaction was monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was poured into 100 mL of water, extracted three times with ethyl acetate (50 mL), washed with brine (50 ml), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 90:10) to obtain the corresponding S -A2 and R -A2, as a white solid.

[0031]

[0032] Using a general synthetic method, through the intermediate S -A1 Preparation S -A2, yield 90%. 1 H NMR (400 MHz, Chloroform-d) δ 8.49 (s, 1H), 7.74 (d, J = 7.6 Hz, 2H), 7.53 (t, J = 7.2 Hz, 2H), 7.40 (d, J = 8.5 Hz, 2H), 7.36 (d, J = 7.4 Hz, 2H), 7.25 (dt, J = 7.4,4.4 Hz, 4H), 5.50 (d, J = 7.3 Hz, 1H), 4.41 (m, 5H), 4.16 (t, J = 6.9 Hz,1H), 1.57-1.63 (m 3H), 0.94 (d, 6H). 13 C NMR (101 MHz, CDCl3) δ 169.90,155.80, 142.58, 142.46, 140.23, 136.25, 134.10, 128.34, 126.75, 126.10,123.93, 119.10, 66.29, 53.35, 45.99, 39.92, 23.73, 21.88, 20.98, 4.74.

[0033]

[0034] Using a general synthetic method, through the intermediate R -A1 Preparation R -A2, yield 90%. 1 H NMR 95%. 1 H NMR (400MHz, CDCl3) δ 8.32 (s, 1H), 7.67 (d, J = 7.6 Hz, 2H), 7.46 (t, J = 6.5 Hz, 2H), 7.38 – 7.26 (m, 4H), 7.20 (d, J = 8.3 Hz, 4H), 5.34 (d, J = 8.2 Hz, 1H), 4.35 (s, 4H), 4.12 – 4.06 (m, 1H), 1.83 – 1.45 (m, 4H), 0.87 (t, J = 7.1 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 169.92, 155.82, 142.59, 142.47, 140.2, 128.35,126.76, 126.11, 126.07, 123.93, 119.11, 118.99, 76.32, 76.01, 75.69, 66.29,53.36, 45.99, 39.92, 28.67, 23.73, 21.87, 20.98, 4.74.

[0035] (3) Compounds S -A3 and R -Synthesis of A3:

[0036] General synthesis method: Under inert gas protection, 3-(4-[(adamantan-2-ylidene)(methoxy)methyl]-2-hydroxy-3-chlorophenyl) acrylate (414 mg, 1.0 mmol) and potassium carbonate (276 mg, 2.0 mmol) were dissolved in N,N-dimethylformamide (8 mL) solution and reacted at room temperature for 30 min. Add the intermediate S -A2 or R -A2 (595 mg, 1.05 mmol), stirred for 5 min, and monitored the reaction by thin layer chromatography (TLC). After the reaction was completed, the mixture was poured into 100 mL of water, extracted three times with ethyl acetate (50 mL), washed with brine (50 ml), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 90:10) to obtain the corresponding S -A3 and R -A3, as a white solid.

[0037]

[0038] Using a general synthetic method, through the intermediate S -A2 Preparation S -A3, yield 88%. 1 H NMR (400 MHz, Chloroform-d) δ 8.14 (s, 1H), 7.88 (d, J = 16.2 Hz, 1H), 7.68 (d, J = 7.5 Hz,2H), 7.49 (d, J = 7.1 Hz, 2H), 7.44 (d, J= 8.1 Hz, 2H), 7.34 (m, J = 7.37-7.28 Hz, 5H), 7.19 (d, J = 6.4 Hz, 2H), 6.99 (d, J = 8.0 Hz, 1H), 6.39 (d, J = 16.2 Hz, 1H), 5.90 (ddt, J = 16.4, 10.8, 5.7 Hz, 1H), 5.29 (dd, J = 16.4,1.6 Hz, 1H), 5.20 (d,J=10.8 Hz 1H), 4.88 (d, J = 4.5 Hz, 2H), 4.63 (dt, J =5.6, 1.5 Hz, 2H), 4.39 (d, J = 8.0 Hz, 2H), 4.23 (s, 1H), 4.13 (t, J = 6.8Hz, 1H), 3.25 (s, 3H), 3.20 (s, 2H), 1.99 (s, 1H), 1.91 – 1.65 (m, 14H), 1.28– 1.10 (m, 1H), 0.90 (d, J = 6.2 Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ 171.45,169.15, 165.23, 155.83, 152.69, 142.61, 140.26, 138.38, 137.94, 137.16,131.86, 131.35, 131.16, 130.73, 128.83, 128.59, 128.56, 126.73, 126.08,124.02, 123.91, 118.98, 118.94, 118.79, 117.26, 116.52, 66.08, 64.49, 64.25,56.21, 54.24, 51.62, 46.10, 39.31, 38.78, 38.17, 38.01, 37.58, 36.03, 31.91,28.68, 27.33, 27.19, 23.85, 21.91, 20.79.

[0039]

[0040] Using a general synthetic method, through the intermediate R -A2 Preparation R -A3, yield 85%. 1 H NMR (400 MHz, CDCl3) δ 8.12 (s, 1H), 7.88 (d, J = 16.2 Hz, 1H), 7.68 (d, J = 7.6 Hz, 2H), 7.53 – 7.41 (m, 4H), 7.39 – 7.27 (m, 5H), 7.23 – 7.16 (m, 3H), 6.99 (d, J =8.0 Hz, 1H), 6.39 (d, J = 16.1 Hz, 1H), 5.91 (ddt, J = 17.3, 10.5, 5.7 Hz,1H), 5.29 (dq, J = 17.2, 1.6 Hz, 1H), 5.24 – 5.16 (m, 2H), 4.89 (d, J = 4.6Hz, 2H), 4.63 (dt, J = 5.7, 1.4 Hz, 2H), 4.39 (t, J = 8.9 Hz, 2H), 4.14 (t, J= 6.7 Hz, 1H), 3.25 (s, 3H), 3.21 (s, 1H), 2.94 – 2.77 (m, 1H), 1.98 (d, J =7.9 Hz, 1H), 1.87 (d, J = 15.8 Hz, 5H), 1.77 – 1.64 (m, 3H), 1.61 (s, 3H), 1.38 – 1.20 (m, 2H), 1.20 – 1.16 (m, 4H), 0.89 (t, J = 6.2 Hz, 6H), 0.85 –0.73 (m, 1H). 13 C NMR (101 MHz, CDCl3) δ 169.28, 165.25, 152.65, 142.60,142.56, 140.31, 138.39, 137.99, 137.18, 131.20, 128.72, 126.77, 126.11,123.92, 119.00, 117.25, 74.68, 64.25, 56.23, 46.14, 38.02, 36.04, 31.92,28.68, 27.33, 27.18, 23.71, 21.93, 20.99.

[0041] (4) Compounds SS -A4, SR -A4, RS -A4, RR -Synthesis of A4:

[0042] General synthesis method: Under inert gas protection, piperidine (0.494 mL, 5.0 mmol) was added to the solution of the intermediate S -A3 or R -A3 (854 mg, 1.0 mmol) in DMF (5 mL). The solution was stirred at room temperature for 30 minutes and the reaction was monitored by thin layer chromatography (TLC). After observing that the fluorenylmethyloxycarbonyl group was completely removed, the mixture was dissolved in ethyl acetate and washed twice with 0.1M hydrochloric acid (50 ml) and brine (50 ml). Drying was performed over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Then, under the protection of inert gas, the deprotected A3, N-fluorenylmethoxycarbonyl-N'-allyloxycarbonyl-L (or D)-lysine (Fmoc-lys(alloc)-OH) (452.5 mg 1.0 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (546 mg, 1.44 mmol) were dissolved in DMF (25.0 mL), N,N-diisopropylethylamine (DIPEA) (0.348 mL, 2.0 mmol) was added, stirred at room temperature for 1 h, and monitored by thin layer chromatography (TLC). After the reaction was completed, the mixture was diluted with ethyl acetate (100 ml), washed with 0.1 M hydrochloric acid (50 ml) and brine (50 ml). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75:25). The corresponding SS -A4, SR -A4, RS -A4, RR -A4, as a white solid.

[0043]

[0044] Using a general synthetic method, through the intermediate S -A3 and N -Fluorenylmethoxycarbonyl- N '-allyloxycarbonyl- L -Lysine ( L -Fmoc-lys(alloc)-OH) SS -A4, yield 70%. 1H NMR (400 MHz, Chloroform-d) δ8.98 (s, 1H), 7.86 (d, J = 16.2 Hz, 1H), 7.60 (d, J = 7.5 Hz, 2H), 7.52 (d, J = 7.9 Hz, 2H), 7.46 – 7.38 (m, 2H), 7.33 (d, J = 8.0 Hz, 1H), 7.26 (d, J =7.8 Hz, 2H), 7.22 (d, J = 7.4 Hz, 2H), 7.12 (t, J = 7.3 Hz, 2H), 6.97 (d, J =7.9 Hz, 1H), 6.37 (d, J = 16.2 Hz, 1H), 6.22 (s, 1H), 5.85 (ddt, J = 16.3,10.9, 5.6 Hz, 1H), 5.73 (ddd, J = 17.0, 10.5, 5.2 Hz, 1H) 5.24 (dd, J = 17.2,1.7 Hz, 1H), 5.19 – 5.07 (m, 3H), 5.02 (d, J = 10.5 Hz, 1H), 4.78 (s, 2H),4.58 (d, J = 5.7 Hz, 2H), 4.37 (d, J = 4.0 Hz, 2H), 4.28 (d, J = 6.4 Hz, 2H),4.23 (t, 1 H), 4.03 (t, J = 7.2 Hz, 1H), 3.21 (s, 3H), 3.19(s, 1H), 2.96 (d, J = 6.5 Hz, 2H), 1.92 (s, 1H), 1.91 – 1.46 (m, 17H) 1.35 – 1.23 (m, 4H), 0.78(dd, J = 6.3, 3.8 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 172.83, 170.71, 166.28,156.81, 156.74, 153.78, 143.68, 141.23, 139.43, 138.97, 138.38, 138.20,133.01, 132.36, 132.18, 131.81, 129.85, 129.55, 127.73, 127.08, 125.06,125.03, 119.97, 119.90, 118.28, 117.44, 75.79, 67.22, 65.43, 65.28, 57.23,54.96, 52.83, 47.07, 40.78, 40.11, 39.20, 39.05, 38.62, 38.61, 37.07, 32.95,31.91, 29.72, 29.26, 28.37, 28.22, 24.87, 22.86, 22.03, 21.08.

[0045]

[0046] Using a general synthetic method, through the intermediate S -A3 and N -Fluorenylmethoxycarbonyl- N '-allyloxycarbonyl- D -Lysine ( D -Fmoc-lys(alloc)-OH) SR -A4, yield 73%. 1 H NMR (400 MHz, CDCl3) δ 8.61 (s,1H), 7.88 (d, J = 16.2 Hz, 1H), 7.65 (d, J = 7.6 Hz, 2H), 7.54 (d, J = 8.0Hz, 2H), 7.46 (t, J = 6.9 Hz, 2H), 7.35 (d, J = 8.1 Hz, 1H), 7.30 (m, 3H), 7.19 (dd, J = 8.0, 5.5 Hz, 2H), 6.99 (d, J = 8.0 Hz, 1H), 6.81 (d, J= 8.1Hz, 1H), 6.39 (d, J = 16.2 Hz, 1H), 5.89 (td, J = 10.8, 5.3 Hz, 2H), 5.76(ddt, J = 15.3, 9.9, 4.9 Hz, 1H), 5.28 (dd, J = 17.2, 1.8 Hz, 1H), 5.19 (d, J = 10.9 Hz, 1H), 5.07 (d, J = 10.4 Hz, 1H), 4.99 (t, J = 6.0 Hz, 1H), 4.83 (s,1H), 4.61 (d, J = 5.7 Hz, 2H), 4.54 (q, J = 7.7 Hz, 1H), 4.41 (s, 1H), 4.35(d, J = 6.9 Hz, 2H), 4.15 (s, 1H), 4.08 (t, J = 6.7 Hz, 1H), 3.24 (s, 3H),3.20 (s, 1H), 3.03 (t, J = 6.6 Hz, 2H), 1.99 (s, 1H), 1.91 – 1.49 (m, 18H),1.39 (d, J = 6.5 Hz, 1H), 1.35 – 1.26 (m, 2H), 0.83 (t, J = 6.7 Hz, 6H). 13CNMR (101 MHz, CDCl3) δ 171.45, 169.15, 165.23, 155.83, 152.70, 142.61,140.26, 138.38, 137.94, 137.16, 131.87, 131.35, 131.16, 130.73, 128.83,128.59, 128.56, 126.73, 126.08, 124.02, 123.92, 118.98, 118.94, 118.79,117.26, 116.52, 66.08, 64.49, 64.25, 56.21, 54.24, 51.62, 46.10, 39.31,38.78, 38.17, 38.01, 37.59, 36.03, 31.91, 28.68, 27.34, 27.19, 23.85, 21.91,20.79.

[0047]

[0048] Using a general synthetic method, through the intermediate R -A3 and N -Fluorenylmethoxycarbonyl- N '-allyloxycarbonyl- L -Lysine ( L -Fmoc-lys(alloc)-OH) RS -A4, yield 73%. 1 H NMR (400 MHz, CDCl3) δ 8.79 (d, J = 9.5 Hz, 1H), 7.86 (d, J = 16.2 Hz, 1H), 7.63 (dd, J = 7.6, 4.9 Hz, 2H),7.53 – 7.40 (m, 4H), 7.38 – 7.24 (m, 4H), 7.17 (dq, J = 7.7, 4.9, 4.3 Hz,2H), 6.98 (d, J = 8.0 Hz, 1H), 6.37 (d, J = 16.2 Hz, 1H), 6.01 (s, 1H), 5.81(dddt, J= 43.8, 16.1, 10.6, 5.5 Hz, 1H), 5.30 – 5.21 (m, 1H), 5.21 – 5.08(m, 2H), 5.09 – 4.92 (m, 1H), 4.81 (d, J = 4.2 Hz, 2H), 4.64 – 4.53 (m, 3H), 4.42 (d, J = 5.6 Hz, 1H), 4.26 (d, J = 7.4 Hz, 2H), 4.11 – 4.05 (m, 2H), 3.23(s, 3H), 3.20 (s, 1H), 3.01 (q, J = 6.9 Hz, 2H), 2.37 – 1.45 (m, 14H), 1.46 –1.02 (m, 7H), 0.94 – 0.69 (m, 6H). 13 C NMR (101 MHz, CDCl3) δ 172.98, 170.36,166.31, 156.78, 156.62, 153.74, 143.76, 143.54, 141.26, 139.43, 138.98,138.22, 132.91, 132.43, 132.18, 129.53, 127.77, 127.14, 127.10, 125.06,119.99, 119.94, 118.30, 117.58, 75.80, 67.33, 65.31, 57.26, 55.43, 52.71,47.02, 40.30, 39.05, 38.63, 37.07, 32.95, 31.46, 29.72, 28.36, 28.22, 24.94,23.04, 22.53, 21.65, 15.24.

[0049]

[0050] Using a general synthetic method, through the intermediate R -A3 and N -Fluorenylmethoxycarbonyl- N '-allyloxycarbonyl- D -Lysine ( D -Fmoc-lys(alloc)-OH) RR -A4, yield 77%. 11H NMR (400 MHz, CDCl3) δ 8.79 (d, J = 9.5 Hz, 1H), 7.86 (d, J = 16.2 Hz, 1H), 7.63 (dd, J = 7.6, 4.9 Hz, 2H), 7.53 – 7.40 (m, 4H), 7.38 – 7.24 (m, 4H), 7.17 (dq, J = 7.7, 4.9, 4.3 Hz, 2H), 6.98 (d, J = 8.0 Hz, 1H), 6.37 (d, J = 16.2 Hz, 1H), 6.01 (s, 1H), 5.81 (dddt, J = 43.8, 16.1, 10.6, 5.5 Hz, 1H), 5.30 – 5.21 (m, 1H), 5.21 – 5.08 (m, 2H), 5.09 – 4.92 (m, 1H), 4.81 (d, J = 4.2 Hz, 2H), 4.64 – 4.53 (m, 3H), 4.42 (d, J = 5.6 Hz, 1H), 4.26 (d, J = 7.4 Hz, 2H), 4.11 – 4.05 (m, 2H), 3.23 (s, 3H), 3.20 (s, 1H), 3.01 (q, J = 6.9 Hz, 2H), 2.37 – 1.45 (m, 14H), 1.46 – 1.02 (m, 7H), 0.94 – 0.69 (m, 6H). 13 13C NMR (101 MHz, CDCl3) δ 172.98, 170.36, 166.31, 156.78, 156.62, 153.74, 143.76, 143.54, 141.26, 139.43, 138.98, 138.22, 132.91, 132.43, 132.18, 129.53, 127.77, 127.14, 127.10, 125.06, 119.99, 119.94, 118.30, 117.58, 75.80, 67.33, 65.31, 57.26, 55.43, 52.71, 47.02, 40.30, 39.05, 38.63, 37.07, 32.95, 31.46, 29.72, 28.36, 28.22, 24.94, 23.04, 22.53, 21.65, 15.24.

[0051] (5) CompoundSSS -A5,S SR -A5, SRS -A5, SRR -A5, RSS -A5, RSR -A5, RRS -A5, RRR -Synthesis of A5:

[0052] General synthesis method: Under inert gas protection, piperidine (0.494 mL, 5 mmol) was added to the solution of the intermediate SS -A4, or SR -A4, or RS -A4, or RR -A4 (1068 mg, 1.0 mmol) in DMF (5 mL). The solution was stirred at room temperature for 30 min and the reaction was monitored by thin layer chromatography (TLC). After observing that the fluorenylmethoxycarbonyl group was completely removed, the mixture was dissolved in ethyl acetate and washed twice with 0.1M hydrochloric acid (50 ml) and brine (50 ml). Drying was performed over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Subsequently, under the protection of inert gas, the deprotected A4 was N -Fluorenylmethoxycarbonyl-L (or D) -4-chlorophenylalanine (Fmoc-Phe (4-Cl) -OH) (422 mg, 1.0 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (546 mg, 1.44 mmol) were dissolved in DMF (25.0 mL), N,N-diisopropylethylamine (DIPEA) (0.348 mL, 2.0 mmol) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography (TLC). After the reaction was completed, the mixture was diluted with ethyl acetate (100 ml), washed with 0.1 M hydrochloric acid (50 ml) and brine (50 ml). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75:25) to obtain the corresponding SSS -A5,S SR -A5, SRS -A5, SRR -A5, RSS -A5, RSR -A5, RRS -A5, RRR -A5, as a white solid.

