Optically pure small peptide chemiluminescent probe and its application in mycobacterium tuberculosis recognition

Abstract

The present application belongs to the field of organic synthesis and medicine, and particularly relates to an optically pure small peptide chemiluminescent probe. The present application discloses the influence of the small peptide chemiluminescent probe on the recognition performance of Mycobacterium tuberculosis based on chirality control and structure transformation, the intensity of chemiluminescence and the chiral structure-activity relationship, and the ability of distinguishing and specifically recognizing clinical sample Mycobacterium 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. Compared with the existing reports, the small peptide chemiluminescent probe developed in the present application creates and innovates the chiral environment of the probe small peptide end recognition fragment from the molecular level, thereby constructing a new three-dimensional structure of the small peptide recognition fragment, and further changing the electronic effect, steric hindrance effect, pi-pi stacking effect, and interaction mode between host and guest of the recognition center, so as to prepare and screen the chemiluminescent probe with higher luminescent intensity and excellent recognition performance than the existing small peptide Mycobacterium tuberculosis probe.

Description

TECHNICAL FIELD

[0001] The present application belongs to the field of organic synthesis and medicine, and specifically relates to an optically pure small peptide chemiluminescent probe and application thereof in Mycobacterium tuberculosis recognition. BACKGROUND

[0002] Chirality plays an extremely important role in the field of drugs, and often has specific interaction with biological targets. Many targets in the body, such as enzymes and receptors, have chirality characteristics. The interaction of different enantiomers (absolute configuration) of chiral drugs with these biological targets can only be perfectly matched with the target when a specific chiral configuration is used, thereby producing the expected pharmacological effect. For example, the antimalarial drug quinine has a specific chiral structure, and only one enantiomer can effectively bind to the related target of Plasmodium, thereby exerting an antimalarial effect. In addition, different enantiomers of chiral drugs can have significant differences in pharmacological activity. One enantiomer can have strong therapeutic activity, while the other enantiomer can have weak or even no activity. For example, the S-enantiomer of the beta-blocker propranolol has about 100 times the activity of the R-enantiomer in blocking beta-receptors, so S-propranolol plays a major pharmacological role in treating cardiovascular diseases and the like. Chiral drugs of different configurations have different pharmacokinetic characteristics. Different enantiomers of chiral drugs can have different pharmacokinetic processes in the body, such as absorption, distribution, metabolism, and excretion. This is because enzymes and transport proteins involved in these processes often also have chiral selectivity. For example, the S-enantiomer of the non-steroidal anti-inflammatory drug naproxen has a higher plasma protein binding rate, and its elimination rate in the body is slower than that of the R-enantiomer, resulting in a longer action time and more stable drug concentration in the body. Chiral drugs also have large differences in toxicity and side effects. One enantiomer can produce serious toxicity and side effects, while the other enantiomer is relatively safe. If chirality is not controlled, it can bring unnecessary risks to patients. For example, thalidomide (thalidomide) has an inhibitory effect on pregnancy, while the S-enantiomer has a strong teratogenic effect, which can cause serious limb malformations in fetuses and other problems.

[0003] Therefore, in the process of drug development, the study and control of the chirality of chiral drugs is a crucial step, which needs to ensure that drugs with a specific chiral configuration are obtained and strictly controlled in quality. Chirality largely determines the safety, effectiveness and quality of drugs, and is crucial in the development, production and clinical application of chiral drugs.

[0004] As a class of bio-chiral active 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 composed of chiral amino acids connected by 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 methods, the introduction of D By combining amino acids with different chiral configurations, small peptides containing different chiral amino acid configurations can be constructed. The introduction of different chiral amino acid configurations alters the spatial conformation of the small peptide, thereby affecting its interaction with and affinity for biological targets. For example, studies of some antimicrobial peptides have found that replacing some L-amino acids with D-amino acids significantly improves the antimicrobial activity and stability of the small peptides. This is because the altered chiral configuration enables the small peptide to better bind to specific targets on the bacterial cell membrane and enhances its resistance to protease degradation.

[0005] Because small peptides composed of amino acids of different configurations exhibit significant differences in pharmacological activity, in-depth research on small peptides based on different chiral amino acid configurations is expected to screen for small peptide drugs with higher activity and efficacy. By systematically varying the chiral configuration of amino acids in small peptides and evaluating their activity, it is possible to precisely identify small peptide structures that bind most tightly to biological targets and produce the strongest pharmacological effects. This will provide more effective drug options for treating various difficult diseases, significantly improving disease treatment outcomes and bringing new hope to patients. Furthermore, studying small peptides composed of different chiral amino acid configurations can clarify the configurational factors that contribute to toxic side effects. This allows for the rational design of small peptide structures to avoid or reduce configurations with potential toxic side effects and improve the safety of small peptide drugs. Furthermore, small peptides containing different chiral amino acid configurations often exhibit distinct interaction patterns with biological targets, thereby revealing new drug mechanisms of action, facilitating in-depth exploration of physiological and pathological processes in vivo, and providing opportunities for the development of drugs with novel 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] On the basis of the previous research and development of small peptide probes, aiming at the single structure of the reported small peptide chemiluminescence probe, without considering the influence, contribution, chiral structure-activity relationship of the absolute configuration of amino acids in small peptide fragments on the reflection intensity and Mycobacterium tuberculosis recognition, a series of novel small peptide probes are designed and synthesized, the main strategies are as follows: (1) the absolute configuration of amino acids in small peptides, (2) the length of the peptide chain, (3) the type of amino acid, (4) the end group of small peptides, a series of optically pure small peptide chemiluminescence probes are synthesized, for the first time, it is innovatively revealed that the influence of the factors such as the absolute configuration of amino acids, the length of the peptide chain, the type of amino acid, the end group of small peptides in small peptide chemiluminescence probes on the recognition performance of Mycobacterium tuberculosis, the intensity of chemiluminescence and the chiral structure-activity relationship, and the ability of distinguishing and specific recognition of clinical sample Mycobacterium. Compared with the single configuration small peptide probe reported by the previous, the small peptide chemiluminescence probe developed in the application changes the absolute configuration of amino acids, introduces different absolute configuration of amino acids, changes the length of the peptide chain, the type of amino acid, the end group of small peptides, creates and innovates the chiral environment of the probe small peptide end recognition fragment from 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, pi-pi stacking effect, weak interaction and mode between the host and the guest of the recognition center, in order to construct the specific recognition of small peptide chemiluminescence probe to Mycobacterium tuberculosis, the intensity of chemiluminescence and the chiral structure-activity relationship, and prepare and screen the chemiluminescence probe with high light intensity and excellent recognition performance, which provides a feasible scheme, experimental basis and theoretical guidance for the development of optically pure small peptide chemiluminescence probe and the research of chiral structure-activity relationship. The method is successfully applied to the specific recognition of clinical sample Mycobacterium.