[0053]

[0054] Using a general synthetic method, through the intermediate SS -A4 and N -Fluorenylmethoxycarbonyl- L -4-Chlorophenylalanine ( L -Fmoc-Phe(4-Cl)-OH) SSS -A5, yield 75%. 1 H NMR (500 MHz, DMSO- d 6) δ 10.07(s,1H), 8.09(d, J =7.6 Hz 2H), 7.82 (d, J = 8.3 Hz), 7.78 – 7.72 (m, 2H), 7.59(m, 4H), 7.36 (d, J = 7.5 Hz, 2H), 7.31 (m, 5H), 7.10 (m, 1H), 7.05 (d, J =8.0 Hz, 1H), 6.65 (d, J = 16.1 Hz, 1H), 5.94 (td, J = 10.9, 5.2 Hz, 1H), 5.82(td, J = 11.0, 5.4 Hz. 1H), 5.31 (d, J = 17.2 Hz, 1H), 5.20 (m, 2H), 5.08 (d, J = 10.5 Hz, 1H), 4.97 – 4.84 (m, 2H), 4.64 (d, J = 5.5 Hz, 2H), 4.48 – 4.36(m, 3H), 4.27 (d, J = 7.7 Hz, 2H), 4.18 – 4.07 (m, 3H), 3.17(s, 3H), 3.14(s,1H), 2.98 (d, J = 13.4 Hz, 1H), 2.92 (t, J = 6.9 Hz, 2H), 1.95-1.75 (m,13H), 1.72-1.5 (m, 9H), 0.87 (dd, J = 6.3 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 170.82,170.18, 169.41, 155.62, 155.30, 152.68, 142.56, 142.40, 140.09, 138.38,137.90, 137.13, 133.91, 131.95, 131.77, 131.31, 131.09, 130.80, 129.68,128.76, 128.50, 128.39, 127.62, 126.64, 125.99, 123.95, 119.15, 118.85,118.74, 8.55, 27.32, 27.18, 23.91, 21.83, 21.40, 21.19, 20.03, 13.17.

[0055]

[0056] Using a general synthetic method, through the intermediate SR -A4 and N -Fluorenylmethoxycarbonyl- L -4-Chlorophenylalanine ( L -Fmoc-Phe(4-Cl)-OH) SRS -A5, yield 69%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.99 (s,1H), 8.31 (dd, J = 8.0, 4.4 Hz, 2H), 7.83 (t, J = 5.7 Hz, 2H), 7.79 (d, J =5.3 Hz, 1H), 7.69 (d, J = 8.8 Hz, 2H), 7.61 (d, J = 7.7 Hz, 1H), 7.59 (d, J=7.8 Hz, 1H), 7.42 – 7.22 (m, 11H), 7.13 (t, J = 5.6 Hz, 1H), 7.09 (d, J = 8.0Hz, 1H), 6.67 (d, J = 16.2 Hz, 1H), 6.02 – 5.93 (m, 1H), 5.93 – 5.84 (m, 1H),5.34 (dd, J = 17.2, 1.6 Hz, 2H), 5.25 (d, J = 12.0 Hz, 1H), 5.15 (d, J = 10.4Hz, 1H), 4.98 – 4.84 (m, 2H), 4.66 (d, J = 5.6 Hz, 2H), 4.44 (dt, J = 5.4,1.6 Hz, 3H), 4.38 – 4.27 (m, 2H), 4.13 (ddd, J = 16.0, 11.5, 6.5 Hz, 3H),3.20 (s, 3H), 3.17 (s, 1H), 2.99 – 2.93 (m, 3H), 2.82 (dd, J = 13.6, 10.0 Hz,1H), 1.92 – 1.66 (m, 13H), 1.65 – 1.47 (m, 9H), 0.86 (dd, J = 6.2 Hz, 6H). 13 CNMR (101 MHz, DMSO- d6) δ 171.99, 171.88, 171.52, 165.98, 162.76, 156.32,156.24, 153.46, 144.23, 144.07, 141.09, 139.75, 139.64, 138.43, 137.88,137.37, 134.29, 133.05, 131.63, 131.49, 130.91, 130.75, 129.88, 129.68,129.19, 128.41, 128.01, 127.42, 126.30, 125.68, 31.76, 31.23, 29.50, 28.16, 28.03,24.76, 23.48, 22.85, 22.56, 21.73, 14.40.

[0057]

[0058] Using a general synthetic method, through the intermediate RS -A4 and N -Fluorenylmethoxycarbonyl- L -4-Chlorophenylalanine ( L -Fmoc-Phe(4-Cl)-OH) RSS -A5, yield 71%. 1 H NMR (400 MHz, CDCl3) δ 9.15 (s,1H), 7.84 (s, 0H), 7.58 (d, J = 7.6 Hz, 1H), 7.39 (t, J = 7.8 Hz, 1H), 7.28(s, 2H), 7.22 – 6.87 (m, 4H), 6.77 (d, J = 7.5 Hz, 1H), 6.34 (d, J = 16.1 Hz,1H), 5.82 (ddt, J = 16.3, 10.7, 5.5 Hz, 1H), 5.63 (ddt, J= 16.2, 10.7, 5.4Hz, 0H), 5.32 – 4.89 (m, 3H), 4.75 (s, 1H), 4.43 (dd, J = 48.9, 5.6 Hz, 2H),4.07 (dt, J = 53.5, 8.3 Hz, 2H), 3.16 (d, J = 17.0 Hz, 5H), 2.31 – 1.41 (m,16H), 0.88 (s, 3H). 13 C NMR (101 MHz, CDCl3) δ 171.07, 170.72, 170.08, 164.96,155.73, 152.88, 142.83, 142.29, 140.19, 140.03, 138.35, 138.25, 137.73,137.07, 136.78, 134.25, 132.10, 131.71, 131.10, 131.07, 129.76, 128.89,128.45, 128.08, 127.52, 126.70, 126.21, 125.99, 124.10, 37.92, 27.31, 27.14, 23.99, 22.39, 21.66, 21.54, 20.93, 29.10, 28.67,28.63, 28.49, 28.33, 28.13, 27.92, 27.31, 27.14, 23.99, 22.39, 21.66, 21.54,20.93, 14.16, 13.10.

[0059]

[0060] Using a general synthetic method, through the intermediate RR -A4 and N -Fluorenylmethoxycarbonyl- L -4-Chlorophenylalanine ( L -Fmoc-Phe(4-Cl)-OH) RRS -A5, yield 70%. 1 H NMR (400 MHz, DMSO-d 6) δ 9.80(s, 1H), 8.43 (d, J = 7.5 Hz, 1H), 8.01 (d, J = 8.0 Hz, 1H), 8.04 – 7.79 (m,5H), 7.81 – 7.55 (m, 4H), 7.52 – 7.25 (m, 10H), 7.16 (dd, J = 6.9, 3.9 Hz,2H), 6.74 (d, J = 16.1 Hz, 1H), 6.10 – 5.86 (m, 2H), 5.53 – 5.14 (m, 4H),5.07 – 4.91 (m, 2H), 4.72 (dt, J = 5.5, 1.6 Hz, 2H), 4.48 (d, J = 5.6 Hz,3H), 4.45 – 4.06 (m, 6H), 3.27 (s, 3H), 2.96 (qd, J = 13.7, 13.1, 4.8 Hz,4H), 2.16 – 1.10 (m, 26H), 0.93 (dd, J = 16.4, 5.9 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 171.40, 170.35, 170.03, 165.35, 155.87, 155.73, 152.63, 142.72,142.17, 140.17, 140.03, 138.42, 138.35, 138.16, 137.46, 137.09, 133.88,131.89, 131.86, 131.20, 131.13, 130.60, 129.72, 128.77, 128.58, 127.66,126.69, 126.09, 126.02, 124.06, 123.88, 118.89, 118.61, 117.20, 116.43,74.54, 66.29, 64.46, 64.20, 56.16, 56.09, 55.36, 52.67, 52.43, 51.23, 45.74,40.64, 39.26, 38.02, 37.59, 36.02, 31.87, 30.89, 28.76, 28.65, 28.32, 27.91,27.31, 27.17, 23.94, 22.04, 21.66, 21.43, 20.74, 14.19, 13.10.

[0061]

[0062] Using a general synthetic method, through the intermediate SS -A4 and N -Fluorenylmethoxycarbonyl- D -4-Chlorophenylalanine ( D -Fmoc-Phe(4-Cl)-OH) SSR -A5, yield 76%. 1 H NMR (500 MHz, DMSO-d6) δ 10.07 (s,1H), 8.09 (dd, J = 7.6 Hz, 2H), 7.82 (d, J = 8.3 Hz, 2H), 7.79 – 7.74 (m,2H), 7.59 (m, 5H), 7.36 (d, J = 7.5 Hz, 2H), 7.31-7.26 (m, 8H), 7.10 (s, 1H),7.05 (d, J = 8.0 Hz, 2H), 6.65 (d,J = 16.1 Hz, 1H), 5.94 (td, J = 10.9, 5.2Hz, 1H), 5.82 (td, J = 11.0, 5.4 Hz, 1H), 5.31 (d, J = 17.2 Hz, 1H), 5.20 (t, J = 13.3 Hz, 1H), 5.08 (d, J = 10.5 Hz, 1H), 4.94 (d, J = 10.6 Hz, 1H), 4.86(d, J = 11.0 Hz, 1H), 4.64 (d, J = 5.5 Hz, 2H), 4.46 – 4.42 (m, , 1H), 4.39(d, J = 5.5 Hz, 2H), 4.27 (d, J = 7.7 Hz, 2H), 4.14 (d, J = 11.7 Hz, 1H),4.10 (s, 1H), 3.17 (s, 3H), 3.14 (s, 1H), 2.98 (d, J = 13.4 Hz, 1H), 2.92 (d, J = 6.6 Hz, 2H), 2.72 (dd, J = 23.6, 10.8 Hz, 1H), 1.90 (s, 1H), 1.97 -1.77(m, 12H), 1.65-1.52 (m, 9H), 0.84 (d, J = 6.3 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 170.82, 170.18, 169.41, 155.62, 155.30, 152.68, 142.56, 142.40,140.09, 138.38, 137.90, 137.13, 137.02, 133.91, 131.95, 131.77, 131.31,131.09, 130.80, 129.68, 128.76, 128.50, 128.39, 127.62, 126.64, 125.99,125.97, 123.95, 123.93, 8.55, 27.32, 27.18, 23.91, 21.83, 21.40,21.19, 20.03, 13.17.

[0063]

[0064] Using a general synthetic method, through the intermediate SR -A4 and N -Fluorenylmethoxycarbonyl- D -4-Chlorophenylalanine ( D -Fmoc-Phe(4-Cl)-OH) SRR -A5, yield 71%. 1 H NMR (400 MHz, CDCl3) δ 9.09 (s,1H), 7.84 (d, J = 16.1 Hz, 1H), 7.67 (d, J = 7.6 Hz, 1H), 7.61 (d, J = 7.7Hz, 2H), 7.48 (d, J = 7.1 Hz, 1H), 7.40 (t, J = 6.8 Hz, 2H), 7.31 (d, J<h2 style=";text-align:left;direction:ltr">= 7.8Hz, 3H), 7.24 – 7.20 (m, 2H), 7.21 – 7.14 (m, 2H), 7.09 (d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 7.7 Hz, 2H), 7.04 – 6.97 (m, 2H), 6.95 (d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 8.0 Hz, 1H), 6.85 – 6.79 (m, 2H), 6.36 (d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 16.2 Hz, 1H), 5.81 (tt,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 10.7, 5.4 Hz, 1H), 5.76 – 5.65 (m, 2H), 5.19(d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 17.2 Hz, 2H), 5.09 (d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 10.1 Hz, 2H), 5.01 (d,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> = 9.6 Hz, 2H),4.78 (s, 2H), 4.51 – 4.37 (m, 4H), 4.25 – 4.14 (m, 3H), 4.02 (t,<h2 style=";text-align:left;direction:ltr"> J <h2 style=";text-align:left;direction:ltr"> J 6.2 Hz, 6H).<h2 style=";text-align:left;direction:ltr"> 13 <h2 style=";text-align:left;direction:ltr"> C NMR (101 MHz, DMSO-<h2 style=";text-align:left;direction:ltr"> d6) δ 172.00, 171.89, 171.52, 165.98, 162.76, 156.33, 156.24,153.46, 144.23, 144.07, 141.09, 139.75, 139.65, 138.44, 137.88, 137.37,134.29, 133.06, 131.64, 130.92, 130.75, 129.88, 129.68, 129.20, 128.41,128.02, 127.43, 126.30, 125.69, 120.53, 31.24, 29.50, 28.17, 28.04, 24.76, 23.48,22.85, 22.57, 21.73, 14.41.

[0065]

[0066] Using a general synthetic method, through the intermediate RS -A4 and N -Fluorenylmethoxycarbonyl- D -4-Chlorophenylalanine ( D -Fmoc-Phe(4-Cl)-OH) RSR -A5, 76%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.03 (s,1H), 8.35 (t, J = 6.8 Hz, 2H), 7.98 – 7.80 (m, 4H), 7.73 (d, J = 8.1 Hz, 3H),7.64 (dd, J = 10.4, 7.5 Hz, 2H), 7.56 – 7.24 (m, 7H), 7.22 – 7.05 (m, 2H), 6.72 (d, J = 16.1 Hz, 1H), 5.97 (dddd, J= 36.6, 22.2, 10.7, 5.5 Hz, 2H),5.41 (d, J = 1.8 Hz, 1H), 5.36 – 5.13 (m, 4H), 4.98 – 4.89 (m, 2H), 4.71 (d, J = 5.4 Hz, 2H), 4.49 (dd, J = 11.2, 5.1 Hz, 3H), 4.41 – 4.06 (m, 5H), 3.25 (s, 3H), 3.11 – 2.78 (m, 4H), 2.12 – 1.16 (m, 27H), 0.91 (dd, J = 15.8, 6.2Hz, 6H). 13 C NMR (101 MHz, CDCl3) δ 170.34, 169.98, 169.29, 165.05, 155.69,155.44, 152.73, 142.65, 142.31, 140.03, 138.41, 137.73, 136.99, 136.68,134.08, 132.12, 131.76, 131.17, 131.09, 130.93, 129.78, 128.73, 128.39,128.13, 127.54, 126.51, 125.98, 125.85, 124.03, 39.86, 38.14, 37.99, 37.56, 36.02, 32.79, 31.87,30.89, 30.61, 28.87, 28.65, 28.59, 28.48, 28.33, 28.12, 27.92, 27.31, 27.17,24.04, 21.67, 14.19, 13.10.

[0067]

[0068] Using a general synthetic method, through the intermediate RR -A4 and N -Fluorenylmethoxycarbonyl- D-4-Chlorophenylalanine ( D -Fmoc-Phe(4-Cl)-OH) RRR -A5, yield 83%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.10 (s,1H), 8.11 (dd, J = 14.1, 7.8 Hz, 2H), 7.88 (d, J = 7.6 Hz, 2H), 7.86 – 7.78(m, 2H), 7.65 – 7.59 (m, 5H), 7.39 (dd, J = 18.5, 11.0 Hz, 4H), 7.33 (s, 1H), 7.12 (dd, J = 10.1, 6.7 Hz, 2H), 6.69 (d, J = 16.2 Hz, 1H), 5.93 (dddt, J =48.0, 16.1, 10.5, 5.4 Hz, 2H), 5.36 (dd, J = 17.2, 1.8 Hz, 1H), 5.30 – 5.19(m, 2H), 5.13 (d, J = 10.4 Hz, 1H), 5.03 – 4.87 (m, 2H), 4.68 (d, J = 5.5 Hz,2H), 4.45 (dd, J = 15.0, 5.6 Hz, 3H), 4.29 (t, J = 8.3 Hz, 2H), 4.25 – 4.11(m, 3H), 3.33 (s, 3H), 3.22 (s, 3H), 3.04 – 2.92 (m, 3H), 2.76 (dd, J = 13.7,10.7 Hz, 1H), 2.51 (s, 2H), 2.17 – 1.49 (m, 19H). 13C NMR (400 MHz, CDCl3) δ170.84, 170.42, 170.20, 169.42, 165.23, 155.64, 155.31, 152.69, 142.57,142.41, 140.11, 138.39, 137.91, 137.14, 133.92, 131.96, 131.78, 131.32,131.10, 129.69, 128.77, 128.51, 128.40, 127.63, 126.65, 126.02, 123.96,119.16, 118.86, 118.75, 117.20, 116.51, 75.73, 74.70, 66.35, 59.40, 56.19,54.96, 52.32, 51.70, 45.82, 40.23, 39.48, 38.16, 38.01, 37.58, 36.02, 31.90,28.67, 28.55, 27.32, 27.18, 23.91, 21.83, 21.40, 21.18, 13.17.

[0069] (6) Compounds SSSS -A6,S SSR -A6, SSRS -A6, SRSS -A6, RSSS -A6, SSRR -A6, RRSS -A6, RSRS -A6, SRSR -A6, RSSR -A6, SRRS -A6, RSRR -A6, RRRS -A6, RRSR -A6, SRRR -A6, RRRR -Synthesis of A6:

[0070] General synthesis method: Under inert gas protection, piperidine (0.494 mL, 5 mmol) was added to the solution of the intermediate SSS -A5 or S SR -A5 or SRS -A5 or SRR -A5 or RSS -A5 or RSR -A5 or RRS -A5 or RRR-A5 (1249 mg, 1.0 mmol) in DMF (5 mL). The solution was stirred at room temperature for 30 min and the reaction was monitored by thin layer chromatography (TLC). After observing that the fluorenylmethyloxycarbonyl group was completely removed, the mixture was dissolved in ethyl acetate and washed twice with 0.1 M hydrochloric acid (50 ml) and brine (50 ml). Drying was performed over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Subsequently, under the protection of inert gas, the deprotected A5 was N -Ac- L (or D )-Indanylglycine (Ac-Igl-OH) (233 mg, 1.0 mmol), benzotriazole-N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (546 mg, 1.44 mmol) were dissolved in DMF (25.0 mL), N,N-diisopropylethylamine (DIPEA) (0.348 mL, 2.0 mmol) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography (TLC). After the reaction was completed, the mixture was diluted with ethyl acetate (100 ml), washed with 0.1M hydrochloric acid (50 ml) and brine (50 ml). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75:25). The corresponding compound was obtained. SSSS -A6,S SSR -A6, SSRS -A6, SRSS -A6, RSSS -A6, SSRR -A6, RRSS -A6, RSRS -A6, SRSR -A6, RSSR -A6, SRRS -A6, RSRR -A6, RRRS -A6, RRSR -A6, SRRR -A6, RRRR -A6, as a white solid.