[0008] In order to achieve the above-mentioned purpose of the application, the application provides the following technical scheme:

[0009] An optically pure small peptide chemiluminescence probe, the structural formula is shown in the following formula (I), (II) or (III): Formula I;

[0010] Formula II;

[0011] Formula III;

[0012] Wherein, the symbol represents a chiral carbon atom, and the configuration is R or S, 1 represents the 1st chiral carbon atom; wherein, the chiral configurations of 1 to 4 can be the same or different;

[0013] Y in formula I represents Or CH2SCH2Ph or other alpha-substituted groups of alpha-amino acids.

[0014] In formula II, X represents an -NH2 protecting group, which is benzyloxycarbonyl, tert-butyloxycarbonyl or fluorenylmethoxycarbonyl; benzyloxycarbonyl (Cbz), tert-butyloxycarbonyl (BOC) and fluorenylmethoxycarbonyl (Fmoc) are the preferred α-NH2 protecting groups in polypeptide synthesis.

[0015] In formula III, X1 represents acetyl, benzyloxycarbonyl, tert-butyloxycarbonyl or fluorenylmethoxycarbonyl; Y1 represents hydrogen or a C1-C3 saturated alkyl group.

[0016] Preferably, X represents fluorenylmethoxycarbonyl.

[0017] Preferably, in formula III, X1 represents an acetyl group or a fluorenylmethoxycarbonyl group; and Y1 represents a hydrogen group or a methyl group.

[0018] 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.

[0019] More preferably, the chiral configurations of 1* to 4* in Formula I are one of the following chiral configurations: SSSR, SSRS, SRSS, SSRR, SRSR.

[0020] Preferably, when Y in formula I represents -CH2SCH2Ph, the chiral configurations 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.

[0021] 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.

[0022] Compared with the prior art, the present invention has the following beneficial effects:

[0023] Compared with existing reports, the present invention creates and innovates the chiral environment of the probe peptide end recognition fragment at the molecular level, thereby constructing a new three-dimensional structure of the small peptide recognition fragment, and then changing the electronic effect, steric effect, π-π stacking effect, weak interaction and pattern between host and guest, etc. of the recognition center, and prepares and screens chemiluminescent probes with higher luminescence intensity and better recognition performance than the existing reported small peptide probes (SSSS-A7). BRIEF DESCRIPTION OF THE DRAWINGS

[0024] Figure 1 A diagram showing the study of Mycobacterium tuberculosis recognition and luminescence intensity using A7 series tetrapeptide probes;

[0025] Figure 2 A diagram showing the study of Mycobacterium tuberculosis recognition and luminescence intensity using the A8 series tetrapeptide probes;

[0026] Figure 3 A diagram showing the study of Mycobacterium tuberculosis recognition and luminescence intensity using tripeptide Fmoc-A5-O2 series probes and tripeptide Ac-A5-O2 series probes;

[0027] Figure 4 Figure 2 shows the ability to differentiate and identify low-concentration clinically isolated tuberculosis strains. DETAILED DESCRIPTION

[0028] The following will clearly and completely describe the technical solutions of the present invention in conjunction with the embodiments of the present invention and comparative examples. Obviously, the embodiments described are only some embodiments of the present invention, not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0029] Unless otherwise specified, the experimental methods used in the following examples are conventional methods; the materials and reagents used are commercially available reagents and materials unless otherwise specified.

[0030] Example 1: Synthesis of optically pure tetrapeptide probe A7 series:

[0031] (1) Compounds S -A1 and R -Synthesis of A1:

[0032] General synthesis method: Under the protection of inert gas, N2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (934 mg, 3.80 mmol) was added to a solution of 1-fluorenylmethyloxycarbonyl-L (or D)-leucine (S-A0 or R-A0, 1020 mg, 2.88 mmol) and 4-aminobenzyl alcohol (400 mg, 3.24 mmol) in tetrahydrofuran (20 mL). 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. 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, a white solid.

[0033]

[0034] 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 = 6.3Hz, 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 = 5.5 Hz, 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). 13C 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.

[0035]

[0036] Using the general synthetic method, compound N - fluorenylmethyloxycarbonyl- D - leucine R - A0) to give R - A1 in 98% yield. 1 H 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, 7.7 Hz, 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 CNMR (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.

[0037] (2) Compound S - A2 and R Synthesis of compound

[0038] General synthetic method: under inert gas protection, intermediateS -A1or 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 5 min of reaction at 0 °C, it was transferred to room temperature and reacted for 1 h in the dark. The reaction was monitored by thin layer chromatography (TLC). After completion of the reaction, the solvent was removed under reduced pressure and the residue was poured into 100 mL of water, extracted with ethyl acetate (50 mL) three times 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 the corresponding S -A2and R -A2, was a white solid.

[0039]

[0040] Using the general synthetic method, intermediate S -A1was prepared 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.

[0041]

[0042] Using the general synthetic method, intermediate R -A1was prepared 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). 13 C 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.

[0043] (3) Compounds S -A3 and R -A3 synthesis:

[0044] 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. The intermediate was added S -A2 or R -A2 (595 mg, 1.05 mmol) was stirred 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 the corresponding S -A3 and R -A3, a white solid.

[0045]

[0046] Using the general synthetic method, intermediate S -A2 was prepared S -A3 in 88% yield. 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 = 6.8 Hz, 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 = 6.2Hz, 6H). 13C 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.

[0047]

[0048] Using the general synthetic method, intermediate R -A2 was prepared R -A3 in 85% yield. 1H NMR (400 MHz, CDC13) 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 = 4.6 Hz, 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, CDC13) 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.

[0049] (4) Compound SS -A4, SR -A4, RS -A4, RR Synthesis of -A4:

[0050] 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 complete removal of the Fmoc group, 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, under the protection of inert gas, the deprotected A3, N-fluorenylmethyloxycarbonyl-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, and the mixture was 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) and 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, a white solid.

[0051]

[0052] Using general synthetic methods, through intermediates S -A3 and N -Fluorenylmethoxycarbonyl- N '-allyloxycarbonyl- L -Lysine ( L -Fmoc-lys(alloc)-OH) SS -A4, yield 70%. 1 H 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 = 7.9Hz, 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.

[0053]

[0054] Using the general synthetic method, intermediate S -A3 was reacted with N -Fluorenylmethoxycarbonyl- N '-Allyloxycarbonyl- D -Lysine ( D -Fmoc-lys(alloc)-OH) to give SR -A4 in 73% yield. 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 = 8.0 Hz, 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 = 8.1 Hz, 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 = 10.4Hz, 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). 13C NMR (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.

[0055]

[0056] Using the general synthetic method, intermediate R -A3 was reacted with N -Fluorenylmethoxycarbonyl- N '-Allyloxycarbonyl- L -Lysine ( L -Fmoc-lys(alloc)-OH) to produce RS -A4 in 73% yield. 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.

[0057]

[0058] Using the general synthetic method, intermediate R -A3 was reacted with N -fluorenylmethoxycarbonyl- N '-allyloxycarbonyl- D -lysine ( D -Fmoc-lys(alloc)-OH) to give RR -A4 in 77% yield. 1H NMR (400 MHz, CDC13) 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, CDC13) 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.

[0059] (5) CompoundSSS - A5, S SR - A5, SRS - A5, SRR - A5, RSS - A5, RSR - A5, RRS - A5, RRR - Synthesis of A5:

[0060] General synthesis procedure: PIPERIDINE (0.494 mL, 5 mmol) was added to 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, monitoring the reaction by thin layer chromatography (TLC). After observing complete removal of fluorenylmethyloxycarbonyl group, the mixture was dissolved in ethyl acetate and washed twice with 0.1 M hydrochloric acid (50 ml) and brine (50 ml). Dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Subsequently, under inert gas protection, the deprotected A4, N - fluorenylmethyloxycarbonyl-L (or D) -4-chlorophenylalanine (Fmoc-Phe(4-Cl)-OH) (422 mg, 1.0 mmol), benzotriazol- N,N,N',N'-tetramethyluronium hexafluorophosphate (HBTU) (546 mg, 1.44 mmol) were dissolved in DMF (25.0 mL), added N,N-diisopropylethylamine (DIPEA) (0.348 mL, 2.0 mmol), stirred at room temperature for 60 min, monitoring the reaction by thin layer chromatography (TLC). After completion of the reaction, the mixture was diluted with ethyl acetate (100 ml) and washed with 0.1 M hydrochloric acid (50 ml) and brine (50 ml). The organic layer was separated, dried over Na2S04and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75:25). The corresponding SSS - A5, S SR - A5, SRS - A5, SRR - A5, RSS - A5, RSR - A5, RRS - A5, RRR - A5, as a white solid.

[0061]

[0062] Using the general synthesis procedure, INTERMEDIATE SS-A4 with N -fluorenylmethyloxycarbonyl- L -4-chlorophenylalanine L -Fmoc-Phe(4-Cl)-OH) to give SSS -A5 in 75% yield. 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, 117.19,116.50, 74.70, 66.35, 64.48, 64.23, 59.39, 56.18, 54.96, 52.32, 51.69, 45.82,40.23, 39.47, 38.16, 38.01, 37.58, 36.02, 31.90, 28.67, 28.55, 27.32, 27.18,23.91, 21.83, 21.40, 21.19, 20.03, 13.17.

[0063]

[0064] Using the general synthetic method, compound SR -A4 was prepared by reaction of the intermediate N -fluorenylmethyloxycarbonyl- L -4-chlorophenylalanine L -Fmoc-Phe(4-Cl)-OH) using the general synthetic method. SRS -A5 in 69% yield. 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= 7.8Hz, 1H), 7.42 – 7.22 (m, 11H), 7.13 (t, J = 5.6 Hz, 1H), 7.09 (d, J = 8.0 Hz,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.