[0071]

[0072] Using a general synthetic method, through the intermediate SSS -A5 and N -Ac- L -Indanylglycine ( N -Ac- L -Igl-OH) SSSS -A6, yield 63%. 1H NMR (400 MHz, DMSO- d 6) δ 10.03 (s, 1H), 9.80 (s,1H), 8.10 (d, J = 16.8, 1H), 7.89 – 7.78 (m, 2H), 7.66 (t, J = 8.1 Hz, 4H),7.35 (m, 4H), 7.08 (m, 6H), 6.70 (d, J = 16.3 Hz, 1H), 5.99 (tt, J = 10.9,5.3 Hz, 1H), 5.88 (tt, J = 10.5, 4.9 Hz, 1H), 5.36 (d, J = 17.3 Hz, 1H), 5.26(m, 2H), 5.14 (d, J = 10.6 Hz, 1H), 4.95 (dd, J = 22.0, 8.2 Hz, 2H), 4.68 (d, J = 5.5 Hz, 2H), 4.65 – 4.54 (m, 1H), 4.43 (m, 3H), 4.32 – 4.11 (m, 2H), 3.23(s, 4H), 3.19 (s, 1H), 3.15 – 3.00 (m, 2H), 3.01 – 2.60 (m, 7H), 2.06 – 1.65(m, 16H), 1.33 – 1.03 (m, 9H), 0.88 (ddd, J = 21.4, 11.4, 4.7 Hz, 6H). 13C NMR(101 MHz, DMSO) δ 172.00, 171.80, 171.59, 171.48, 170.08, 169.95, 166.00,156.30, 153.47, 142.88, 142.75, 142.57, 142.51, 139.79, 138.49, 137.89,134.31, 133.08, 131.68, 131.58, 130.73, 129.98, 129.21, 128.45, 128.08,126.54, 126.32, 124.60, 119.40, 118.44, 117.24, 75.95, 65.14, 64.56, 56.99,56.45, 55.36, 54.62, 53.34, 42.18, 41.81, 38.85, 38.55, 37.29, 36.93, 35.84,35.65, 35.33, 28.18, 28.05, 24.78, 23.47, 23.44, 22.97, 22.09, 21.85.

[0073]

[0074] Using a general synthetic method, through the intermediate RSS -A5 and N -Ac- L -Indanylglycine ( N -Ac- L -Igl-OH) RSSS -A6, yield 73%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.99 (s, 1H), 8.55 (d, J = 8.5 Hz, 1H), 8.20 (dd, J = 7.8, 2.6 Hz, 2H), 8.11 (d, J = 7.3 Hz, 1H),7.88 – 7.75 (m, 2H), 7.70 (d, J = 8.2 Hz, 2H), 7.40 – 7.29 (m, 4H), 7.16 –7.02 (m, 2H), 6.69 (d, J = 16.1 Hz, 1H), 5.94 (dddd, J= 32.3, 11.9, 10.5,5.2 Hz, 2H), 5.46 – 5.33 (m, 1H), 5.31 – 5.27 (m, 1H), 5.16 (dq, J = 10.5,1.6 Hz, 1H), 5.02 – 4.87 (m, 2H), 4.67 (dt, J = 5.6, 1.5 Hz, 2H), 4.46 (dt, J = 5.7, 1.8 Hz, 3H), 4.26 (t, J = 8.1 Hz, 2H), 3.22 (s, 3H), 3.18 (s, 1H), 3.02 – 2.60 (m, 6H), 1.99 – 1.54 (m, 25H), 0.90 (dd, J = 14.6, 6.3 Hz, 6H). 13 C NMR (101 MHz, DMSO) δ 172.07, 171.85, 171.61, 170.25, 165.98, 156.35,153.46, 142.73, 142.53, 139.77, 139.70, 138.48, 137.89, 137.61, 134.30,133.06, 131.63, 131.47, 130.91, 130.74, 129.96, 129.72, 129.21, 128.45,128.07, 126.54, 126.29, 124.61, 124.40, 35.90, 35.35, 32.86, 31.90, 29.63, 29.50, 28.18,28.04, 24.81, 23.55, 23.16, 22.77, 21.71.

[0075]

[0076] Using a general synthetic method, through the intermediate SRS -A5 and N -Ac- L-Indanylglycine ( N -Ac- L -Igl-OH) SRSS -A6, yield 66%. 1 H NMR (500 MHz, DMSO- d 6) δ 10.02 (d, J = 39.3 Hz,1H), 8.57 (d, J = 9.0 Hz, 1H), 8.17 – 8.05 (m, 3H), 7.98 – 7.92 (m, 1H), 7.78(dt, J = 19.2, 6.3 Hz, 2H), 7.61 (d, J = 8.3 Hz, 2H), 7.31 (t, J = 7.6 Hz,4H), 7.22 (s, 1H), 7.06 (dd, J = 19.3, 11.5 Hz, 6H), 6.65 (d, J = 16.3 Hz,1H), 5.94 (ddt, J = 16.5, 10.7, 5.5 Hz, 1H), 5.84 (ddt, J = 16.6, 10.7, 5.5Hz, 1H), 5.31 (d, J = 17.2 Hz, 1H), 5.22 (d, J = 10.7 Hz, 2H), 5.13 – 5.06(m, 1H), 4.95 (d, J = 10.5 Hz, 1H), 4.87 (d, J = 10.7 Hz, 1H), 4.63 (d, J =5.8 Hz, 3H), 4.58 (d, J = 11.5 Hz, 1H), 4.43 (dd, J = 20.9, 10.6 Hz, 4H), 4.32 – 4.18 (m, 2H), 3.17 (s, 3H), 3.13 (s, 1H), 3.04 (d, J = 11.6 Hz, 1H), 3.02 – 2.60 (m, 7H), 2.07 – 1.47 (m, 25H), 0.91 – 0.83 (m, 6H). 13 C NMR (126MHz, DMSO- d6) δ 172.06, 171.97, 171.79, 171.61, 171.15, 170.14, 169.94,166.05, 156.40, 153.52, 143.02, 142.82, 142.61, 139.85, 138.56, 137.95,134.38, 133.13, 131.72, 130.89, 130.09, 129.79, 129.30, 128.52, 126.64,124.68, 120.60, 119.45, 118.48, 117.32, 76.08, 65.20, 64.65, 57.03, 56.26,54.52, 53.61, 52.54, 42.34, 41.27, 38.99, 38.60, 36.99, 35.38, 32.93, 29.73,29.56, 28.25, 28.09, 24.85, 23.54, 22.09.

[0077]

[0078] Using a general synthetic method, through the intermediate SSR -A5 and N -Ac- L -Indanylglycine ( N -Ac- L -Igl-OH) SSRS -A6, yield 63%. 1 H NMR (500 MHz, DMSO- d 6) δ 10.06 (s, 1H), 9.98 (s, 1H), 8.14 (d, J = 8.1 Hz, 1H), 8.10 – 8.06 (m, 2H), 7.95 (dd, J = 12.9,7.8 Hz, 1H), 7.79 (d, J = 16.2 Hz, 1H), 7.76 (d, J = 8.2 Hz, 1H), 7.61 (d, J = 8.2 Hz, 2H), 7.31 (d, J = 7.4 Hz, 4H), 7.23 (m, 2H), 7.07 (m, 6H), 6.65 (d, J= 16.2 Hz, 1H), 5.94 (ddt, J = 16.3, 10.7, 5.5 Hz, 1H), 5.84 (ddt, J =15.9, 10.5, 5.2 Hz, 1H), 5.32 (d, J = 17.2 Hz, 1H), 5.22 (d, J = 10.1 Hz,2H), 5.10 (dd, J = 10.7, 5.0 Hz, 1H), 4.98 – 4.84 (m, 2H), 4.64 (d, J = 5.5Hz, 2H), 4.57 (dd, J = 15.9, 7.7 Hz, 1H), 4.41 (t, J = 5.7 Hz, 4H), 4.25(ddd, J = 17.4, 11.8, 6.3 Hz, 2H), 3.18 (s, 3H), 3.14 (s, 1H), 3.08 – 3.01(m, 1H), 2.97 – 2.65 (m, 7H), 1.93 – 1.56 (m, 25H), 0.87 (d, J = 6.3 Hz, 6H). 13 C NMR (126 MHz, DMSO- d6) δ 172.06, 171.95, 171.80, 171.61, 171.15, 170.14,169.94, 166.06, 156.41, 153.53, 143.02, 142.82, 142.66, 139.85, 138.56,137.95, 137.75, 134.37, 133.13, 131.55, 130.80, 130.07, 129.80, 129.30,128.40, 126.60, 124.67, 120.60, 119.43, 118.48, 117.32, 65.20, 64.65, 57.03,54.08, 53.60, 52.55, 52.53, 42.35, 41.32, 38.99, 38.59, 36.98, 35.92, 35.71,35.61, 35.38, 32.93, 29.74, 29.56, 28.25, 28.11, 24.84, 23.52, 22.83, 22.10.

[0079]

[0080] Using a general synthetic method, through the intermediate RRS -A5 and N -Ac- L -Indanylglycine ( N -Ac- L -Igl-OH) RRSS -A6, yield 69%. 1 H NMR (400 MHz, Chloroform- d ) δ 7.88 (d, J = 16.2Hz, 1H), 7.66 – 7.59 (m, 2H), 7.35 (dd, J = 8.2, 3.9 Hz, 3H), 7.19 (s, 1H), 7.14 – 6.96 (m, 5H), 6.40 (d, J = 16.1 Hz, 1H), 5.96 – 5.71 (m, 1H), 5.27(dq, J = 17.4, 1.6 Hz, 2H), 5.22 – 5.12 (m, 1H), 5.08 (d, J= 10.5 Hz, 4H), 4.87 (s, 2H), 4.78 (s, 1H), 4.60 (dd, J = 5.8, 1.6 Hz, 2H), 4.42 (d, J = 5.6Hz, 2H), 3.24 (s, 3H), 3.20 (s, 1H), 3.04 – 2.86 (m, 7H), 2.04 – 1.73 (m, 25H), 0.82 (dd, J = 25.0, 6.4 Hz, 6H). 13 C NMR (101 MHz, DMSO) δ 172.75,172.00, 171.42, 170.44, 166.00, 162.74, 156.30, 153.47, 139.79, 139.61,138.48, 137.90, 136.84, 134.30, 133.08, 131.60, 131.52, 130.92, 130.75,129.95, 129.71, 129.21, 128.47, 128.09, 126.31, 120.55, 119.41, 118.46,117.25, 80.22, 75.97, 65.39, 65.17, 64.58, 64.26, 56.99, 55.17, 53.60, 52.45,38.92, 38.86, 38.69, 38.55, 37.16, 36.92, 36.23, 32.87, 31.98, 31.76, 31.22,29.87, 29.46, 29.17, 28.18, 28.04, 24.83, 23.49, 22.95, 22.69, 21.77.

[0081]

[0082] Using a general synthetic method, through the intermediate SRR -A5 and N -Ac- L -Indanylglycine ( N -Ac- L -Igl-OH) SRRS -A6, yield 69%. 1 H NMR (500 MHz, DMSO- d6) δ 10.10 (s, 1H), 10.03(s, 1H), 8.13 (dt, J = 15.9, 7.5 Hz, 3H), 7.99 (d, J = 7.0 Hz, 1H), 7.77 (t, J = 11.9 Hz, 3H), 7.61 (d, J = 7.9 Hz, 3H), 7.30 (t, J = 8.0 Hz, 4H), 7.06(dd, J = 20.3, 12.5 Hz, 5H), 6.65 (d, J = 16.2 Hz, 1H), 5.94 (ddt, J = 16.4,10.7, 5.5 Hz, 1H), 5.88 – 5.80 (m, 1H), 5.31 (d, J = 17.2 Hz, 1H), 5.22 (d, J = 9.2 Hz, 2H), 5.10 (d, J = 8.2 Hz, 1H), 5.00 – 4.82 (m, 2H), 4.63 (d, J =5.5 Hz, 2H), 4.59 – 4.52 (m, 1H), 4.40 (t, J = 5.7 Hz, 4H), 4.30 – 4.15 (m,2H), 3.17 (s, 3H), 3.14 (s, 1H) 1.97 – 1.50 (m, 25H), 0.89 (d, J = 6.3 Hz,6H). 13 C NMR (126 MHz, DMSO- d6) δ 172.05, 171.65, 171.17, 170.12, 169.97,166.07, 156.39, 153.51, 143.02, 142.82, 142.66, 140.92, 139.87, 138.55,137.94, 134.37, 133.13, 131.69, 131.46, 130.78, 130.06, 129.79, 129.28,128.14, 126.60, 126.57, 126.38, 124.67, 120.59, 119.44, 118.51, 117.33,76.02, 65.21, 64.64, 57.04, 41.31, 38.61, 38.59, 36.98, 35.61, 34.72, 34.46,32.92, 29.72, 29.55, 29.01, 28.24, 28.08, 26.87, 25.31, 24.83, 23.52, 22.97,22.09, 21.11.

[0083]

[0084] Using a general synthetic method, through the intermediate RSR -A5 and N -Ac- L -Indanylglycine ( N -Ac- L -Igl-OH) RSRS -A6, yield 68%. 1H NMR (400 MHz, DMSO-d6) δ 10.03 – 9.98 (s, 1H),8.34 – 8.28 (t, J = 8.0 Hz, 1H), 8.23 – 8.17 (d, J = 7.9 Hz, 1H), 8.13 – 8.07(d, J = 7.8 Hz, 2H), 7.86 – 7.77 (m, 2H), 7.71 – 7.65 (d, J = 8.3 Hz, 2H),7.37 – 7.25 (m, 6H), 7.18 – 7.02 (m, 7H), 6.73 – 6.65 (d, J = 16.2 Hz, 1H),6.06 – 5.82 (dddt, J = 5.4, 10.5, 16.1, 39.0 Hz, 1H), 5.40 – 5.31 (d, J =17.2 Hz, 1H), 5.30 – 5.22 (m, 2H), 5.19 – 5.12 (d, J = 10.4 Hz, 1H), 5.03 –4.82 (m, 2H), 4.73 – 4.64 (d, J = 5.5 Hz, 2H), 4.53 – 4.39 (t, J = 6.5 Hz,3H), 4.33 – 4.20 (t, J = 7.4 Hz, 2H), 3.21 – 3.17 (s, 1H), 3.06 – 2.92 (m,3H), 2.89 – 2.74 (m, 2H), 2.66 – 2.54 (q, J = 12.1 Hz, 3H), 2.17 – 1.11 (m,39H), 0.93 – 0.84 (dd, J = 6.1, 13.9 Hz, 6H). 13C NMR (101 MHz, DMSO-d6) δ171.89, 171.59, 171.56, 171.18, 170.06, 166.01, 156.33, 153.47, 142.91,142.57, 139.79, 139.71, 138.49, 137.90, 137.08, 134.32, 133.09, 131.57,131.48, 130.88, 130.74, 129.96, 129.73, 129.21, 128.40, 128.09, 126.49 ,126.33, 37.35, 36.93,35.74 , 32.87, 32.17, 31.76, 29.34 , 28.11 , 24.80, 23.51, 22.88, 21.72.

[0085]

[0086] Using a general synthetic method, through the intermediate RRR -A5 and N -Ac- L -Indanylglycine ( N -Ac- L -Igl-OH) RRRS -A6, yield 62%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.12 (s, 1H), 8.15 (dd, J = 8.2, 3.9 Hz, 2H), 8.09 (d, J = 7.8 Hz, 1H), 7.90 – 7.80 (m, 1H), 7.68 (dd, J = 8.3, 4.9 Hz, 2H), 7.46 – 7.28 (m, 6H), 7.24 – 7.05 (m, 3H), 6.73 (d, J = 16.2 Hz, 1H), 6.08 – 5.85 (m, 2H), 5.40 (dd,J = 17.2, 1.7 Hz,1H), 5.34 – 5.26 (m, 2H), 5.19 (dt, J = 10.0, 1.7 Hz, 1H), 5.00 (d, J = 22.4Hz, 2H), 4.72 (dt, J = 5.5, 1.5 Hz, 2H), 4.49 (d, J = 5.5 Hz, 3H), 4.30 (td, J = 8.2, 5.1 Hz, 1H), 3.27 (s, 3H), 3.23 (s, 1H), 3.09 – 2.87 (m, 6H), 2.11 –1.40 (m, 25H), 0.95 (dd, J = 12.4, 6.2 Hz, 6H). 13 C NMR (101 MHz, DMSO) δ171.96, 171.64, 171.56, 169.73, 165.99, 156.34, 153.45, 139.78, 138.49,137.88, 137.47, 134.31, 133.08, 131.48, 131.40, 130.82, 130.73, 130.01,129.73, 129.21, 128.37, 128.08, 126.54, 126.31, 120.53, 119.37, 118.43,117.28, 75.95, 65.14, 64.58, 56.98, 54.13, 53.07, 52.41, 41.28, 38.87, 38.55,37.25, 36.92, 32.86, 31.75, 29.50, 29.45, 28.18, 24.77, 23.47, 22.87, 22.07.

[0087]

[0088] Using a general synthetic method, through the intermediate SSS -A5 and N -Ac- D -Indanylglycine ( N -Ac- D -Igl-OH) SSSR -A6, yield 65%. 1H NMR (500 MHz, DMSO- d 6) δ 10.12 (s, 1H), 10.05(s, 1H), 8.62 (d, J = 8.3 Hz, 1H)8.23 – 8.13 (m, 3H), 8.03 (t, J = 7.3 Hz,1H), 7.84 – 7.72 (m, 3H), 7.63 (d, J = 8.0 Hz, 2H), 7.34 – 7.28 (m, 2H), 7.25– 7.21 (m, 2H), 7.10 – 7.00 (m, 6H), 6.64 (d, J = 16.2 Hz, 1H), 5.94 (ddt, J = 16.2, 10.7, 5.5 Hz, 1H), 5.84 (ddt, J = 15.9, 10.4, 5.3 Hz, 1H), 5.31 (d, J = 17.2 Hz, 1H), 5.24 – 5.17 (m, 2H), 5.09 (dd, J = 10.7, 5.0 Hz, 1H), 4.96 –4.84 (m, 2H), 4.63 (d, J = 5.5 Hz, 2H), 4.57 (dt, J = 8.7, 5.5 Hz, 1H), 4.40(d, J = 6.2 Hz, 3H), 4.28 – 4.22 (m, 2H), 3.17 (s, 3H), 3.12 (s, 1H), 2.92(q, J = 6.8 Hz, 2H), 2.80 – 2.57 (m, 7H), 1.93 – 1.49 (m, 25H), 0.87 (d, J =6.3 Hz, 3H), 0.85 (d, J = 6.2 Hz, 3H). 13 C NMR (126 MHz, DMSO- d6) δ 172.00,171.83, 171.76, 171.66, 171.22, 170.22, 170.08, 166.06, 166.06, 156.42,153.51, 143.01, 142.82, 142.65, 142.61, 139.84, 138.55, 137.95, 134.34,133.10, 131.69, 131.48, 130.81, 130.02, 129.78, 129.29, 128.48, 128.37,121.85, 120.49, 119.43, 118.46, 117.30, 76.02, 65.20, 64.64, 57.01, 41.30,38.98, 38.58, 36.97, 35.93, 34.70, 34.45, 32.92, 31.49, 29.69, 29.56, 29.00,28.26, 28.10, 26.86, 25.30, 23.53, 23.50, 22.10, 21.07.