[0065]

[0066] Using general synthetic methods, through intermediates 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, 5.4 Hz, 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, 123.88, 118.94, 116.84, 116.37,113.05, 74.70, 66.39, 64.46, 63.98, 56.12, 45.96, 40.27, 38.12, 37.96, 37.59,37.50, 36.01, 32.80, 31.84, 30.90, 30.61, 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.

[0067]

[0068] Using the general synthetic method, compound RR -A4 was prepared by reaction of intermediate N -Fluorenylmethyloxycarbonyl- L -4-chlorophenylalanine L -Fmoc-Phe(4-Cl)-OH) according to the general synthetic method RRS -A5, yield 70%. 1 H NMR (400 MHz, DMSO- d6) 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.

[0069]

[0070] Using the general synthetic method, compound SS -A4 was prepared by reaction of intermediate N -Fluorenylmethyloxycarbonyl- D -4-Chlorophenylalanine D -Fmoc-Phe(4-Cl)-OH) using the general synthetic method. SSR -A5 in 76% yield. 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, 5.2 Hz, 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, 119.15, 118.85,118.74, 117.19, 116.50, 74.70, 66.35, 64.48, 64.23, 59.39, 56.18, 54.96,52.32, 51.69, 45.82, 40.23, 39.47, 38.16, 38.01, 37.58, 36.02, 31.90, 28.67,28.55, 27.32, 27.18, 23.91, 21.83, 21.40, 21.19, 20.03, 13.17.

[0071]

[0072] Using the general synthetic method, compound SR -A4 was prepared by reaction of the intermediate N -Fluorenylmethyloxycarbonyl- D -4-chlorophenylalanine D -Fmoc-Phe(4-Cl)-OH) using the general synthetic method. 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 = 7.7 Hz, 2H),7.48 (d, J = 7.1 Hz, 1H), 7.40 (t, J = 6.8 Hz, 2H), 7.31 (d, J= 7.8 Hz, 3H), 7.24– 7.20 (m, 2H), 7.21 – 7.14 (m, 2H), 7.09 (d, J = 7.7 Hz, 2H), 7.04 – 6.97 (m,2H), 6.95 (d, J = 8.0 Hz, 1H), 6.85 – 6.79 (m, 2H), 6.36 (d, J = 16.2 Hz, 1H),5.81 (tt, J = 10.7, 5.4 Hz, 1H), 5.76 – 5.65 (m, 2H), 5.19 (d, J = 17.2 Hz, 2H),5.09 (d, J = 10.1 Hz, 2H), 5.01 (d, J = 9.6 Hz, 2H), 4.78 (s, 2H), 4.51 – 4.37(m, 4H), 4.25 – 4.14 (m, 3H), 4.02 (t, J = 7.3 Hz, 1H), 3.20 (s, 3H), 3.16 (s,1H), 3.11 – 2.95 (m, 3H), 2.88 (s, 1H), 1.98 – 1.68 (m, 13H), 1.66 – 1.34 (m,9H), 0.87 – 0.84 (dd, J = 6.2 Hz, 6H). 13 C NMR (101 MHz, DMSO- 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, 120.47, 119.54, 118.44, 117.28, 75.91, 66.20, 65.14, 64.58, 56.98, 56.44, 53.16, 52.23, 46.99, 41.10, 38.70, 37.60, 36.91, 36.24, 32.86, 32.18, 31.24, 29.50, 28.17, 28.04, 24.76, 23.48, 22.85, 22.57, 21.73, 14.41.

[0073]

[0074] Using the general synthetic method, the intermediate RS -A4 was reacted with N -fluorenylmethyloxycarbonyl- D -4-chlorophenylalanine D -Fmoc-Phe(4-Cl)-OH) to produce 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 = 1.8Hz, 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, 6.2 Hz, 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, 123.88, 119.62, 118.74, 117.09, 116.41, 113.06, 74.60, 66.47, 64.47, 64.14, 63.57, 56.14, 54.33, 52.43, 52.04, 51.40, 45.73, 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.

[0075]

[0076] Using the general synthetic method, intermediate RR -A4 with N -Fluorenylmethoxycarbonyl- D -4-Chlorophenylalanine (D Prepared from Fmoc-Phe(4-Cl)-OH) reaction 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) δ 170.84, 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.

[0077] (6) Compound 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:

[0078] General synthesis procedure: piperidine (0.494 mL, 5 mmol) was added to a solution of intermediate SSS -A5 or S SR -A5 or SRS -A5 or SRR -A5 or RSS -A5 or RSR -A5 or RRS -A5 or RRRA5 (1249 mg, 1.0 mmol) in DMF (5 mL). The solution was stirred at room temperature for 30 min, monitoring the reaction by thin layer chromatography (TLC). After observing complete removal of fluorenylmethyloxycarbonyl group, the mixture was dissolved in ethyl acetate and washed twice with 0.1 M hydrochloric acid (50 ml) and brine (50 ml). Dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Subsequently, the deprotected A5, N -Ac- L (Or D Indaneglycine (Ac-Igl-OH) (233 mg, 1.0 mmol), benzotriazol-1-yl-N,N,N',N'- tetramethyluronium hexafluorophosphate (HBTU) (546 mg, 1.44 mmol) were dissolved in DMF (25.0 mL), added N,N-diisopropylethylamine (DIPEA) (0.348 mL, 2.0 mmol), stirred at room temperature for 60 min, monitoring the reaction by thin layer chromatography (TLC). After completion of the reaction, the mixture was diluted with ethyl acetate (100 ml) and washed with 0.1 M hydrochloric acid (50 ml) and brine (50 ml). The organic layer was separated, dried over Na2S04and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75:25). The corresponding compound 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, was obtained as a white solid.