[0089]

[0090] Using a general synthetic method, through the intermediate RSS -A5 and N -Ac- D -Indanylglycine ( N -Ac- D -Igl-OH) RSSR -A6, yield 70%. 1H NMR (400 MHz, DMSO-d6) δ 10.11 – 9.87 (s, 1H),8.50 – 8.36 (d, J = 8.1 Hz, 1H), 8.19 – 8.03 (m, 3H), 7.82 – 7.78 (d, J = 8.6Hz, 1H), 7.74 – 7.69 (m, 2H), 7.41 – 7.30 (m, 3H), 7.29 – 7.21 (s, 4H), 7.19– 7.14 (q, J = 3.4, 4.5, 4.5 Hz, 1H), 7.13 – 7.02 (m, 4H), 6.76 – 6.65 (d, J= 16.1 Hz, 1H), 6.06 – 5.84 (m, 1H), 5.39 – 5.37 (q, J = 1.7, 1.7, 1.7 Hz,1H), 5.34 – 5.32 (q, J = 1.6, 1.6, 1.7 Hz, 2H), 5.30 – 5.26 (dp, J = 1.4,1.4, 1.6, 1.6, 4.4 Hz, 1H), 5.26 – 5.23 (q, J = 1.8, 1.8, 2.1 Hz, 1H), 5.18 –5.13 (dq, J = 1.5, 1.5, 1.5, 10.5 Hz, 1H), 5.02 – 4.86 (m, 2H), 4.70 – 4.66(m, 2H), 4.61 – 4.55 (td, J = 4.3, 8.7, 8.8 Hz, 1H), 4.52 – 4.41 (m, 3H),4.38 – 4.29 (t, J = 6.9, 6.9 Hz, 2H), 4.26 – 4.20 (t, J = 6.5, 6.5 Hz, 1H),3.28 – 3.22 (s, 3H), 3.10 – 2.93 (m, 4H), 2.86 – 2.75 (dd, J = 10.0, 13.9 Hz,2H), 2.71 – 2.60 (m, 3H), 2.56 – 2.49 (p, J = 1.8, 1.8, 1.8, 1.8 Hz, 2H),1.97 – 1.51 (m, 23H), 0.99 – 0.84 (ddd, J = 1.7, 7.0, 15.2 Hz, 6H). 13C NMR(101 MHz, DMSO-d6) δ 171.96, 171.62, 171.22, 169.89, 167.42, 166.00, 156.36,153.48, 142.95, 142.58, 140.70, 139.79, 139.68, 139.28, 138.48, 137.90,137.11, 134.31, 133.08, 131.45, 130.74, 129.93, 129.22, 128.35, 126.51,126.32, 124.65, 120.56, 35.61 ,34.84, 32.87, 31.99, 29.88, 28.11, 24.83, 23.58, 22.56, 21.63.

[0091]

[0092] Using a general synthetic method, through the intermediate SRS -A5 and N -Ac- D -Indanylglycine ( N -Ac- D -Igl-OH) SRSR A6, yield 73% 。 1 H NMR (500 MHz, DMSO- d 6) δ 10.07 (s, 1H), 9.99 (s, 1H), 8.58 (d, J = 8.4 Hz, 1H), 8.15 (d, J = 7.8 Hz, 1H), 8.12 – 8.06 (m, 2H), 8.01 – 7.93 (m, 1H), 7.83 – 7.73 (m, 2H), 7.61 (d, J = 8.2 Hz, 2H), 7.30 (m,5H), 7.16 – 7.13 (m, 1H), 7.10 – 7.08 (m, 1H), 7.07 – 7.00 (m, 5H), 6.65 (d,J = 16.1 Hz, 1H), 5.94 (ddt, J = 16.3, 10.7, 5.5 Hz, 1H), 5.84 (ddt, J =21.0, 10.6, 5.3 Hz, 1H), 5.31 (dq, J = 17.2, 1.7 Hz, 1H), 5.23 (dd, J = 3.9,2.2 Hz, 1H), 5.22 – 5.18 (m, 1H), 5.13 – 5.07 (m, 1H), 4.97 – 4.84 (m, 2H),4.64 (dd, J = 5.3, 1.7 Hz, 2H), 4.40 (t, J = 5.8 Hz, 1H), 4.25 (ddd, J =18.2, 11.6, 6.2 Hz, 4H), 3.17 (s, 3H), 3.14 (s, 1H), 2.96 – 2.59 (m, 9H),2.07 – 1.69 (m, 16H), 1.62 – 1.25 (m, 9H), 0.87 (ddd, J = 15.8, 6.5, 3.6 Hz,6H). 13 C NMR (126 MHz, DMSO- d6) δ 172.07, 171.95, 171.61, 171.15, 170.14,169.95, 166.06, 156.39, 153.52, 143.02, 142.82, 142.61, 139.85, 138.56,137.95, 137.74, 137.31, 134.37, 133.13, 131.73, 131.68, 130.80, 130.08,129.80, 129.30, 128.52, 128.40, 128.14, 126.60, 126.36, 36.98, 32.92, 29.56, 28.10, 24.84,23.55, 22.95, 22.82, 22.14, 22.09.

[0093]

[0094] Using a general synthetic method, through the intermediate SSR -A5 and N -Ac- D -Indanylglycine ( N -Ac- D -Igl-OH) SSRR -A6, yield 63%. 1 H NMR (500 MHz, DMSO- d 6) δ 10.10 (s, 1H), 10.02 (s, 1H), 8.61 (d, J = 8.4 Hz, 1H), 8.18 (d, J = 8.6 Hz, 1H), 8.11 (d, J = 7.2Hz, 2H), 7.99 (dd, J = 12.8, 7.7 Hz, 1H), 7.81 (dd, J = 17.8, 12.1 Hz, 2H),7.65 (d, J = 8.1 Hz, 2H), 7.34 (d, J= 7.5 Hz, 2H), 7.10 (dd, J = 19.7, 11.8Hz, 6H), 6.69 (d, J = 16.1 Hz, 1H), 5.98 (ddt, J = 16.2, 10.6, 5.5 Hz, 1H),5.88 (ddt, J = 15.7, 10.3, 5.1 Hz, 1H), 5.35 (d, J = 17.2 Hz, 1H), 5.26 (d, J = 10.0 Hz, 2H), 5.14 (dd, J = 10.9, 4.9 Hz, 1H), 5.01 – 4.87 (m, 2H) , 4.68 –4.56 (m, 3H), 4.44 (t, J = 5.8 Hz, 3H), 4.29 (dt, J = 26.3, 8.0 Hz, 2H), 3.21(s, 3H), 3.18 (s, 1H), 3.08 (d, J = 12.8 Hz, 1H), 3.01 – 2.62 (m, 7H), 2.07(s, 1H), 1.97 – 1.63 (m, 24H), 0.91 (dd, J = 15.0, 5.8 Hz, 6H). 13 C NMR (126MHz, DMSO- d6) δ 171.95, 171.61, 171.15, 170.13, 169.94, 166.06, 156.39,153.53, 143.02, 142.82, 142.66, 142.61, 139.85, 138.56, 137.95, 134.36,133.15, 131.55, 130.80, 130.07, 129.80, 129.30, 128.52, 128.40, 126.60,126.36, 124.67, 120.60, 119.43, 118.48, 117.32, 76.03, 65.20, 64.65, 57.03,54.52, 54.08, 53.60, 53.12, 52.56, 42.35, 41.27, 38.92, 38.60, 36.98, 35.92,35.38, 32.93, 29.74, 29.56, 28.10, 24.86, 23.55, 23.38, 22.96, 22.15, 22.10.

[0095]

[0096] Using a general synthetic method, through the intermediate RRS -A5 and N -Ac- D -Indanylglycine ( N -Ac- D -Igl-OH) RRSR -A6, yield 64%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.04 (s, 1H), 9.80 (s, 1H), 8.61 (d, J = 8.4 Hz, 1H), 8.10 (td, J = 10.5, 10.0, 7.6 Hz, 2H), 7.88 –7.78 (m, 2H), 7.66 (t, J = 8.1 Hz, 2H), 7.43 – 7.31 (m, 3H), 7.16 – 6.96 (m,4H), 6.70 (dd, J = 16.2, 2.1 Hz, 1H), 6.09 – 5.79 (m, 2H), 5.37 (dd, J=17.2, 1.8 Hz, 2H), 5.30 – 5.25 (m, 3H), 5.14 (ddd, J = 10.5, 3.1, 1.6 Hz,2H), 4.95 (dd, J = 21.5, 8.2 Hz, 3H), 4.72 – 4.67 (m, 4H), 4.44 (t, J = 4.9Hz, 5H), 4.34 – 4.13 (m, 4H), 3.23 (s, 1H), 3.17 (S, 1H), 3.09 – 2.65 (m,7H), 1.95 – 1.67 (m, 25H), 0.98 – 0.82 (m, 6H). 13 C NMR (101 MHz, DMSO) δ172.00, 171.80, 171.59, 171.46, 170.06, 169.93, 165.99, 156.30, 153.47,142.89, 142.76, 142.57, 139.79, 139.68, 138.49, 137.89, 137.62, 136.87,134.31, 133.08, 131.68, 131.48, 130.73, 129.96, 129.73, 129.21, 128.42,128.08, 126.55, 31.99, 31.59, 30.28, 29.88, 29.51, 28.05, 24.77, 23.44, 22.83, 22.78, 21.85.

[0097]

[0098] Using a general synthetic method, through the intermediate SRR -A5 and N -Ac- D -Indanylglycine ( N -Ac- D -Igl-OH) SRRR -A6, yield 73%。 1 H NMR (500 MHz, DMSO- d 6) δ 10.11 (s, 1H), 10.02(s, 1H), 8.61 (d, J = 8.4 Hz, 1H), 8.18 (t, J = 6.2 Hz, 1H), 8.12 (dt, J =8.4, 4.3 Hz, 2H), 7.99 (dd, J = 12.1, 7.7 Hz, 1H), 7.83 (d, J = 16.2 Hz, 1H),7.79 (d, J = 8.1 Hz, 1H), 7.65 (d, J = 8.1 Hz, 2H), 7.36 – 7.32 (m, 5H), 7.18(q, J = 4.7, 3.7 Hz, 1H), 7.13 (t, J = 3.2 Hz, 1H), 7.11 – 7.04 (m, 5H), 6.69(d, J = 16.1 Hz, 1H), 5.98 (ddt, J = 17.2, 10.7, 5.5 Hz, 1H), 5.88 (ddd, J =17.1, 10.6, 5.3 Hz, 1H), 5.35 (dd, J = 17.2, 1.7 Hz, 1H), 5.27 (q, J = 1.6Hz, 1H), 5.23 (dd, J = 4.0, 2.0 Hz, 1H), 5.14 (ddt, J = 10.5, 4.9, 1.5 Hz,1H), 5.01 – 4.88 (m, 2H), 4.67 (dt, J = 5.5, 1.5 Hz, 2H), 4.65 – 4.57 (m,1H), 4.44 (t, J= 5.6 Hz, 3H), 4.33 – 4.23 (m, 2H), 3.21 (s, 3H), 3.17 (s,1H), 3.01 – 2.62 (m, 9H), 1.95 – 1.84 (m, 16H), 1.77 – 1.51 (m, 9H), 0.91(ddd, J = 15.7, 6.5, 3.6 Hz, 6H). 13 C NMR (126 MHz, DMSO- d 6) δ 172.07, 171.95,171.61, 171.15, 170.15, 169.96, 166.06, 156.41, 153.52, 143.02, 142.61,139.84, 137.95, 134.36, 133.13, 131.73, 131.55, 130.80, 130.08, 129.79,129.30, 128.52, 128.40, 126.61, 124.71, 120.59, 119.43, 118.49, 117.33,76.02, 65.21, 64.66, 57.03, 56.25, 54.51, 54.08, 52.52, 46.86, 42.35, 38.99,38.60, 36.98, 36.09, 35.70, 32.92, 31.83, 29.56, 28.24, 28.10, 27.38, 24.84,23.52, 22.95, 22.82, 22.09.

[0099]

[0100] Using a general synthetic method, through the intermediate RSR -A5 and N -Ac- D -Indanylglycine ( N -Ac- D -Igl-OH) RSRR -A6, yield 71%. 1H NMR (400 MHz, DMSO-d6) δ 10.13 – 10.01 (d, J =12.5 Hz, 1H), 8.69 – 8.62 (d, J = 8.6 Hz, 0H), 8.46 – 8.38 (d, J = 8.0 Hz,0H), 8.37 – 8.28 (d, J = 7.7 Hz, 1H), 8.28 – 8.20 (t, J = 7.8, 7.8 Hz, 1H),8.16 – 8.05 (d, J = 7.3 Hz, 1H), 7.90 – 7.80 (m, 2H), 7.79 – 7.68 (dd, J =8.1, 13.2 Hz, 2H), 7.47 – 7.24 (dt, J = 8.9, 8.9, 26.9 Hz, 6H), 7.26 – 7.03(tdd, J = 6.5, 12.8, 20.6, 20.6 Hz, 4H), 6.81 – 6.66 (dd, J = 4.6, 16.1 Hz,1H), 6.05 – 5.83 (ddd, J = 5.9, 11.7, 36.2 Hz, 1H), 5.44 – 5.35 (d, J = 17.2Hz, 1H), 5.34 – 5.24 (m, 2H), 5.23 – 5.16 (d, J = 10.4 Hz, 1H), 5.07 – 4.91(d, J = 20.8 Hz, 2H), 4.78 – 4.65 (d, J = 5.4 Hz, 2H), 4.53 – 4.44 (dd, J =6.0, 15.5 Hz, 3H), 4.37 – 4.29 (t, J = 7.1, 7.1 Hz, 1H), 4.27 – 4.15 (t, J =7.7, 7.7 Hz, 1H), 3.27 – 3.24 (s, 2H), 3.24 – 3.20 (s, 1H), 3.05 – 2.91 (tt,J = 7.2, 7.2, 7.8, 12.1 Hz, 2H), 2.84 – 2.70 (ddd, J = 3.9, 12.4, 25.5 Hz,1H), 2.33 – 2.16 (dd, J = 9.0, 15.9 Hz, 5H), 2.03 – 1.50 (ddd, J = 22.4,34.8, 49.7 Hz, 20H), 1.02 – 0.80 (dq, J = 5.3, 6.0, 6.0, 11.9 Hz, 6H).

[0101] 13 C NMR (101 MHz, DMSO-d6) δ 172.05, 171.79, 171.61, 171.52, 170.28,169.78, 166.01, 156.33, 153.48, 142.71, 142.56, 139.80, 139.70, 138.49,137.90, 137.68, 134.31, 133.09, 131.65, 131.53, 129.97, 129.74, 129.22,128.49, 128.42, 126.56, 126.32, 124.62, 37.65, 36.92, 36.02, 35.28, 32.87, 32.08, 31.74, 29.50, 24.80, 23.54.

[0102]

[0103] Using a general synthetic method, through the intermediate RRR -A5 and N -Ac- D -Indanylglycine ( N -Ac- D -Igl-OH) RRRR -A6, yield 66%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.07 (d, J = 33.0 Hz,1H), 8.62 (d, J = 8.3 Hz, 1H), 8.20 (d, J = 7.7 Hz, 1H), 8.12 (dd, J = 8.0,4.4 Hz, 1H), 8.05 – 7.97 (m, 1H), 7.95 – 7.82 (m, 1H), 7.68 (d, J= 8.2 Hz,2H), 7.42 – 7.35 (m, 2H), 7.14 (td, J = 13.0, 12.2, 6.6 Hz, 5H) , 6.72 (d, J = 16.2 Hz, 1H), 6.07 – 5.88 (m, 2H), 5.45 – 5.35 (m, 1H), 5.33 – 5.25 (m,2H), 5.21 – 5.13 (m, 1H), 5.06 – 4.93 (m, 2H), 4.71 (d, J = 1.8 Hz, 1H), 4.65(dt, J = 10.9, 2.8 Hz, 1H), 4.48 (t, J = 5.3 Hz, 3H), 4.41 – 4.25 (m, 2H),3.25 (s, 3H), 3.22 (s, 1H), 3.05 – 2.55 (m, 7H). 2.03 – 1.51 (m, 25H), 0.95(ddd, J = 12.8, 6.4, 2.9 Hz, 6H). 13 C NMR (101 MHz, DMSO) δ 171.98, 171.88,170.07, 169.86, 165.99, 156.34, 153.46, 142.94, 142.74, 142.54, 139.78,138.49, 137.89, 137.66, 137.22, 134.30, 133.07, 131.65, 131.49, 130.84,130.73, 130.01, 129.73, 129.23, 128.45, 128.33, 128.07, 126.54, 126.30,120.54, 119.37, 118.41, 117.26, 75.95, 65.38, 65.13, 64.59, 56.97, 56.78,56.17, 54.00, 53.51, 52.44, 42.26, 41.22, 36.92, 35.65, 35.32, 32.87, 31.76,29.68, 29.50, 28.05, 24.78, 23.48, 22.88, 22.76, 22.09, 22.04, 15.62.

[0104] (7) Optically pure tetrapeptide probe compounds SSSS -A7,S SSR -A7, SSRS -A7, SRSS -A7, RSSS -A7, SSRR -A7, RRSS -A7, RSRS -A7, SRSR -A7, RSSR -A7, SRRS -A7, RSRR -A7, RRRS -A7, RRSR -A7, SRRR -A7, RRRR -Synthesis of A7:

[0105] General synthesis method: Under inert gas protection, SSSS -A6 or S SSR -A6 or SSRS -A6 or SRSS -A6 or RSSS -A6, SSRR -A6 or RRSS -A6 or RSRS -A6 or SRSR -A6 or RSSR -A6 or SRRS -A6 or RSRR -A6 or RRRS -A6 or RRSR -A6 or SRRR -A6 or RRRR -A6 (300 mg, 0.24 mmol), 1,3-dimethylbarbituric acid (DMBA) (150 mg, 0.96 mmol) and tetrakis(triphenylphosphine)palladium (30 mg, 0.024 mmol). The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane (20 ml) and methylene blue (1.0 mg, 0.003 mmol) were added. Under irradiation with a tungsten lamp (300w), oxygen was bubbled for 50 min and monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel (dichloromethane: methanol = 50:1) to obtain the corresponding target tetrapeptide probe compound. SSSS -A7,S SSR -A7, SSRS -A7, SRSS -A7, RSSS -A7, SSRR -A7, RRSS -A7, RSRS-A7, SRSR -A7, RSSR -A7, SRRS -A7, RSRR -A7, RRRS -A7, RRSR -A7, SRRR -A7, RRRR -A7, as a white solid.

[0106]

[0107] Using a general synthetic method, through the intermediate SSSS -A6 preparation SSSS -A7, yield 80%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.21 – 10.07 (m, 1H), 8.71 – 8.40 (m, 3H), 8.08 (d, J = 10.3 Hz,1H), 7.74 – 7.60 (m, 4H), 7.42 (m, 1H), 7.33 (d, J = 8.6 Hz, 1H), 7.26 (d,2H), 7.14 – 7.05 (m, 4H), 6.51 (dd, J = 17.0, 8.7 Hz, 1H), 4.90 (m, 2H), 4.66– 4.56 (m, 1H), 4.45 (d, J = 7.4 Hz, 1H), 4.36 (t, J = 7.6 Hz, 1H), 4.29 (s,1H), 4.14 (t, J = 4.6 Hz, 1H), 3.22 (s, 3H), 3.12 (s, 1H), 3.03 – 2.67 (m,9H), 1.98 – 1.52 (m, 25H), 0.89 (d, J = 8.9 Hz, 6H). 13C NMR (101 MHz, DMSO) δ171.83, 171.74, 171.53, 170.86, 169.82, 167.43, 153.05, 142.94, 142.79,142.57, 140.04, 139.50, 137.26, 135.95, 132.17, 132.04, 131.64, 131.38,130.31, 129.82, 129.11, 128.41, 128.29, 127.92, 126.52, 125.63, 124.65,75.55, 67.86, 60.71, 56.87, 56.32, 54.02, 52.94, 52.71, 42.08, 38.98, 38.93,38.55, 36.95, 35.63, 32.85, 32.20, 32.11, 31.70, 30.89, 30.26, 29.49, 28.83,28.17, 28.07, 27.64, 27.31, 24.83, 23.71, 23.37, 22.86, 22.55, 22.44, 22.11,14.36, 11.27. HRMS (ESI) calcd for C 62 H 75 Cl2N6O 11 + [M+H] + 1149.4865, found1149.4866.

[0108]

[0109] Using the general synthesis method, under the protection of inert gas, RSSS -A6, (300 mg, 0.24 mmol), 1,3-dimethylbarbituric acid (DMBA) (150 mg, 0.96 mmol) and tetrakis(triphenylphosphine)palladium (30 mg, 0.024 mmol). The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane (20 ml) and methylene blue (1.0 mg, 0.003 mmol) were added. Under irradiation of a tungsten lamp (300w), oxygen was bubbled for 50 min and monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel (dichloromethane: methanol = 50:1) to obtain the corresponding target tetrapeptide probe compound. RSSS -A7, a white solid, yield 56%. 1H NMR (500 MHz, DMSO- d 6) δ 8.35 (d, J = 7.9 Hz, 1H), 8.19 – 8.08 (m, 2H), 7.76 (d, J = 8.4 Hz,1H), 7.70 – 7.66 (m, 2H), 7.64 – 7.62 (m, 3H), 7.52 (dd, J = 8.9, 4.2 Hz,2H), 7.38 – 7.35 (m, 1H), 7.22 (d, J = 3.5 Hz, 2H), 7.10 – 7.08 (m, 2H), 7.03(m, 3H), 5.71 (s, 1H), 4.88 (d, J = 10.7 Hz, 1H), 4.80 (d, J = 10.6 Hz, 2H), 4.54 (s, 1H), 4.45 – 4.37 (m, 1H), 4.25 (s, 3H), 4.09 (t, J = 5.4 Hz, 1H),3.83 (s, 3H), 3.80 (s, 1H), 2.76 – 2.65 (m, 9H), 2.07 – 1.61 (m, 25H), 0.83(t, J = 7.3 Hz, 6H). HRMS (ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+H] + 1149.4865, found1149.4866.

[0110]

[0111] Using a general synthetic method, through the intermediate SRSS -A6 preparation SRSS -A7, yield 87%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.15 (d, J = 17.2 Hz, 1H), 8.60 – 8.22 (m, 3H), 8.08 (d, J= 10.3Hz, 1H), 7.72 – 7.59 (m, 4H), 7.44 – 7.39 (m, 1H), 7.33 (d, J = 8.6 Hz, 1H),7.31 – 7.21 (m, 2H), 7.16 – 6.99 (m, 4H), 6.50 (d, J = 16.1 Hz, 1H), 5.04 –4.77 (m, 2H), 4.58 (p, J = 6.0, 5.1 Hz, 1H), 4.45 (d, J = 7.4 Hz, 1H), 4.36(t, J = 7.6 Hz, 1H), 4.29 (s, 1H), 4.14 (t, J = 4.6 Hz, 1H), 3.22 (s, 3H), 3.17 (s, 1H), 2.85 – 2.74 (m, 9H), 2.02 – 1.63 (m, 25H), 0.89 (dd, J = 16.1,8.9 Hz, 6H). MS (ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+H] + 1149.49, found 1149.47.

[0112]

[0113] Using a general synthetic method, through the intermediate SSRS -A6 preparation SSRS -A7, yield 69%. 1 H NMR (500 MHz, DMSO- d 6) δ 9.82 (s, 1H), 8.50 – 8.44 (m, 1H), 8.40 (d, J = 7.2 Hz, 2H), 7.64(d, J = 8.2 Hz, 2H), 7.59 (d, J = 7.8 Hz, 1H), 7.55 (s, 2H), 7.37 (d, J = 8.3Hz, 3H),7.26 – 7.18 (m, 5H), 6.97 (d, J= 7.7 Hz, 1H), 6.50 (d, J = 15.9 Hz,1H), 4.99 – 4.76 (m, 4H), 4.48 (q, J = 7.5 Hz, 2h), 4.33 (s, 2H), 4.14 (d, J = 7.0 Hz, 1H), 3.14 (s, 3H), 3.13 (s, 3H), 3.11(s, 1H), 3.02 – 2.94 (m, 1H),2.89 – 2.84 (m, 1H), 2.77 (t, J = 6.7 Hz, 1H), 2.65 (t, J = 7.5 Hz, 3H), 1.90– 1.53 (m, 25H), 0.84 (d, J = 6.0 Hz, 3H), 0.80 (d, J = 6.0 Hz, 3H). 13 C NMR(126 MHz, DMSO- D 6) δ 172.30, 172.02, 171.65, 171.27, 170.28, 169.90, 152.81,140.10, 139.50, 137.09, 135.66, 131.82, 131.69, 131.60, 131.56, 131.44,131.42, 130.86, 130.27, 129.86, 129.85, 129.00, 128.46, 128.38, 127.91,125.60, 119.53, 75.49, 72.79, 60.78, 56.84, 54.95, 53.21, 52.94, 49.12,38.98, 38.57, 32.87, 32.04, 31.55, 29.46, 28.25, 28.09, 27.60, 24.89, 23.45,22.78, 22.63, 22.57, 22.08, 21.92. MS (ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+H] + 1149.49, found 1149.41.

[0114]

[0115] Using a general synthetic method, through the intermediate SSSR -A6 preparation SSSR -A7, yield 81%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.16 (s, 1H), 8.83 (s, 1H), 8.67 (d, J = 6.5 Hz, 1H), 7.89 (d, J= 7.2 Hz, 2H), 7.68 (m, 8H), 7.40 (m, 6H), 7.06 (d, J = 7.7 Hz, 1H), 6.62 (d,J = 15.7 Hz, 1H), 4.87 (s, 2H), 4.46 (s, 2H), 4.34 (d, J = 8.8 Hz, 2H), 4.23(d, J = 5.7 Hz, 1H), 4.15 (t, J = 6.0 Hz, 3H), 3.22 (m, 3H), 3.13 (s, 3H), 3.04 (s, 1H), 2.00 – 1.38 (m, 25H), 0.90 (t, J = 7.0 Hz, 6H). 13 C NMR (101MHz, DMSO- d 6) δ 172.21, 171.98, 171.70, 156.31, 152.83, 144.18, 144.12,141.09, 139.93, 139.49, 137.88, 137.61, 134.50, 134.41, 131.53, 131.22,129.70, 129.37, 129.04, 128.38, 128.04, 127.72, 127.46, 125.64, 121.81,120.46, 119.48, 75.47, 66.12, 66.07, 56.82, 56.41, 56.25, 55.35, 53.61,52.76, 49.06, 46.99, 38.55, 36.99, 36.94, 32.82, 31.75, 30.83, 30.28, 29.46,29.39, 29.15, 28.20, 28.19, 28.04, 27.36, 27.34, 24.84, 23.51, 22.65, 21.74.MS (ESI) calcd for C 62 H 74Cl2N6O 11 + [M+H] + 1149.49, found 1149.43.

[0116]

[0117] Using a general synthetic method, through the intermediate SSRR -A6 preparation SSRR -A7, yield 83%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.11 (d, J = 17.7 Hz, 1H), 8.59 (d, J = 8.2 Hz, 1H), 8.37 – 8.03(m, 3H), 7.87 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 8.6 Hz, 1H), 7.66 (t, J =12.0 Hz, 3H), 7.49 – 7.24 (m, 4H), 7.11 (d, J = 21.3 Hz, 3H), 6.59 (d, J =16.8 Hz, 1H), 5.75 (s, 1H), 4.87 (t, J = 15.2 Hz, 1H), 4.62 (d, J = 11.1 Hz,1H), 4.45-4.29 (m, 2H), 3.18 (s, 2H), 3.12 (s, 3H), 3.01 (s, 1H), 2.89 (s,1H), 2.72 (d, J = 12.6 Hz, 3H), 2.62 (s, 2H), 1.95 – 1.43 (m, 25H), 0.89 (t, J = 5.7 Hz, 6H). 13C NMR (101 MHz, CDCl3) δ 171.33, 170.42, 170.13, 165.27,155.78, 152.69, 142.74, 142.15, 140.14, 140.01, 138.41, 138.08, 137.36,137.09, 134.02, 131.94, 131.79, 131.13, 130.73, 129.74, 128.54, 127.62,126.67, 126.08, 124.08, 118.95, 117.12, 116.35, 74.56, 33.94, 31.88, 30.89, 30.48, 29.12,28.65, 27.32, 27.18, 23.93, 21.98, 21.66, 20.88, 19.99, 14.19, 13.16. MS(ESI) calculation for C 62 H 74 Cl2N6O 11 + [M+H] + 1149.49, found 1149.40.

[0118]

[0119] Using a general synthetic method, through the intermediate SRSR -A6 preparation SRSR -A7, yield 76%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.88 (d, J = 14.4 Hz, 1H), 8.37 – 8.08 (m, 1H), 7.95 (d, J = 18.7Hz, 2H), 7.66 – 7.47 (m, 2H), 7.36 (t, J = 14.6 Hz, 4H), 7.25 – 7.04 (m, 5H), 6.30 (d, J= 16.2 Hz, 1H), 4.60 (dt, J = 35.2, 11.5 Hz, 1H), 4.36 (d, J =26.3 Hz, 4H), 4.23 – 3.95 (m, 4H), 2.86 (d, J = 13.4 Hz, 6H), 2.57 – 2.15 (m,5H), 1.63 – 1.27 (m, 25H), 0.66 (d, J = 19.1 Hz, 6H). 13 C NMR (101 MHz, DMSO- d 6) δ 171.84, 171.63, 171.54, 171.40, 170.53, 169.94, 142.75, 142.54, 139.48,137.62, 137.20, 131.73, 131.68, 131.38, 129.96, 129.35, 128.68, 128.41,128.29, 126.54, 124.63, 124.41, 119.62, 111.75, 95.85, 75.57, 56.55, 56.37,52.99, 49.80, 42.34, 42.04, 37.05, 36.90, 36.34, 35.61, 35.30, 33.75, 33.48,32.16, 31.30, 29.44, 27.58, 25.98, 25.67, 24.80, 23.37, 22.79, 22.08. MS(ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+H] + 1149.49, found 1149.37.

[0120]

[0121] Using a general synthetic method, through the intermediate RRSS -A6 preparation RRSS -A7, yield 77%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.11 (d, J = 17.7 Hz, 1H), 8.59 (d, J= 8.2 Hz, 1H), 8.37 – 8.03(m, 3H), 7.87 (d, J = 8.2 Hz, 1H), 7.77 (d, J = 8.6 Hz, 1H), 7.66 (t, J =12.0 Hz, 3H), 7.49 – 7.24 (m, 4H), 7.11 (d, J = 21.3 Hz, 3H), 6.59 (d, J =16.8 Hz, 1H), 5.75 (s, 1H), 4.87 (t, J = 15.2 Hz, 1H), 4.62 (d, J = 11.1 Hz,1H), 4.45-4.29 (m, 2H), 3.18 (s, 2H), 3.12 (s, 3H), 3.01 (s, 1H), 2.89 (s,1H), 2.72 (d, J = 12.6 Hz, 3H), 2.62 (s, 2H), 1.95 – 1.43 (m, 25H), 0.89 (t, J = 5.7 Hz, 6H). MS (ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+2+H] + 1151.49, found1151.46.

[0122]

[0123] Using a general synthetic method, through the intermediate RSRS -A6 preparation RSRS -A7, yield 73%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.88 (d, J = 14.4 Hz, 1H), 8.37 – 8.08 (m, 1H), 7.95 (d, J = 18.7Hz, 2H), 7.66 – 7.47 (m, 2H), 7.36 (t, J = 14.6 Hz, 4H), 7.25 – 7.04 (m, 5H), 6.30 (d,J = 16.2 Hz, 1H), 4.60 (dt, J = 35.2, 11.5 Hz, 1H), 4.36 (d, J =26.3 Hz, 4H), 4.23 – 3.95 (m, 4H), 2.86 (d, J = 13.4 Hz, 6H), 2.57 – 2.15 (m,5H), 1.63 – 1.27 (m, 25H), 0.66 (d, J = 19.1 Hz, 6H). MS (ESI) calcd forC 62 H 74 Cl2N6O 11 + [M+H] + 1149.49, found 1149.54.

[0124]

[0125] Using a general synthetic method, through the intermediate SRRS -A6 preparation SRRS -A7, yield 75%. 1 H NMR (500 MHz, CDCl3) δ 9.40 (s, 1H), 8.29 (s, 1H), 7.65 (d, J = 16.0 Hz, 2H), 7.56 (d, J =7.9 Hz, 4H), 7.42 – 7.35 (m, 4H), 7.10 – 7.03 (m, 4H), 6.97 (d, J = 7.8 Hz,2H), 6.47 (d, J 0.82 (d, J = 6.2Hz, 6H). MS (ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+H] +1149.49, found 1149.36.

[0126]

[0127] Using a general synthetic method, through the intermediate RSSR -A6 preparation RSSR -A7, yield 75%. 1 H NMR (500 MHz, CDCl3) δ 9.40 (s, 1H), 8.29 (s, 1H), 7.65 (d, J = 16.0 Hz, 2H), 7.56 (d, J =7.9 Hz, 4H), 7.42 – 7.35 (m, 4H), 7.10 – 7.03 (m, 4H), 6.97 (d, J = 7.8 Hz,2H), 6.47 (d, J 0.82 (d, J = 6.2Hz, 6H). MS (ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+2+H] + 1151.49, found 1151.43.

[0128]

[0129] Using a general synthetic method, through the intermediate SRRR -A6 preparation SRRR -A7, yield 81%. 1 H NMR (500 MHz, DMSO- d 6) δ 8.35 (d, J = 7.9 Hz, 1H), 8.19 – 8.08 (m, 2H), 7.76 (d, J = 8.4Hz, 1H), 7.70 – 7.66 (m, 2H), 7.64 – 7.62 (m, 3H), 7.52 (dd, J= 8.9, 4.2 Hz,2H), 7.38 – 7.35 (m, 1H), 7.22 (d, J = 3.5 Hz, 2H), 7.10 – 7.08 (m, 2H), 7.03(m, 3H), 5.71 (s, 1H), 4.88 (d, J = 10.7 Hz, 1H), 4.80 (d, J = 10.6 Hz, 2H), 4.54 (s, 1H), 4.45 – 4.37 (m, 1H), 4.25 (s, 3H), 4.09 (t, J = 5.4 Hz, 1H),3.83 (s, 3H), 3.80 (s, 1H), 2.76 – 2.65 (m, 9H), 2.07 – 1.61 (m, 25H), 0.83(t, J = 7.3 Hz, 6H). MS (ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+H] + 1149.49, found1149.47.

[0130]

[0131] Using a general synthetic method, through the intermediate RSRR -A6 Preparation R SRR -A7, yield 68%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.15 (d, J = 17.2 Hz, 1H), 8.60 – 8.22 (m, 3H), 8.08 (d, J = 10.3Hz, 1H), 7.72 – 7.59 (m, 4H), 7.44 – 7.39 (m, 1H), 7.33 (d, J = 8.6 Hz, 1H),7.31 – 7.21 (m, 2H), 7.16 – 6.99 (m, 4H), 6.50 (d, J = 16.1 Hz, 1H), 5.04 –4.77 (m, 2H), 4.58 (p, J= 6.0, 5.1 Hz, 1H), 4.45 (d, J = 7.4 Hz, 1H), 4.36(t, J = 7.6 Hz, 1H), 4.29 (s, 1H), 4.14 (t, J = 4.6 Hz, 1H), 3.22 (s, 3H), 3.17 (s, 1H), 2.85 – 2.74 (m, 9H), 2.02 – 1.63 (m, 25H), 0.89 (dd, J = 16.1,8.9 Hz, 6H). MS (ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+H] + 1149.49, found 1149.47.

[0132]

[0133] Using a general synthetic method, through the intermediate RRSR -A6 preparation RRSR -A7, yield 82%. 1 H NMR (500 MHz, DMSO- d 6) δ 9.82 (s, 1H), 8.50 – 8.44 (m, 1H), 8.40 (d, J = 7.2 Hz, 2H), 7.64(d, J = 8.2 Hz, 2H), 7.59 (d, J = 7.8 Hz, 1H), 7.55 (s, 2H), 7.37 (d, J = 8.3Hz, 3H),7.26 – 7.18 (m, 5H), 6.97 (d, J = 7.7 Hz, 1H), 6.50 (d, J = 15.9 Hz,1H), 4.99 – 4.76 (m, 4H), 4.48 (q, J = 7.5 Hz, 2h), 4.33 (s, 2H), 4.14 (d, J= 7.0 Hz, 1H), 3.14 (s, 3H), 3.13 (s, 3H), 3.11(s, 1H), 3.02 – 2.94 (m, 1H), 2.89 – 2.84 (m, 1H), 2.77 (t, J = 6.7 Hz, 1H), 2.65 (t, J = 7.5 Hz, 3H), 1.90– 1.53 (m, 25H), 0.84 (d, J = 6.0 Hz, 3H), 0.80 (d, J = 6.0 Hz, 3H). 13 C NMR (126 MHz, DMSO- D 6) δ 172.30, 172.02, 171.65, 171.27, 170.28, 169.90, 152.81,140.10, 139.50, 137.09, 135.66, 131.82, 131.69, 131.60, 131.56, 131.44,131.42, 130.86, 130.27, 129.86, 129.85, 129.00, 128.46, 128.38, 127.91,125.60, 119.53, 75.49, 72.79, 60.78, 56.84, 54.95, 53.21, 52.94, 49.12,38.98, 38.57, 32.87, 32.04, 31.55, 29.46, 28.25, 28.09, 27.60, 24.89, 23.45,22.78, 22.63, 22.57, 22.08, 21.92. MS (ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+H] + 1149.49, found 1149.42.