[0079]

[0080] Using the general synthetic procedure, compound SSS -A5 was reacted with N -Ac- L indaneglycine (Ac-Igl-OH) to produce N -Ac- L -Igl-OH) to produce SSSS -A6, in 63% yield. 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.

[0081]

[0082] Using the general synthetic method, intermediate RSS -A5 was reacted with N -Ac- L -Indanoylglycine N -Ac- L -Igl-OH) to give RSSS -A6 in 73% yield. 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, 120.53, 119.45, 118.40, 117.28, 75.94, 65.12, 64.59, 60.21, 56.97, 55.34, 54.27, 53.73, 52.21, 42.11, 41.03, 39.34, 38.89, 36.92, 36.74, 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.

[0083]

[0084] Using the general synthetic method, intermediate SRS -A5 was reacted with N -Ac- L- indanyl glycine N - Ac- L - Igl-OH) to produce 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, 5.5 Hz, 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.

[0085]

[0086] Using the general synthetic method, intermediate SSR -A5 was reacted with N -Ac- L -Indane glycine N -Ac- L -Igl-OH) to give SSRS -A6 in 63% yield. 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, 7.8Hz, 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, 5.0Hz, 1H), 4.98 – 4.84 (m, 2H), 4.64 (d, J = 5.5 Hz, 2H), 4.57 (dd, J = 15.9, 7.7Hz, 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.

[0087]

[0088] Using the general synthetic method, intermediate RRS -A5 was reacted with N -Ac- L -Indanoyl glycine N -Ac- L -Igl-OH) to give RRSS -A6 in 69% yield. 1 H NMR (400 MHz, Chloroform- d ) δ 7.88 (d, J = 16.2 Hz, 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.

[0089]

[0090] Using the general synthetic method, intermediate SRR -A5 was reacted with N -Ac- L -Indane glycine N -Ac- L -Igl-OH) to give SRRS -A6 in 69% yield. 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.

[0091]

[0092] Using the general synthetic method, intermediate RSR -A5 was reacted with N -Ac- L -Indanoyl glycine N -Ac- L -Igl-OH) to give RSRS -A6 in 68% yield. 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) δ 171.89, 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, 124.71, 120.56, 119.45, 118.45, 117.28, 75.92, 65.14, 64.58, 56.99, 56.42, 54.35, 53.17, 52.28, 51.46, 41.86, 41.11, 38.89, 38.55, 37.35, 36.93, 35.74, 32.87, 32.17, 31.76, 29.34, 28.11, 24.80, 23.51, 22.88, 21.72.

[0093]

[0094] Using the general synthetic method, intermediate RRR -A5 was reacted with N -Ac- L -indanoyl glycine N -Ac- L -Igl-OH) to give RRRS -A6 in 62% yield. 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 = 22.4 Hz, 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) δ 171.96, 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.

[0095]

[0096] Using the general synthetic method, intermediate SSS -A5 was reacted with N -Ac- D -indanoylglycine N -Ac- D -Igl-OH) to give SSSR -A6 in 65% yield. 1 H 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 = 6.2Hz, 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.

[0097]

[0098] Using the general synthetic method, intermediate RSS -A5 was reacted with N -Ac- D -indanoyl glycine N -Ac- D -Igl-OH) to give RSSR -A6 in 70% yield. 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 = 8.6 Hz, 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, 119.49, 118.43, 117.30, 116.04, 115.05, 75.96, 65.48, 65.14, 56.98, 56.14, 53.92, 53.36, 52.22, 42.08, 41.06, 38.71, 36.92, 35.61, 34.84, 32.87, 31.99, 29.88, 28.11, 24.83, 23.58, 22.56, 21.63.

[0099]

[0100] Using the general synthetic method, intermediate SRS -A5 was reacted with N -Ac- D -Indanoyl glycine N -Ac- D -Igl-OH) to produce SRSR A6 in 73% yield 。 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, 124.67, 124.48, 120.59, 119.43, 118.49, 117.33, 76.01, 65.21, 64.65, 57.03, 56.25, 54.52, 54.07, 53.59, 53.11, 52.53, 46.86, 42.36, 36.98, 32.92, 29.56, 28.10, 24.84, 23.55, 22.95, 22.82, 22.14, 22.09.

[0101]

[0102] Using the general synthetic method, intermediate SSR -A5 with N -Ac- D -Indanoyl glycine N -Ac- D -Igl-OH) to give SSRR -A6 in 63% yield. 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 = 7.2 Hz, 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, 11.8 Hz, 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 = 5.8Hz, 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 (126 MHz, 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.

[0103]

[0104] Using the general synthetic method, intermediate RRS -A5 was reacted with N -Ac- D -Indanoyl glycine N -Ac- D -Igl-OH) to give RRSR -A6 in 64% yield. 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 = 4.9 Hz, 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) δ 172.00, 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, 126.32, 124.60, 119.41, 118.45, 117.24, 75.95, 65.14, 64.56, 56.98, 54.76, 54.62, 53.05, 52.55, 42.19, 41.82, 36.92, 35.85, 35.31, 32.87, 31.99, 31.59, 30.28, 29.88, 29.51, 28.05, 24.77, 23.44, 22.83, 22.78, 21.85.

[0105]

[0106] Using the general synthetic method, intermediate SRR -A5 was reacted with N -Ac- D -Indanoylglycine N -Ac- D -Igl-OH) to give 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, 4.3Hz, 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, 3.7Hz, 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 = 1.6 Hz, 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.

[0107]

[0108] Using the general synthetic method, intermediate RSR -A5 was reacted with N -Ac- D -Indanoyl glycine N -Ac- D -Igl-OH) to give RSRR -A6 in 71% yield. 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 = 17.2 Hz, 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).