[0134]

[0135] Using the general synthesis method, under the protection of inert gas, RRRS-A6, (300 mg, 0.24 mmol), 1,3-dimethylbarbituric acid (DMBA) (150 mg, 0.96 mmol) and tetrakis(triphenylphosphine)palladium (30 mg, 0.024 mmol). The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane (20 ml) and methylene blue (1.0 mg, 0.003 mmol) were added. Under irradiation of a tungsten lamp (300w), oxygen was bubbled for 50 minutes and monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (dichloromethane: methanol = 50:1) to obtain the corresponding target tetrapeptide probe compound. RRRS -A7, as a white solid, yield 58%. 1 H NMR (400 MHz, DMSO- d 6) δ 10.16 (s, 1H), 8.83 (s, 1H), 8.67 (d, J = 6.5Hz, 1H), 7.89 (d, J = 7.2 Hz, 2H), 7.68 (m, 8H), 7.40 (m, 6H), 7.06 (d, J =7.7 Hz, 1H), 6.62 (d, J = 15.7 Hz, 1H), 4.87 (s, 2H), 4.46 (s, 2H), 4.34 (d,J = 8.8 Hz, 2H), 4.23 (d, J = 5.7 Hz, 1H), 4.15 (t, J = 6.0 Hz, 3H), 3.22 (m,3H), 3.13 (s, 3H), 3.04 (s, 1H), 2.00 – 1.38 (m, 25H), 0.90 (t, J = 7.0 Hz,6H). MS (ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+H] + 1149.49, found 1149.45.

[0136]

[0137] Using a general synthetic method, through the intermediate RRRR -A6 preparation RRRR -A7, yield 69%. 1 H NMR (400 MHz, DMSO- d 6) δ 9.88 (d, J= 14.4 Hz, 1H), 8.37 – 8.19 (m, 1H), 7.95 (d, J = 18.7Hz, 2H), 7.62 – 7.47 (m, 2H), 7.41 – 7.01 (m, 8H), 6.30 (d, J = 16.2 Hz, 1H),4.63 (dd, J = 27.7, 13.4 Hz, 1H), 4.36 (d, J = 26.3 Hz, 1H), 4.20 – 3.92 (m,3H), 2.91 – 2.53 (m, 5H), 1.72 – 1.39 (m, 30H), 0.66 (d, J = 19.1 Hz, 7H). 13 CNMR (101 MHz, DMSO) δ 171.84, 171.63, 171.40, 170.53, 169.94, 142.90, 142.75,142.54, 139.48, 137.62, 137.20, 135.31, 133.57, 131.73, 131.66, 131.44,129.94, 129.35, 128.41, 128.29, 126.99, 126.54, 125.80, 124.69, 124.68,124.41, 119.62, 119.57, 111.75, 95.85, 75.57, 56.55, 56.37, 53.03, 49.80,35.61, 35.59, 32.29, 32.14, 27.63, 25.98, 25.65, 24.80, 23.37, 22.79, 22.08,14.23. MS (ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+H] + 1149.49, found 1149.38.

[0138] Example 2: Synthesis of optically pure tetrapeptide probe A8 series:

[0139] General synthesis method: Under inert gas protection, piperidine (0.494 mL, 5 mmol) was added to DMF (5 mL) dissolved in A5 (1248 mg, 1.0 mmol). The solution was stirred at room temperature for 30 min and the reaction was monitored by thin layer chromatography (TLC) (Hex: EtOAc = 50: 50). After observing that Fmoc was completely deprotected, the mixture was dissolved in ethyl acetate and washed twice with 0.1M hydrochloric acid (50 mL) and brine (50 mL). Dry over anhydrous sodium sulfate and remove the solvent under reduced pressure. Then, under inert gas protection, the deprotected A5, N -acetylbenzylcysteine ​​(253 mg, 1.0 mmol), benzotriazole- N, N, N, N -Tetramethyluronium hexafluorophosphate (HBTU) (546 mg, 1.44 mmol) was dissolved in DMF (25 mL), N,N-diisopropylethylamine (DIPEA) (0.348 mL, 2.0 mmol) was added, and stirred at room temperature for 60 min. It was monitored by thin layer chromatography (TLC) (Hex: EtOAc = 50:50). After the reaction was completed, the mixture was diluted with ethyl acetate (100 mL), washed with 0.1M hydrochloric acid (50 mL) and brine (50 mL). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75:25). A white solid intermediate was obtained. Under the protection of inert gas, the intermediate (630 mg, 0.5 mmol), 1,3-dimethylbarbituric acid (DMBA) (313 mg, 0.96 mmol) and tetrakis(triphenylphosphine)palladium (62.5 mg, 0.05 mmol) were added. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane (30 mL) and methylene blue (2.0 mg, 0.006 mmol) were added. The reaction mixture was heated under a tungsten lamp (300 w). ） Under irradiation, oxygen was bubbled for 1.0 h and monitored by thin layer chromatography (TLC) (Hex: EtOAc = 50: 50). After the reaction was completed, the solvent was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain the tetrapeptide probe A8 series as a white solid.

[0140]

[0141] The general synthesis method is: SSS -A5 and N -Acetyl- D SSSR-A9 was prepared by reaction of -benylcysteine ​​with a yield of 80%. 1H NMR (500 MHz, DMSO- d 6) δ 10.18 (s, 1H), 8.53 (d, J = 14.2 Hz, 1H),8.27 – 8.06 (m, 3H), 7.64 (dd, J = 8.2, 3.6 Hz, 4H), 7.46 – 7.38 (m, 2H),7.31 – 7.26 (m, 5H), 7.22 (m, 5H), 7.04 (d, J = 8.0 Hz, 1H), 6.55 – 6.42 (m,1H), 4.99 (dd, J = 28.1, 6.4 Hz 1H), 4.92 – 4.71 (m 2H), 4.58 – 4.40 (m 4H),4.33 (t, J = 7.1 Hz 1H), 3.71 (d, J = 4.0 Hz 2H), 3.64 – 3.61 (m 1H), 3.21(s, 3H), 3.18 (s, 1H), 3.17 (s, 1H), 3.02 (d, J = 5.4 Hz, 2H), 2.98 (s, 1H),2.66 – 2.61 (m, 1H), 2.45 (dd, J = 13.5, 8.5 Hz, 1H), 2.00 – 1.72 (m, 17H),1.63 (d, J = 13.2 Hz, 2H), 1.56 – 1.50 (m, 2H), 1.30 (m, , 1H), 0.92 (d, J =6.4 Hz, 3H), 0.88 (dd, J = 6.5, 2.4 Hz, 1H). 13 C NMR (126 MHz, DMSO- d6) δ171.84, 171.73, 170.77, 170.61, 169.88, 169.80, 162.84, 153.01, 149.80,140.12, 139.90, 139.58, 138.99, 138.86, 137.30, 137.06, 135.80, 131.76,131.45, 131.39, 130.32, 129.87, 129.42, 128.97, 128.86, 128.31, 127.97,127.30, 119.57, 75.56, 72.80, 63.56, 60.77, 56.90, 55.44, 53.02, 52.71,40.84, 39.02, 38.63, 37.01, 36.31, 35.72, 35.41, 33.42, 32.90, 29.54, 28.12,27.66, 24.89, 23.45, 22.98, 22.63, 22.18, 22.14.

[0142]

[0143] The general synthesis method is adopted: under the protection of inert gas, the deprotected SSS -A5, N -Acetyl- L -Benzylcysteine, benzotriazole- N, N, N, N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75: 25). A white solid intermediate was obtained. Under the protection of inert gas, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under a tungsten lamp (300 w ） Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the reaction is completed, the solvent is concentrated under reduced pressure, and the crude product is purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a tetrapeptide probe SSSS - A8 is a white solid.

[0144]

[0145] The general synthesis method is adopted: under the protection of inert gas, the deprotected RSS -A5, N -Acetyl- L -Benzylcysteine, benzotriazole- N, N, N, N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75: 25). A white solid intermediate was obtained. Under the protection of inert gas, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under a tungsten lamp (300 w ） Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the reaction is completed, the solvent is concentrated under reduced pressure, and the crude product is purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a tetrapeptide probe RSSS - A8 is a white solid.

[0146]

[0147] The general synthesis method is adopted: under the protection of inert gas, the deprotected SRS -A5, N -Acetyl- L -Benzylcysteine, benzotriazole- N, N, N, N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75: 25). A white solid intermediate was obtained. Under the protection of inert gas, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under a tungsten lamp (300 w ） Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the reaction is completed, the solvent is concentrated under reduced pressure, and the crude product is purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a tetrapeptide probe SSSS - A8 is a white solid.

[0148]

[0149] The general synthesis method is adopted: under the protection of inert gas, the deprotected SSR -A5, N -Acetyl- L -Benzylcysteine, benzotriazole- N, N, N, N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75: 25). A white solid intermediate was obtained. Under the protection of inert gas, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under a tungsten lamp (300 w ） Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the reaction is completed, the solvent is concentrated under reduced pressure, and the crude product is purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a tetrapeptide probe SSSS - A8 is a white solid.

[0150]

[0151] The general synthesis method is adopted: under the protection of inert gas, the deprotected RSS -A5, N -Acetyl- D -Benzylcysteine, benzotriazole- N, N, N, N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75: 25). A white solid intermediate was obtained. Under the protection of inert gas, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under a tungsten lamp (300 w ） Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the reaction is completed, the solvent is concentrated under reduced pressure, and the crude product is purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a tetrapeptide probeSSSS - A8 is a white solid.

[0152]

[0153] The general synthesis method is adopted: under the protection of inert gas, the deprotected SSR -A5, N -Acetyl- D -Benzylcysteine, benzotriazole- N, N, N, N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75: 25). A white solid intermediate was obtained. Under the protection of inert gas, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under a tungsten lamp (300 w ） Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the reaction is completed, the solvent is concentrated under reduced pressure, and the crude product is purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a tetrapeptide probe SSSS - A8 is a white solid.

[0154]

[0155] The general synthesis method is adopted: under the protection of inert gas, the deprotected RRS -A5, N -Acetyl- L -Benzylcysteine, benzotriazole- N, N, N, N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75: 25). A white solid intermediate was obtained. Under the protection of inert gas, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under a tungsten lamp (300 w ）Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the reaction is completed, the solvent is concentrated under reduced pressure, and the crude product is purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a tetrapeptide probe SSSS - A8 is a white solid.

[0156]

[0157] The general synthesis method is adopted: under the protection of inert gas, the deprotected SRR -A5, N -Acetyl- L -Benzylcysteine, benzotriazole- N, N, N, N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75: 25). A white solid intermediate was obtained. Under the protection of inert gas, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under a tungsten lamp (300 w ） Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the reaction is completed, the solvent is concentrated under reduced pressure, and the crude product is purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a tetrapeptide probe SSSS - A8 is a white solid.

[0158]

[0159] The general synthesis method is adopted: under the protection of inert gas, the deprotected RSR -A5, N -Acetyl- L -Benzylcysteine, benzotriazole- N, N, N, N-Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75: 25). A white solid intermediate was obtained. Under the protection of inert gas, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under a tungsten lamp (300 w ） Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the reaction is completed, the solvent is concentrated under reduced pressure, and the crude product is purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a tetrapeptide probe SSSS - A8 is a white solid.

[0160]

[0161] The general synthesis method is adopted: under the protection of inert gas, the deprotected SRS -A5, N -Acetyl- D -Benzylcysteine, benzotriazole- N, N, N, N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75: 25). A white solid intermediate was obtained. Under the protection of inert gas, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under a tungsten lamp (300 w ） Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the reaction is completed, the solvent is concentrated under reduced pressure, and the crude product is purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a tetrapeptide probe SSSS - A8 is a white solid.

[0162]

[0163] The general synthesis method is adopted: under the protection of inert gas, the deprotected RSR -A5, N-Acetyl- D -Benzylcysteine, benzotriazole- N, N, N, N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75: 25). A white solid intermediate was obtained. Under the protection of inert gas, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under a tungsten lamp (300 w ） Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the reaction is completed, the solvent is concentrated under reduced pressure, and the crude product is purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a tetrapeptide probe SSSS - A8 is a white solid.

[0164]

[0165] The general synthesis method is adopted: under the protection of inert gas, the deprotected SRR -A5, N -Acetyl- D -Benzylcysteine, benzotriazole- N, N, N, N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75: 25). A white solid intermediate was obtained. Under the protection of inert gas, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under a tungsten lamp (300 w ） Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the reaction is completed, the solvent is concentrated under reduced pressure, and the crude product is purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a tetrapeptide probe SSSS - A8 is a white solid.

[0166]

[0167] The general synthesis method is adopted: under the protection of inert gas, the deprotected RRS -A5, N -Acetyl- D -Benzylcysteine, benzotriazole- N, N, N, N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75: 25). A white solid intermediate was obtained. Under the protection of inert gas, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under a tungsten lamp (300 w ） Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the reaction is completed, the solvent is concentrated under reduced pressure, and the crude product is purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a tetrapeptide probe SSSS - A8 is a white solid.

[0168]

[0169] The general synthesis method is adopted: under the protection of inert gas, the deprotected RRR -A5, N -Acetyl- L -Benzylcysteine, benzotriazole- N, N, N, N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75: 25). A white solid intermediate was obtained. Under the protection of inert gas, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under a tungsten lamp (300 w ） Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the reaction is completed, the solvent is concentrated under reduced pressure, and the crude product is purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a tetrapeptide probe SSSS - A8 is a white solid.

[0170]

[0171] The general synthesis method is adopted: under the protection of inert gas, the deprotected RRR -A5, N -Acetyl- D -Benzylcysteine, benzotriazole- N, N, N, N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75: 25). A white solid intermediate was obtained. Under the protection of inert gas, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under a tungsten lamp (300 w ） Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the reaction is completed, the solvent is concentrated under reduced pressure, and the crude product is purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a tetrapeptide probe SSSS - A8 is a white solid.

[0172] Example 3: Synthesis of optically pure tripeptide probe Fmoc-A5-O2 series:

[0173] General synthesis method: Under the protection of inert gas, A5 (416 mg, 0.3 mmol), 1,3-dimethylbarbituric acid (DMBA) (150 mg, 0.96 mmol) and tetrakis(triphenylphosphine)palladium (38 mg, 0.03 mmol) were added. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane (20 mL) and methylene blue (1.0 mg, 0.003 mmol) were added. Under tungsten lamp (300 w), oxygen was bubbled for 50 min and monitored by thin layer chromatography (TLC) (CH2Cl2: MeOH = 30: 1). After the reaction was completed, the solvent was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a white solid Fmoc-A5-O 2。

[0174]

[0175] Using the general synthesis method: Fmoc-SSS-A5 was prepared 5-O2, yield 88%. 1 H NMR (500 MHz, DMSO- d 6) δ 10.13 (s, 1H), 8.70 (s, 1H), 7.82 (m, 3H), 7.73 (d, J = 8.5 Hz, 1H), 7.59 (m, 5H), 7.55 (d, J = 7.7 Hz, 1H), 7.39 – 7.35 (m, 2H), 7.34 – 7.28 (m, 4H), 7.24 (d, J = 7.5 Hz, 1H), 7.22 (d, J = 2.0 Hz, 2H), 7.21 – 7.18 (m, 3H), 7.13 (d, J = 7.7 Hz, 2H), 6.97 (d, J = 7.9 Hz, 1H), 4.87 – 4.68 (m, 2H), 4.36 (s, 1H), 4.30 – 4.22 (m, 2H), 4.16 – 4.03 (m, 3H), 3.12 (s, 3H), 3.04 (s, 1H), 2.76 – 2.64 (m, 3H), 1.92 – 1.30 (m, 24H), 0.80 (t, J = 7.0 Hz, 6H). 13 C NMR (126 MHz, DMSO- D6) δ 172.44, 172.10, 171.84, 171.82, 156.39, 144.25,144.21, 143.09, 141.17, 141.16, 140.06, 139.94, 139.55, 137.95, 137.87,131.61, 131.46, 129.76, 129.43, 128.73, 128.44, 128.12, 127.82, 127.54,125.84, 121.91, 120.56, 119.50, 110.30, 75.48, 66.18, 56.86, 56.56, 47.03,39.10, 38.91, 38.59, 37.00, 32.87, 29.51, 28.25, 28.10, 27.56, 24.90, 23.57,23.55, 22.72, 21.80, 21.58, 19.09, 15.53.

[0176]

[0177] The general synthesis method was adopted: Fmoc-SSR-A5-O2 was prepared from SSR-A5 with a yield of 85%. 1 H NMR (500 MHz, DMSO- d 6) δ 7.84 (d, J = 7.6 Hz, 2H), 7.80 (d, J = 7.5 Hz, 2H), 7.76 (d, J =8.0 Hz, 1H), 7.65 – 7.60 (m, 3H), 7.41 (d, J = 8.1 Hz, 1H), 7.37 (t, J = 7.5Hz, 2H), 7.37 – 7.25 (m, 4H), 7.20 (m, 5H), 7.13 (d, J = 8.1 Hz, 2H), 7.00(t, J = 6.3 Hz, 1H), 6.44 (d, J = 16.0 Hz, 1H), 4.34 (m, 3H), 4.15 (d, J=6.4 Hz, 2H), 3.17 (s, 3H), 3.14(s, 1H), 2.96 – 2.82 (m, 2H), 2.64 (m, 2H),1.94 – 1.54 (m, 24H), 0.83 (d, J = 6.9 Hz, 6H). 13 C NMR (126 MHz, DMSO- D 6) δ174.34, 172.23, 171.57, 170.18, 152.91, 143.09, 139.94, 137.99, 137.94,137.87, 131.77, 131.70, 131.36, 130.39, 129.82, 129.81, 129.45, 128.73,128.44, 128.34, 127.82, 125.84, 121.91, 120.56, 119.51, 119.50, 110.29,56.91, 56.14, 55.44, 52.71, 39.08, 38.93, 38.60, 38.58, 36.99, 32.90, 32.87,29.52, 28.25, 28.22, 28.13, 28.09, 27.33, 24.92, 23.67, 23.56, 22.55, 21.81,21.57, 21.51.

[0178]

[0179] The general synthesis method is adopted: under the protection of inert gas, SRR -A5, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred at room temperature for reaction. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under the irradiation of a tungsten lamp (300 W), oxygen was bubbled for 50 min and monitored by thin layer chromatography. After the reaction was completed, the solvent was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain the tripeptide probe Fmoc- SRR - A5-O2 is a white solid.

[0180]

[0181] The general synthesis method is adopted: under the protection of inert gas, SRS-A5, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred at room temperature for reaction. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under the irradiation of a tungsten lamp (300 W), oxygen was bubbled for 50 min and monitored by thin layer chromatography. After the reaction was completed, the solvent was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain the tripeptide probe Fmoc- SRS - A5-O2 is a white solid.

[0182]

[0183] The general synthesis method is adopted: under the protection of inert gas, RSS -A5, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred at room temperature for reaction. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under the irradiation of a tungsten lamp (300 W), oxygen was bubbled for 50 min and monitored by thin layer chromatography. After the reaction was completed, the solvent was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain the tripeptide probe Fmoc- RSS - A5-O2 is a white solid.

[0184]

[0185] The general synthesis method is adopted: under the protection of inert gas, RSR -A5, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred at room temperature for reaction. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under the irradiation of a tungsten lamp (300 W), oxygen was bubbled for 50 min and monitored by thin layer chromatography. After the reaction was completed, the solvent was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain the tripeptide probe Fmoc- RSR- A5-O2 is a white solid.

[0186]

[0187] The general synthesis method is adopted: under the protection of inert gas, RRR-A5, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred at room temperature for reaction. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under the irradiation of a tungsten lamp (300 W), oxygen was bubbled for 50 min and monitored by thin layer chromatography. After the reaction was completed, the solvent was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain the tripeptide probe Fmoc- RRR- A5-O2 is a white solid.