[0109] 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, 124.41, 120.55, 119.52, 118.45, 117.27, 75.96, 65.15, 64.57, 56.98, 55.36, 54.59, 53.48, 52.24, 41.94, 41.06, 38.90, 38.55, 37.65, 36.92, 36.02, 35.28, 32.87, 32.08, 31.74, 29.50, 24.80, 23.54.

[0110]

[0111] Using the general synthetic method, intermediate RRR -A5 was reacted with N -Ac- D -indanoyl glycine N -Ac- D -Igl-OH) to give RRRR -A6 in 66% yield. 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, 2.8Hz, 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, 2.9Hz, 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.

[0112] (7) optically pure tetrapeptide probe compoundsSSSS -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:

[0113] General synthetic procedure: under inert gas protection, -A6or S SSSS -A6or S SSR -A6or SSRS -A6or SRSS -A6or RSSS -A6, SSRR -A6or RRSS -A6or RSRS -A6or SRSR -A6or RSSR -A6or SRRS -A6or RSRR -A6or RRRS -A6or RRSR -A6or SRRR -A6or 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 removed sufficiently, dichloromethane (20 ml) and methylene blue (1.0 mg, 0.003 mmol) were added. Oxygen was bubbled into the reaction under the irradiation of a tungsten lamp (300w) for 50 min, which 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 (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, a white solid.

[0114]

[0115] Using general synthetic methods, through intermediates SSSS -A6 preparation SSR -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) δ 171.83, 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, found 1149.4866.

[0116]

[0117] General synthetic procedure was adopted. Under inert gas protection, the reaction was carried out by mixing of the corresponding amino acid SSRS -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 removed completely, dichloromethane (20 ml) and methylene blue (1.0 mg, 0.003 mmol) were added. Under the irradiation of tungsten lamp (300w), oxygen was bubbled into the reaction for 50 min, which was monitored by thin layer chromatography (TLC). After the reaction was completed, the solvent was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (dichloromethane:methanol = 50:1) to obtain the corresponding target tetrapeptide probe compound SRSS -A7, was a white solid with a yield of 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, found 1149.4866.

[0118]

[0119] Using the general synthetic method, intermediate RSSS -A6 was prepared SSRR -A7 in 87% yield. 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= 10.3 Hz,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.

[0120]

[0121] Using general synthetic methods, through intermediates RRSS -A6 preparation RSRS -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 = 8.3 Hz, 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.

[0122]

[0123] Using the general synthetic method, intermediate SRSR -A6 was prepared RSSR -A7 in 81% yield. 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 (101 MHz, 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.

[0124]

[0125] Using the general synthetic method, intermediate SRRS was prepared from -A6 RSRR -A7 in 83% yield. 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, 66.28, 64.41, 64.17, 62.03, 59.36, 56.13, 53.88, 52.44, 51.24, 50.66, 45.75, 43.79, 39.50, 38.00, 37.58, 36.02, 35.46, 34.64, 33.95, 33.40, 32.78, 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) calcd for C 62 H 74 Cl2N6O 11 + [M+H] + 1149.49, found 1149.40.

[0126]

[0127] Using the general synthetic method, intermediate RRRS -A6 was prepared RRSR -A7 in 76% yield. 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 = 18.7 Hz,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.

[0128]

[0129] Using the general synthetic method, intermediate SRRR -A6 was prepared RRRR -A7 in 77% yield. 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)calcdfor C 62 H 74 Cl2N6O 11 + [M+2+H] + 1151.49, found 1151.46.

[0130]

[0131] Using the general synthetic method, intermediate SSSS -A6 was prepared SSSS -A7 in 73% yield. 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 = 18.7 Hz,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 for C 62 H 74 Cl2N6O 11 + [M+H] + 1149.49, found 1149.54.

[0132]

[0133] Using the general synthetic method, intermediate RSSS -A6 was prepared RSSS -A7 in 75% yield. 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 = 7.9Hz, 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 = 16.1 Hz, 1H), 4.84 (m, 3H), 4.46 (m,4H), 4.24 (m, 2H), 3.25 (s,3H), 3.21 (s, 1H), 2.91 (m,9H), 2.04 – 1.77 (m, 25H), 0.82 (d, J = 6.2 Hz, 6H).MS (ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+H] + 1149.49, found 1149.36.

[0134]

[0135] Using the general synthetic method, intermediate SRSS -A6 was prepared SRSS -A7 in 75% yield. 1 H NMR (500 MHz, CDC13) 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 = 16.1 Hz, 1H), 4.84 (m, 3H), 4.46 (m, 4H), 4.24 (m, 2H), 3.25 (s, 3H), 3.21 (s, 1H), 2.91 (m, 9H), 2.04 - 1.77 (m, 25H), 0.82 (d, J = 6.2 Hz, 6H). MS (ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+2+H] + 1151.49, found 1151.43.

[0136]

[0137] Using the general synthetic method, intermediate SSRS -A6 was prepared SSRS -A7 in 81% yield. 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 = 8.4 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).MS (ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+H] + 1149.49, found 1149.47.

[0138]

[0139] Using the general synthetic method, R SSSR -A6 was prepared from intermediate SSSR -A7 in 68% yield. 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 = 10.3 Hz,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.

[0140]

[0141] Using the general synthetic method, intermediate SSRR -A6 was prepared SSRR -A7 in 82% yield. 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 = 8.3 Hz,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.