[0188]

[0189] The general synthesis method is adopted: under the protection of inert gas, RRS -A5, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred at room temperature for reaction. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under the irradiation of a tungsten lamp (300 W), oxygen was bubbled for 50 min and monitored by thin layer chromatography. After the reaction was completed, the solvent was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain the tripeptide probe Fmoc- RRS- A5-O2 is a white solid.

[0190] Example 4: Synthesis of optically pure tripeptide probe Ac-A5-O2 series:

[0191] General synthesis method: Under inert gas protection, piperidine (0.494 mL, 5 mmol) was added to a solution of SS -A4 (1068 mg, 1.0 mmol) in DMF (5 mL). The solution was stirred at room temperature for 30 min and the reaction was monitored by thin layer chromatography (TLC). After observing that Fmoc was completely removed, the mixture was dissolved in ethyl acetate and washed twice with 0.1 M hydrochloric acid (50 mL) and brine (50 mL). Drying was performed over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Then, the deprotected SS -A4, N -Vinyl- L -4-Chlorophenylalanine ( S -Ac-Phe(4-Cl)-OH) (422 mg, 1.0 mmol), benzotriazole- N, N,N,N-Tetramethyluronium hexafluorophosphate (HBTU) (546 mg, 1.44 mmol) was dissolved in DMF (25 mL), and N,N-diisopropylethylamine (DIPEA) (0.348 mL, 2.0 mmol) was added. The mixture was stirred at room temperature for 60 min and monitored by thin layer chromatography (TLC). After the reaction was completed, the mixture was diluted with ethyl acetate (100 mL), washed with 0.1M hydrochloric acid (50 mL) and brine (50 mL). The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75:25) to obtain a white solid intermediate.

[0192]

[0193] The general synthesis method was adopted: taking SS-A4 as an example, the intermediate Ac-SSS-A5 was prepared with a yield of 75%. 1 H NMR (500 MHz, DMSO- d 6) δ 9.51 (s, 1H), 8.38 (dd, J = 18.1, 7.1 Hz, 2H), 8.16 (d, J = 8.1 Hz, 1H), 7.90 (d, J = 9.1 Hz, 1H), 7.76 (d, J = 9.7 Hz, 2H), 7.64 (d, J = 8.6 Hz, 2H), 7.31 (d, J = 8.7 Hz, 2H), 7.28 (dd, J = 8.4, 1.7 Hz, 2H),7.23 (d, J = 8.6 Hz, 2H), 7.20 (d, J = 8.4 Hz, 2H), 7.06 (d, J = 8.1 Hz, 1H),6.65 (d, J = 16.3 Hz, 1H), 5.95 (ddt, J = 17.3, 10.7, 5.5 Hz, 1H), 5.84 (ddt, J = 17.2, 10.5, 5.3 Hz, 1H), 5.32 (dd, J = 17.2, 1.7 Hz, 1H), 5.25 – 5.17 (m,2H), 5.10 (dt,J = 10.5, 1.6 Hz, 1H), 4.98 – 4.80 (m,2H), 4.64 (dt, J = 5.5,1.5 Hz, 2H), 4.43 (d, J = 7.4 Hz, 1H), 4.41 – 4.38 (m, 2H), 4.38 – 4.27 (m,2H), 4.01 (s, 1H), 3.17(s, 3H), 3.13(s, 1H), 2.99 (dd, J = 13.8, 4.9 Hz, 1H),2.90 – 2.84 (m, 2H), 1.90 – 1.58 (m, 25H), 0.86 (d, J = 6.2 Hz, 1H), 0.82 (d, J = 6.4 Hz, 1H). 13 C NMR (126 MHz, DMSO- d 6) δ 173.50, 172.83, 172.09, 171.51,170.54, 169.80, 166.09, 162.84, 156.38, 153.52, 139.84, 139.67, 138.54,137.96, 137.26, 136.89, 134.35, 133.13, 131.67, 131.59, 131.48, 130.98,130.04, 129.77, 129.28, 128.54, 128.16, 126.37, 120.60, 119.47, 118.54,117.33, 65.23, 64.64, 57.05, 55.22, 53.79, 38.59, 37.21, 36.58, 32.92, 31.29,30.32, 29.51, 28.08, 24.88, 23.55, 22.83, 21.81.

[0194] General synthesis method: Under the protection of inert gas, the intermediate obtained in the above reaction (343 mg, 0.3 mmol), 1,3-dimethylbarbituric acid (DMBA) (150 mg, 0.96 mmol) and tetrakis(triphenylphosphine)palladium (38 mg, 0.03 mmol) were added. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane (20 mL) and methylene blue (1.0 mg, 0.003 mmol) were added. Under tungsten lamp (300 w), oxygen was bubbled for 50 min and monitored by thin layer chromatography (TLC) (CH2Cl2: MeOH = 30: 1). After the reaction was completed, the solvent was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain the optically pure tripeptide probe Ac-A5-O2 series.

[0195]

[0196] The general synthesis method is: Ac-SSS-A5 is used to prepare Ac- SSS -A5-O2, yield 62%. 1 H NMR (500 MHz, DMSO- d 6) δ 10.26 (s, 1H), 8.70 (s, 1H), 8.37 (s, 1H), 8.21 (d, J = 8.1 Hz,1H), 7.63 (m, 4H), 7.39 (d, J = 8.2 Hz, 2H), 7.25 (m, 3H), 7.02 (d, J = 7.8Hz, 1H), 6.53 (d, J = 15.9 Hz, 1H), 5.00 – 4.68 (m, 2H), 4.54 (td, J = 9.0,3.9 Hz, 1H), 4.43 (q, J = 7.8 Hz, 2H), 4.34 (q, J = 7.2 Hz, 2H), 3.49 (t, J =5.2 Hz, 1H), 3.42 (t, J = 5.2 Hz, 1H), 3.19 (s, 3H), 3.17 (s, 1H), 2.70 (m,3H), 1.95 (s, 1H), 1.90 – 1.52 (m, 24H), 0.90 (d, J= 6.1 Hz, 3H), 0.86 (d, J = 6.2 Hz, 3H). 13 C NMR (126 MHz, DMSO- D 6) δ 172.02, 171.77, 171.56, 171.33,169.82, 152.90, 140.13, 139.61, 137.55, 135.64, 131.86, 131.66, 131.56,131.43, 131.35, 130.27, 129.83, 129.78, 128.96, 128.38, 127.90, 125.57,119.54, 72.81, 60.78, 56.86, 55.43, 54.29, 53.12, 52.85, 49.13, 40.85, 40.42,40.25, 38.90, 38.54, 37.37, 36.86, 32.84, 32.09, 29.51, 28.30, 28.10, 27.72,24.87, 23.43, 22.92, 22.60, 22.10.

[0197]

[0198] Using the above general synthesis method, SS -A4 and N -Vinyl- D -4-Chlorophenylalanine ( R -Ac-Phe(4-Cl)-OH), through two steps of reaction, to prepare Ac- SSR -A5-O2, yield 65%. 1 H NMR (500 MHz, DMSO- d 6) δ 9.82 (s,1H), 8.49 – 8.44 (m, 1H), 8.40 (d, J = 7.2 Hz, 2H), 7.66 – 7.52 (m, 5H), 7.37(d, J = 8.4 Hz, 2H), 7.25 – 7.19 (m, 4H), 6.99 – 6.96 (m, 1H), 6.49 (d, J =15.9Hz, 1H), 4.91 – 4.74 (m, 2H), 4.48 (t, J= 7.5 Hz, 1H), 4.33 (s, 1H), 4.14 (d, J = 6.8 Hz, 1H), 3.45 (t, J = 5.2 Hz, 1H), 3.38 (t, J = 5.2 Hz, 1H), 3.14 (s, 3H), 3.11 (s, 1H), 2.87 (d, J = 7.7 Hz, 1H), 2.76 (dd, J = 13.5, 8.6Hz, 1H), 2.65 (t, J = 7.5 Hz, 2H), 1.95 – 1.45 (m, 25H), 0.84 (d, J = 6.0 Hz,3H), 0.80 (d, J = 5.9 Hz, 3H). 13 C NMR (126 MHz, DMSO- D 6) δ 172.02, 171.77,171.56, 171.33, 169.82, 152.90, 140.13, 139.61, 137.55, 135.64, 131.86,131.66, 131.56, 131.43, 131.35, 130.27, 129.83, 129.78, 128.96, 128.38,127.90, 125.57, 119.54, 72.81, 60.78, 56.86, 55.43, 54.29, 53.12, 52.85,49.13, 40.85, 40.42, 40.25, 38.90, 38.54, 37.37, 36.86, 32.84, 32.09, 29.51,28.30, 28.10, 27.72, 24.87, 23.43, 22.92, 22.60, 22.10.

[0199]

[0200] Using the above general synthesis method, SR -A4 and N -Vinyl- D -4-Chlorophenylalanine ( R -Ac-Phe(4-Cl)-OH), through two steps of reaction, to prepare Ac- SRR-A5-O2: Under the protection of inert gas, piperidine is added to the solution SR -A4 in DMF. The solution was stirred at room temperature for 30 min and the reaction was monitored by thin layer chromatography (TLC). After observing that Fmoc was completely removed, the mixture was dissolved in ethyl acetate and washed twice with 0.1 M hydrochloric acid and brine. It was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Then, the deprotected SR -A4, N -Vinyl- D -4-Chlorophenylalanine ( R -Ac-Phe(4-Cl)-OH), benzotriazole- N,N,N,N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography (TLC). After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75: 25) to obtain a white solid intermediate. Under the protection of inert gas, the intermediate obtained by the above reaction, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under tungsten lamp (300 W), oxygen was bubbled for 50 min and monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain an optically pure tripeptide probe Ac- SRR -A5-O2.

[0201]

[0202] Using the above general synthesis method, SR -A4 and N -Vinyl- L -4-Chlorophenylalanine ( S -Ac-Phe(4-Cl)-OH), through two steps of reaction, to prepare Ac- SRS -A5-O2: Under the protection of inert gas, piperidine is added to the solution SR -A4 in DMF. The solution was stirred at room temperature for 30 min and the reaction was monitored by thin layer chromatography (TLC). After observing that Fmoc was completely removed, the mixture was dissolved in ethyl acetate and washed twice with 0.1 M hydrochloric acid and brine. It was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Then, the deprotected SR -A4, N -Vinyl-L -4-Chlorophenylalanine ( S -Ac-Phe(4-Cl)-OH), benzotriazole- N,N,N,N -Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography (TLC). After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75: 25) to obtain a white solid intermediate. Under the protection of inert gas, the intermediate obtained by the above reaction, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under tungsten lamp (300 W), oxygen was bubbled for 50 min and monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain an optically pure tripeptide probe Ac- SRS -A5-O2.

[0203]

[0204] Using the above general synthesis method, RS -A4 and N -Vinyl- L -4-Chlorophenylalanine ( S -Ac-Phe(4-Cl)-OH), through two steps of reaction, to prepare Ac- RSS -A5-O2: Under the protection of inert gas, piperidine is added to the solution RS -A4 in DMF. The solution was stirred at room temperature for 30 min and the reaction was monitored by thin layer chromatography (TLC). After observing that Fmoc was completely removed, the mixture was dissolved in ethyl acetate and washed twice with 0.1 M hydrochloric acid and brine. It was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Then, the deprotected RS -A4, N -Vinyl- L -4-Chlorophenylalanine ( S -Ac-Phe(4-Cl)-OH), benzotriazole- N,N,N,N-Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography (TLC). After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75: 25) to obtain a white solid intermediate. Under the protection of inert gas, the intermediate obtained by the above reaction, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under tungsten lamp (300 W), oxygen was bubbled for 50 min and monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain an optically pure tripeptide probe Ac- RSS -A5-O2.

[0205]

[0206] Using the above general synthesis method, RS -A4 and N -Vinyl- D -4-Chlorophenylalanine ( R -Ac-Phe(4-Cl)-OH), through two steps of reaction, to prepare Ac- RSR -A5-O2: Under the protection of inert gas, piperidine is added to the solution SR -A4 in DMF. The solution was stirred at room temperature for 30 min and the reaction was monitored by thin layer chromatography (TLC). After observing that Fmoc was completely removed, the mixture was dissolved in ethyl acetate and washed twice with 0.1 M hydrochloric acid and brine. It was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Then, the deprotected RS -A4, N -Vinyl- D -4-Chlorophenylalanine ( R -Ac-Phe(4-Cl)-OH), benzotriazole- N,N,N,N-Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography (TLC). After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75: 25) to obtain a white solid intermediate. Under the protection of inert gas, the intermediate obtained by the above reaction, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under tungsten lamp (300 W), oxygen was bubbled for 50 min and monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain an optically pure tripeptide probe Ac- RSR -A5-O2.

[0207]

[0208] Using the above general synthesis method, RR -A4 and N -Vinyl- D -4-Chlorophenylalanine ( R -Ac-Phe(4-Cl)-OH), through two steps of reaction, to prepare Ac- RRR -A5-O2: Under the protection of inert gas, piperidine is added to the solution RR -A4 in DMF. The solution was stirred at room temperature for 30 min and the reaction was monitored by thin layer chromatography (TLC). After observing that Fmoc was completely removed, the mixture was dissolved in ethyl acetate and washed twice with 0.1 M hydrochloric acid and brine. It was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Then, the deprotected RR -A4, N -Vinyl- D -4-Chlorophenylalanine ( R -Ac-Phe(4-Cl)-OH), benzotriazole- N,N,N,N-Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography (TLC). After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75: 25) to obtain a white solid intermediate. Under the protection of inert gas, the intermediate obtained by the above reaction, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under tungsten lamp (300 W), oxygen was bubbled for 50 min and monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain an optically pure tripeptide probe Ac- RRR -A5-O2.

[0209]

[0210] Using the above general synthesis method, RR -A4 and N -Vinyl- L -4-Chlorophenylalanine ( R -Ac-Phe(4-Cl)-OH), through two steps of reaction, to prepare Ac- RRS -A5-O2: Under the protection of inert gas, piperidine is added to the solution RR -A4 in DMF. The solution was stirred at room temperature for 30 min and the reaction was monitored by thin layer chromatography (TLC). After observing that Fmoc was completely removed, the mixture was dissolved in ethyl acetate and washed twice with 0.1 M hydrochloric acid and brine. It was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Then, the deprotected RR -A4, N -Vinyl- L -4-Chlorophenylalanine ( S -Ac-Phe(4-Cl)-OH), benzotriazole- N,N,N,N-Tetramethyluronium hexafluorophosphate (HBTU) was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography (TLC). After the reaction was completed, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75: 25) to obtain a white solid intermediate. Under the protection of inert gas, the intermediate obtained by the above reaction, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under tungsten lamp (300 W), oxygen was bubbled for 50 min and monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was concentrated under reduced pressure and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain an optically pure tripeptide probe Ac- RRS -A5-O2.

[0211] Example 4: Synthesis of optically pure single amino acid probes: (1) Synthesis of optically pure amino acid probe Fmoc-Lue-A9:

[0212] General synthesis method: Under the protection of inert gas, A3 (427 mg, 0.5 mmol), 1,3-dimethylbarbituric acid (DMBA) (313 mg, 0.96 mmol) and tetrakis(triphenylphosphine)palladium (62.5 mg, 0.05 mmol) were added. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane (30 mL) and methylene blue (2.0 mg, 0.006 mmol) were added. Under tungsten lamp (300 w), oxygen was bubbled for 1.0 h and monitored by thin layer chromatography (TLC) (Hex: EtOAc = 50: 50). After the reaction was completed, the solvent was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain white solid Fmoc-Lue-A9.

[0213]

[0214] Using a general synthetic method, S- A3 Synthesis of Fmoc- S -Lue-A9 isolated yield 82%. 1 H NMR (500 MHz, Chloroform- d ) δ 8.40 (s, 1H), 7.94 (d, J= 16.2 Hz, 1H), 7.73 (d, J = 7.6 Hz,2H), 7.57 – 7.49 (m, 4H), 7.38 (dt, J = 24.6, 7.7 Hz, 5H), 7.30 – 7.21 (m,2H), 7.06 (d, J = 8.0 Hz, 1H), 6.45 (d, J = 16.2 Hz, 1H), 5.48 (d, J = 8.4Hz, 1H), 4.93 (d, J = 6.8 Hz, 2H), 4.68 (dt, J = 5.7, 1.5 Hz, 2H), 4.46 –4.26 (m, 3H), 4.17 (t, J = 6.9 Hz, 1H), 3.34 (s, 3H), 3.27 (s, 1H), 2.05 (d, J = 3.6 Hz, 1H), 2.00 – 1.70 (m, 12H), 0.95 (dd, J = 6.6, 3.4 Hz, 6H). 13 C NMR (126 MHz, Chloroform- d ) δ 170.64, 166.36, 153.78, 143.64, 141.39, 139.49,139.08, 138.27, 132.29, 129.95, 129.71, 127.87, 127.21, 125.15, 125.06,120.10, 119.94, 118.36, 75.80, 65.36, 57.34, 47.19, 39.28, 39.12, 37.14,34.76, 33.02, 31.68, 29.79, 28.44, 28.29, 27.00, 25.37, 24.83, 23.05.

[0215]

[0216] Using a general synthetic method, R- A3 Synthesis of Fmoc- R -Lue-A9. Under the protection of inert gas, R-A3, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred at room temperature for reaction. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added, and oxygen was bubbled under tungsten lamp (300 w) for 1.0 h, and monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a white solid Fmoc- R -Lue-A9.

[0217] (2) Synthesis of optically pure amino acid probe Ac-Lue-A9:

[0218] Under the protection of inert gas, N 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (311 mg, 1.26 mmol) was added to a tetrahydrofuran solution (5 mL) of 1-acetylleucine (173 mg, 1 mmol) and 4-aminobenzyl alcohol (133 mg, 1.32 mmol), and the mixture was reacted at room temperature and monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was dissolved in ethyl acetate (50 mL), washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 60:40) to obtain white solid Ac-A1 with an isolated yield of 93%.

[0219]

[0220] Under the protection of inert gas, Ac-A1 (556 mg, 2.0 mmol) and sodium iodide (900 mg, 6.0 mmol) were dissolved in acetonitrile (30 mL) solution. Trimethylsilyl chloride (1.5 mL) was added at 0 °C, and after reacting at 0 °C for 5 min, it was moved to room temperature and protected from light for 1 h. The reaction was monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was poured into 100 mL of water, extracted three times with ethyl acetate (50 mL) and washed with brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 90:10) to obtain a white solid Ac - A2, isolated yield 86%.

[0221]

[0222] General method: Under inert gas protection, 3-(4-[(adamantan-2-ylidene)(methoxy)methyl]-2-hydroxy-3-chlorophenyl) acrylate (414 mg, 1.0 mmol) and potassium carbonate (276 mg, 2.0 mmol) were dissolved in N,N-dimethylformamide (8 mL) solution and reacted at room temperature for 30 min. Ac-A2 (595 mg, 1.05mmol) was added and reacted for 5 min. The reaction was monitored by thin layer chromatography (TLC). After the reaction was completed, the mixture was poured into 100 mL of water, extracted three times with ethyl acetate (50 mL), washed with brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 90:10) to obtain white solid Ac-A3 with an isolated yield of 87%.

[0223]

[0224] General method: Under the protection of inert gas, Ac-A3 (333 mg, 0.5 mmol), 1,3-dimethylbarbituric acid (DMBA) (313 mg, 0.96 mmol) and tetrakis(triphenylphosphine)palladium (62.5 mg, 0.05 mmol) were added. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane (30 mL) and methylene blue (2.0 mg, 0.006 mmol) were added. Under tungsten lamp (300 w), oxygen was bubbled for 1.0 h and monitored by thin layer chromatography (TLC) (Hex: EtOAc = 50: 50). After the reaction was completed, the solvent was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain white solid Ac-Lue-A9.

[0225]

[0226] According to the above general method, Ac- S -A3 Synthesis of Ac- S -Lue-A9, isolated yield 76%. 1 H NMR (400 MHz, Chloroform- d ) δ 8.67 (s, 1H), 7.87 (d, J = 16.1 Hz, 1H), 7.48 (d, J = 8.5 Hz,2H), 7.42 – 7.26 (m, 3H), 6.99 (d, J = 8.0 Hz, 1H), 6.38 (d,J = 16.1 Hz,1H), 6.10 (d, J = 8.2 Hz, 1H), 4.88 (d, J = 4.5 Hz, 2H), 4.64 (dt, J = 5.7,1.4 Hz, 2H), 2.01 – 1.95 (m, 4H), 1.93 – 1.52 (m, 12H), 0.90 (dd, J = 8.9,6.4 Hz, 6SH).

[0227]

[0228] Using the general synthetic method, Ac- R -A3 Synthesis of Ac- R -Lue-A9: Under the protection of inert gas, Ac- R -A3,1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred at room temperature for reaction. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added, and oxygen was bubbled under tungsten lamp (300 W) for 1.0 h. The reaction was monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a white solid Ac- R -Lue-A9.

[0229] (3) Optically pure amino acid probe Ac-MeLue-A 10 Synthesis of: Synthesis of compound Ac-MeLue-A1:

[0230] Under the protection of inert gas, N 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (934 mg, 3.80 mmol) was added to a tetrahydrofuran solution (20 mL) of -vinylmethylleucine (538 mg, 2.88 mmol) and 4-aminobenzyl alcohol (400 mg, 3.24 mmol), and the mixture was reacted at room temperature and monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was dissolved in ethyl acetate (100 mL), washed with saturated brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 60:40) to obtain white solid Ac-MeLue-A1 with an isolated yield of 95%.

[0231] Synthesis of Compound Ac-MeLue-A2

[0232] Under the protection of inert gas, Ac-MeLue-A1 (374 mg, 2.0 mmol) and sodium iodide (900 mg, 6.0 mmol) were dissolved in acetonitrile (30 mL) solution. Trimethylsilyl chloride (1.5 mL) was added at 0 °C, and the reaction was carried out at 0 °C for 5 min, and then moved to room temperature to react in the dark for 1 h. The reaction was monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was removed by concentration under reduced pressure, and the residue was poured into 100 mL of water, extracted three times with ethyl acetate (50 mL), washed with brine (50 mL), dried with anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 90:10) to obtain white solid Ac-MeLue-A2 with an isolation yield of 90%.

[0233] Synthesis of Compound Ac-MeLue-A3

[0234] Under the protection of inert gas, 3-(4-[(adamantan-2-ylidene)(methoxy)methyl]-2-hydroxy-3-chlorophenyl) acrylate (414 mg, 1.0 mmol) and potassium carbonate (276 mg, 2.0 mmol) were dissolved in N,N-dimethylformamide (8 mL) solution and reacted at room temperature for 30 min. A2 (422 mg, 1.05 mmol) was added and reacted for 5 min, and the reaction was monitored by thin layer chromatography (TLC). After the reaction was completed, the mixture was poured into 100 mL of water, extracted three times with ethyl acetate (50 mL), washed with brine (50 mL), dried over anhydrous sodium sulfate, and the solvent was removed under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 90:10) to obtain white solid Ac-MeLue-A3 with an isolated yield of 88%.

[0235] Compound Ac-MeLue-A 10 Synthesis

[0236] General synthesis method: Under the protection of inert gas, Ac-MeLue-A3 (344 mg, 0.5 mmol), 1,3-dimethylbarbituric acid (DMBA) (313 mg, 0.96 mmol) and tetrakis(triphenylphosphine)palladium (62.5 mg, 0.05 mmol) were added. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane (30 mL) and methylene blue (2.0 mg, 0.006 mmol) were added. Under tungsten lamp (300 w), oxygen was bubbled for 1.0 h and monitored by thin layer chromatography (TLC) (Hex: EtOAc = 50: 50). After the reaction was completed, the solvent was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain white solid Ac-MeLue-A 10 ,

[0237] General synthesis method: Synthesis of Ac-S-MeLue-A from Ac-S-MeLue-A3 10 , isolated yield 76%. 1 H NMR (400MHz, Chloroform- d ) δ 8.14 (s, 1H), 7.90 (d, J = 16.2 Hz, 1H), 7.69 (d, J =7.5 Hz, 2H), 7.52 (d, J = 7.5 Hz, 2H), 7.43 (d, J = 8.6 Hz, 2H), 7.40 – 7.30(m, 4H), 7.23 (d, J = 7.5 Hz, 2H), 7.00 (d, J = 8.0 Hz, 1H), 6.40 (d, J =16.2 Hz, 1H), 5.54 (s, 1H), 4.90 (d, J = 4.4 Hz, 2H), 4.64 (dt, J = 5.7, 1.4Hz, 2H), 4.40 (dd, J = 6.7, 3.9 Hz, 2H), 4.15 (t, J = 6.5 Hz, 1H), 3.25 (s,3H), 3.21 (s, 1H), 2.00 (s, 1H), 1.88 – 1.62 (m, 12H), 0.83 (dd,J = 11.3,6.0 Hz, 6H).

[0238]

[0239] General synthetic method, from Ac- R -MeLue-A 10 Synthesis of Ac- R -MeLue-A 10 :Under the protection of inert gas, Ac- R -MeLue-A3, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred and reacted at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added, and the reaction was carried out under tungsten lamp (300 w) irradiation, oxygen was bubbled for 1.0 h, and monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain a white solid Ac- R -MeLue-A 10 .

[0240] Application Example Probe Recognition of Mycobacterium tuberculosis and Chemiluminescence Intensity: Experimental methods: 1. Preparation of probe: (1) Prepare 0.01% Triton X-100 in PBS:DMSO = 1:1 buffer solution; (2) Weigh a certain amount of probe powder, dissolve it in DMSO solution, and dilute it with buffer solution to prepare a probe solution concentration of 800 μmol / L; (3) Store the probe solution at 4°C away from light and use it on the same day.

[0241] 2. Preparation of bacterial solution: (1) Use an inoculation loop to pick up a loop of Mycobacterium tuberculosis / non-tuberculous mycobacteria from the Roselle tube and add it to the 7H9 / OADC liquid culture medium. Ultrasonicate and disperse the bacterial solution. (2) Use the culture medium to adjust the OD value of the bacterial solution. 600 The value is 1.1-1.2, which is used as working bacterial solution.

[0242] 3. Probe recognition of Mycobacterium tuberculosis and luminescence intensity test: (1) Take a 96-well plate (white plate), add 100 μL of the above working bacterial solution and 6 μL of the probe to each well, and start timing. (2) The luminescence value should be measured within 5 minutes after addition, with an integration time of 1 second per well and a 96-well plate scanning time of 5 minutes. (3) The luminescence value should be detected every 10 minutes for at least 1.5 hours.

[0243] 4. In experimental research, probe SSSS -A7 is a small peptide probe reported in the literature, used as a comparative example and active reference substance of the present invention:

[0244] As a probe for comparative examples and reference products Results and structure-activity relationship: 1. Probes involved in the present invention SSSS -A7, as a comparative example and reference substance, has good recognition ability for Mycobacterium tuberculosis. However, during the synthesis process, indanyl glycine was introduced at the end to prepare the probe SSSS -A7, racemization is likely to occur, and the stereoselectivity is poor, so the optical purity of the product is affected. SSSS -A7 is insoluble in water and generally has poor solubility in organic solvents, which is not conducive to its later application as a probe. Therefore, it is urgent to develop probes with good selectivity and high luminescence intensity. Considering that small peptides are short-chain molecules formed by connecting chiral amino acids through peptide bonds, and amino acids are the basic building blocks of small peptides, their chiral configuration has a decisive influence on the overall structure and function of small peptides. Naturally occurring amino acids are L - configuration. However, through artificial synthesis or modification, D -configuration amino acids can be used to construct small peptides containing different chiral amino acid configurations. The introduction of different chiral amino acid configurations will change the spatial conformation of the small peptide, thereby affecting the interaction mode and affinity of the small peptide with the biological target.

[0245] Therefore, the present invention synthesized a series of optically pure small peptide chemiluminescent probes by changing the absolute configuration of amino acids on the peptide chain; the length of the peptide chain; the type of amino acids; and the end-capping group of the peptide, revealing the influence of small peptide chemiluminescent probes on the recognition performance of Mycobacterium tuberculosis based on chiral control and structural transformation, the intensity of chemiluminescence and the chiral structure-activity relationship, as well as the ability to distinguish and specifically recognize mycobacteria in clinical samples.

[0246] 2. In the A7 series probes, the present invention synthesized 16 optically pure tetrapeptide probes by changing the configuration of 1*-4* amino acids: SSSS -A7,S SSR -A7, SSRS -A7, SRSS -A7, RSSS -A7, SSRR -A7, RRSS -A7, RSRS -A7, SRSR -A7, RSSR -A7, SRRS -A7, RSRR -A7, RRRS -A7, RRSR -A7, SRRR -A7, RRRR -A7. Among them, SSSS-A7 is a reported probe, which is used as a positive control and comparative example of the present invention. The 16 synthesized tetrapeptide probes were used to study the recognition and luminescence intensity of Mycobacterium tuberculosis using Mycobacterium tuberculosis H37Ra and BCG (Mycobacterium bovis Bacille Calmette – Guérin, BCG). The results of the change of the luminescence intensity (ordinate) of the probe over time (abscissa) are shown in Figure 1 (BCG: bacillus Calmette-Guérin; Ra: Mycobacterium tuberculosis H37Ra).

[0247] The results showed that the tetrapeptide probe SSSS -A7,S SSR -A7, SSRS -A7, SRSS -A7, SSRR -A7, SRSR -A7, can recognize Mycobacterium tuberculosis, interact with Mycobacterium tuberculosis and produce chemiluminescence of different intensities. SRRR -A7, SRRS -A7, RSSS -A7, RRSS -A7, RSRS -A7, RSRR -A7, RRRS -A7, RRSR -A7, RRRR -A7. RSSR -A7, cannot recognize Mycobacterium tuberculosis, and thus does not produce chemiluminescence. This indicates that the absolute configuration of the amino acids in 1* is S The tetrapeptide probes with the same configuration can recognize Mycobacterium tuberculosis H37Ra and distinguish Mycobacterium tuberculosis from BCG. Under the same conditions, the absolute configuration of the amino acid at position 1 is R The tetrapeptide probes with the same configuration cannot recognize Mycobacterium tuberculosis H37Ra. S The absolute configuration and chiral structure-activity relationship of the configuration were studied and it was found that the absolute configuration of the amino acids at the 2*, 3*, and 4* positions is changeable. However, the absolute configuration of the amino acids at the 2* and 3* positions is R configuration at the same time, resulting in the compound SRRR -A7 and SRRS -A7 lost the ability to recognize Mycobacterium tuberculosis H37Ra. SSSS -A7 has the strongest recognition ability for Mycobacterium tuberculosis in the A7 series. However, the introduction of the indanyl amino acid at the 4* position leads to a sharp decrease in the solubility of the target tetrapeptide, and the introduction of the indanyl amino acid to construct the peptide bond leads to racemization at the 4* position, thereby reducing the chiral purity and yield of the tetrapeptide, which is not conducive to later applications.

[0248] 3. Based on the absolute configuration and chiral structure-activity relationship of the A7 series tetrapeptide probes, it was found that the tetrapeptide probes have spatial structural modifiability. In order to solve the problem of reduced solubility and chiral purity caused by the introduction of the indanyl amino acid at the 4* position, the present invention converted the amino acid at the 4* position into benzylmethionine and synthesized the A8 series tetrapeptide probe compounds. The synthesized A8 series tetrapeptide probes were used to study the recognition and luminescence intensity of Mycobacterium tuberculosis. The results of the change of the luminescence intensity (vertical axis) of the probe with time (horizontal axis) are shown in Figure 2 (MTB: Mycobacterium tuberculosis; KSS: Mycobacterium kansasii OR: Mycobacterium fortuitum).

[0249] The results show that the tetrapeptide probe synthesized by converting the amino acid at position 4 into benzylmethionine has a significantly higher chemiluminescence intensity than the control after identifying Mycobacterium tuberculosis. SSSS -A7. The chiral structure-activity relationship of the A8 series tetrapeptide probes is similar to the overall rule of the A7 series tetrapeptide probes, that is, when the amino acid at position 1* is R When the amino acid at position 1* is S When the leucine of the 4* position is substituted, it can generally react with Mycobacterium tuberculosis to produce chemiluminescence. The absolute configuration of the 4* benzylmethionine has a similar effect on the luminescence intensity, for example, SSSS -A8 and SSSR -A8 has similar luminous intensity and is stronger than SSSS -A7 tetrapeptide probe, the probe has good discrimination and recognition ability for Mycobacterium tuberculosis and non-tuberculous mycobacteria KSS and RO. It shows that the amino acid at position 4* is modifiable and replaceable. The A8 series tetrapeptide probe has good solubility and is not prone to racemization at position 4* during the synthesis process. It has good application prospects.

[0250] 4. Based on the chiral structure-activity relationship, luminescence intensity and replaceability of amino acid at position 4 of A7 and A8 series tetrapeptide probes, the present invention further removes the chiral amino acid at position 4 and synthesizes 8 optically pure tripeptide Fmoc-A5-O2 series probes and 8 optically pure tripeptide Ac-A5-O2 series probes. The necessity of amino acid at position 4 and the influence of tripeptide capping group were studied. The recognition and luminescence intensity of Mycobacterium tuberculosis of the synthesized tripeptide probe were studied. The results of the change of the luminescence intensity (vertical axis) of the probe over time (horizontal axis) are shown in Figure 3 .

[0251] The research results show that in the tripeptide probe synthesized by removing the amino acid at position 4*, the amino acid fragment at position 1* is in R configuration and still cannot react with Mycobacterium tuberculosis. However, when the amino acid fragment at position 1* is in S configuration, it can react with Mycobacterium tuberculosis. Among them, the tripeptide with three amino acid fragments in S configuration exhibits good luminescence performance after reacting with Mycobacterium tuberculosis. Among them, Ac- SSS -A5-O2 has a stronger chemiluminescence intensity than SSSS -A7 tetrapeptide probe. The end groups of this series of tripeptide probes are Fmoc (fluorenyloxycarbonyl) and Ac (acetyl) respectively. The results show that when the Ac-end-tripeptide probe acts on Mycobacterium tuberculosis, the chemiluminescence intensity is higher than that of the corresponding Fmoc-end-tripeptide probe. For example, Ac- SSS -A5-O2 has a stronger chemiluminescence intensity than Fmoc- SSS -Chemieluminescence intensity of A5-O2.

[0252] It is worth noting that Ac- SSS The -A5-O2 series of tripeptide probes are easy to synthesize, have good discrimination and recognition capabilities for Mycobacterium tuberculosis and KSS\RO, and have relatively good solubility, and have good application potential.

[0253] 5. In order to further study the effects of the 1* amino acid and the blocking group on the probe recognition ability and luminescence intensity, the present invention designed and synthesized three series of single amino acid probes: Fmoc-Lue-A9; Ac-Lue-A9; Ac-MeLue-A 10 The three series of single amino acid probes were used to study the recognition and luminescence intensity of Mycobacterium tuberculosis. The results showed that none of the three series of single amino acid probes could interact with Mycobacterium tuberculosis to produce chemiluminescence.

[0254] 6. Study on the ability of synthetic small peptide probes to distinguish and identify low-concentration clinically isolated tuberculosis strains. The results of the change of the luminescence intensity (ordinate) of the probe over time (abscissa) are shown in Figure 4 , where the red curve represents clinically isolated tuberculosis strains (a total of 10 strains, numbered 190, 179, 170, 177, 96, 115, 125, 153, 106, and 98), and the green curve represents clinically isolated nontuberculous mycobacteria (a total of 10 strains, numbered 2, 6, 10, 13, 14, 28, 30, 45, 51, and 70).

[0255] The experimental results show that under the same conditions, the preferred tripeptide probe (Ac- SSS -A5-O2) and tetrapeptide probes ( SSSR-A8) has good clinical mycobacterial differentiation and recognition ability, and the luminescence intensity is better than the reported positive control probe S SSS -A7, indicating potential clinical application prospects.

[0256] Obviously, the above embodiments of the present invention are only examples for clearly illustrating the technical solution of the present invention, and are not intended to limit the specific implementation methods of the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the claims of the present invention shall be included in the protection scope of the claims of the present invention.

Claims

1. An optically pure small peptide chemiluminescent probe, characterized in that: The structural formula is shown in the following formula (I) or (II): ； ； Among them, the * represents a chiral carbon atom, and its configuration is R or S , 1* represents chiral carbon atom No. 1, 2* represents chiral carbon atom No. 2, 3* represents chiral carbon atom No. 3, and 4* represents chiral carbon atom No. 4; In formula I, Y represents -CH2SCH2Ph; In formula II, X represents an -NH2 protecting group, which is an acetyl group, a benzyloxycarbonyl group, a tert-butyloxycarbonyl group or a fluorenylmethoxycarbonyl group.

2. The optically pure small peptide chemiluminescent probe according to claim 1, characterized in that: X represents an acetyl group or a fluorenylmethoxycarbonyl group.

3. The optically pure small peptide chemiluminescent probe according to claim 1, characterized in that: When Y in formula I represents -CH2SCH2Ph, the chiral configuration of 1* to 4* is one of the following chiral configurations: SSSS, SRRR, SSRR, SSSR, SRRS, SSRS, SRSR, SRSS, RSSS, RSRR, RSSR, RRRR, RSRS, RRSR, RRRS, RRSS.

4. The optically pure small peptide chemiluminescent probe according to claim 1, characterized in that: The chiral configurations of 1* to 3* in Formula II are one of the following chiral configurations, respectively: SSS, SSR, SRR, SRS, RSS, RSR, RRR, RRS.

5. Use of the optically pure small peptide chemiluminescent probe according to claim 1 in the preparation of Mycobacterium tuberculosis identification.

6. The use according to claim 5, characterized in that: The optically pure small peptide chemiluminescent probe is used for preparing a preparation for identifying Mycobacterium tuberculosis.

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