[0142]

[0143] Using the general synthetic method, under inert gas protection, compound 1-1 was reacted with compound 2-1 to give compound 3-1. SRSR-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 removed completely, dichloromethane (20 ml) and methylene blue (1.0 mg, 0.003 mmol) were added. Oxygen was bubbled into the reaction under the irradiation of a tungsten lamp (300w) for 50 min, which 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 (dichloromethane:methanol = 50:1) to obtain the corresponding target tetrapeptide probe compound SRSR -A7, was a white solid with a yield of 58%. 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). MS (ESI) calcd for C 62 H 74 Cl2N6O 11 + [M+H] + 1149.49, found 1149.45.

[0144]

[0145] The general synthetic method was used to prepare RRSS -A6 from intermediate RRSS -A7, with a yield of 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 = 18.7 Hz,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 C NMR (101MHz, 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.

[0146] Example 2: Synthesis of optically pure tetrapeptide probe A8 series:

[0147] General synthetic procedure: Prolinium (0.494 mL, 5 mmol) was added to A5 (1248 mg, 1.0 mmol) dissolved in DMF (5 mL) under inert gas protection. 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 complete deprotection of Fmoc, the mixture was dissolved in ethyl acetate and washed twice with 0.1 M hydrochloric acid (50 mL) and brine (50 mL). Dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Then, the deprotected A5, N - acetyl propyl cysteine (253 mg, 1.0 mmol), benzotriazole- N, RSRS - tetramethyl urea hexafluorophosphate (HBTU) (546 mg, 1.44 mmol) was dissolved in DMF (25 mL), N,N-diisopropyl ethylamine (DIPEA) (0.348 mL, 2.0 mmol) was added, stirred at room temperature for 60 min, and monitored by thin layer chromatography (TLC) (Hex: EtOAc = 50:50). After completion of the reaction, the mixture was diluted with ethyl acetate (100 mL) and washed with 0.1 M hydrochloric acid (50 mL) and brine (50 mL). The organic layer was separated, dried over Na2S04, 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 inert gas protection, 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 completely removed, dichloromethane (30 mL) and methylene blue (2.0 mg, 0.006 mmol) were added. Under irradiation with a tungsten lamp (300 w ） oxygen was bubbled for 1.0 h, monitored by thin layer chromatography (TLC) (Hex: EtOAc = 50:50), and after completion of the reaction, the solvent was concentrated under reduced pressure. 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.

[0148]

[0149] The general synthetic procedure was used: SSSR-A9 was prepared from RSRS -A5 and N - acetyl- D propyl cysteine reaction 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) δ 171.84, 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.

[0150]

[0151] The general synthetic method was used: The deprotected tetrapeptide probe was dissolved in DMF under inert gas protection. The intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were added. The reaction was stirred at room temperature. After the allyl protecting group was completely removed, dichloromethane and methylene blue were added. The reaction was irradiated with a tungsten lamp (300 w SRRS - A5, N - acetyl- L - benzyl cysteine, benzotriazole- SRRS - tetramethyl urea hexafluorophosphate (HBTU) was dissolved in DMF, N,N- diisopropylethylamine (DIPEA) was added, and it was stirred at room temperature for 60 min, which was monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate and 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). The obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were added under inert gas protection. The reaction was stirred at room temperature. After the allyl protecting group was completely removed, dichloromethane and methylene blue were added. The reaction was irradiated with a tungsten lamp (300 w ） for 1.0 h, which was 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 tetrapeptide probe RSSR A8, was a white solid.

[0152]

[0153] The general synthetic procedure was followed: The deprotected RSSR -A5, N - acetyl- L - benzyl cysteine, benzotriazole- SRRR - tetramethyl urea hexafluorophosphate (HBTU) was dissolved in DMF, N,N- diisopropylethylamine (DIPEA) was added and the reaction was stirred at room temperature for 60 min, monitored by TLC. After completion of the reaction, the mixture was diluted with ethyl acetate and washed with 0.1 M hydrochloric acid and brine. The organic layer was separated, dried over Na2S04and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75:25). White solid intermediate was obtained. The obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were added under inert gas protection. The reaction was stirred at room temperature. After the allyl protecting group was removed sufficiently, dichloromethane and methylene blue were added. The reaction was irradiated with a tungsten lamp (300 w ） for 1.0 h with oxygen bubbling, monitored by TLC. After completion of the reaction, 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 SRRR A8, as a white solid.

[0154]

[0155] The general synthetic procedure was followed: The deprotected RSRR -A5, N - acetyl- L - benzyl cysteine, benzotriazole- SRR - tetramethyl urea hexafluorophosphate (HBTU) was dissolved in DMF, N,N- diisopropylethylamine (DIPEA) was added and the reaction was stirred at room temperature for 60 min, monitored by TLC. After completion of the reaction, the mixture was diluted with ethyl acetate and washed with 0.1 M hydrochloric acid and brine. The organic layer was separated, dried over Na2S04and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75:25). White solid intermediate was obtained. The obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were added under inert gas protection. The reaction was stirred at room temperature. After the allyl protecting group was removed sufficiently, dichloromethane and methylene blue were added. The reaction was irradiated with a tungsten lamp (300 w ） for 1.0 h with oxygen bubbling, monitored by TLC. After completion of the reaction, 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 RRSR A8, as a white solid.

[0156]

[0157] Using the general synthetic procedure: The deprotected tetrapeptide probe RRSR -A5, N -acetyl- L -benzyloxycarbonyl cysteine, benzotriazole- RRRS -HBTU was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added and the reaction was stirred at room temperature for 60 min, monitored by TLC. After completion of the reaction, the mixture was diluted with ethyl acetate and washed with 0.1 M hydrochloric acid and brine. The organic layer was separated, dried over Na2S04and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75:25). White solid intermediate was obtained. The obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were added under inert gas protection. The reaction was stirred at room temperature. After the allyl protecting group was removed sufficiently, dichloromethane and methylene blue were added. The reaction was irradiated with a tungsten lamp (300 w ） for 1.0 h with oxygen bubbling, monitored by TLC. After completion of the reaction, 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 RRRS A8, as a white solid.

[0158]

[0159] Using the general synthetic procedure: The deprotected tetrapeptide probe RRRR -A5, N -acetyl- D -benzyloxycarbonyl cysteine, benzotriazole- RRRR -HBTU was dissolved in DMF, N,N-diisopropylethylamine (DIPEA) was added and the reaction was stirred at room temperature for 60 min, monitored by TLC. After completion of the reaction, the mixture was diluted with ethyl acetate and washed with 0.1 M hydrochloric acid and brine. The organic layer was separated, dried over Na2S04and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75:25). White solid intermediate was obtained. The obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were added under inert gas protection. The reaction was stirred at room temperature. After the allyl protecting group was removed sufficiently, dichloromethane and methylene blue were added. The reaction was irradiated with a tungsten lamp (300 w ） for 1.0 h with oxygen bubbling, monitored by TLC. After completion of the reaction, 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 probeN,N,N A8 is a white solid.

[0160]

[0161] The general synthesis method is adopted: under the protection of inert gas, the deprotected SSS -A5, N -acetyl- D -benzylcysteine, benzotriazole- SSS HBTU was dissolved in DMF, and N,N-diisopropylethylamine (DIPEA) was added. The mixture was stirred at room temperature for 60 minutes and monitored by thin-layer chromatography. After the reaction was complete, the mixture was diluted with ethyl acetate and 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 inert gas protection, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA), and tetrakis(triphenylphosphine)palladium were added. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. The reaction was carried out under a tungsten lamp (300 W). ） Irradiation, oxygen bubbling reaction for 1.0 h, monitoring by thin layer chromatography, after the completion of the reaction, 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 tetrapeptide probe N,N,N,N A8 is a white solid.

[0162]

[0163] The general synthesis method is adopted: under the protection of inert gas, the deprotected SSSS- -A5, N -acetyl- L -benzylcysteine, benzotriazole- RSS HBTU was dissolved in DMF, and N,N-diisopropylethylamine (DIPEA) was added. The mixture was stirred at room temperature for 60 minutes and monitored by thin-layer chromatography. After the reaction was complete, the mixture was diluted with ethyl acetate and 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 inert gas protection, the obtained intermediate, 1,3-dimethylbarbituric acid (DMBA), and tetrakis(triphenylphosphine)palladium were added. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. The reaction was carried out under a tungsten lamp (300 W). ）Irradiation, oxygen bubbling reaction for 1.0 h, monitored by TLC, after completion of the reaction, the solvent was concentrated under reduced pressure, the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain the tetrapeptide probe N,N,N,N A8, was a white solid.

[0164]

[0165] Using the general synthetic method: under inert gas protection, the deprotected RSSS- -A5, N - acetyl- L - benzyl cysteine, benzotriazole- SRS - tetramethyl urea hexafluorophosphate (HBTU) was dissolved in DMF, N,N- diisopropyl ethylamine (DIPEA) was added, stirred at room temperature for 60 min, monitored by TLC. After completion of the reaction, the mixture was diluted with ethyl acetate, washed with 0.1M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75:25). The obtained intermediate, 1,3-dimethyl barbituric acid (DMBA) and tetrakis (triphenylphosphine) palladium were obtained under inert gas protection. The reaction was stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under the irradiation of a tungsten lamp (300 w ） Irradiation, oxygen bubbling reaction for 1.0 h, monitored by TLC, after completion of the reaction, the solvent was concentrated under reduced pressure, the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to obtain the tetrapeptide probe N,N,N,N A8, was a white solid.

[0166]

[0167] Using the general synthetic method: under inert gas protection, the deprotected SSSS- -A5, N - acetyl- L - benzyl cysteine, benzotriazole- SSR- Tetramethyl urea hexafluorophosphate (HBTU) was dissolved in DMF, N,N- diisopropylethylamine (DIPEA) was added, and the mixture was stirred at room temperature for 60 min, which was monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate and washed with 0.1 M hydrochloric acid and brine. The organic layer was separated, dried over Na2S04, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75:25). The obtained intermediate was a white solid. The obtained intermediate, 1,3-dimethylbarbituric acid (DMBA), and tetrakis(triphenylphosphine)palladium were stirred at room temperature under inert gas protection. After the allyl protecting group was completely removed, dichloromethane and methylene blue were added. The reaction was irradiated with a tungsten lamp (300 w ） for 1.0 h under oxygen bubbling, which was 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 tetrapeptide probe N,N,N,N A8, was a white solid.

[0168]

[0169] The general synthetic method was used: the deprotected SSSS- -A5, N - acetyl- D - benzyl cysteine, benzotriazole- RSS - Tetramethyl urea hexafluorophosphate (HBTU) was dissolved in DMF, N,N- diisopropylethylamine (DIPEA) was added, and the mixture was stirred at room temperature for 60 min, which was monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate and washed with 0.1 M hydrochloric acid and brine. The organic layer was separated, dried over Na2S04, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75:25). The obtained intermediate was a white solid. The obtained intermediate, 1,3-dimethylbarbituric acid (DMBA), and tetrakis(triphenylphosphine)palladium were stirred at room temperature under inert gas protection. After the allyl protecting group was completely removed, dichloromethane and methylene blue were added. The reaction was irradiated with a tungsten lamp (300 w ） for 1.0 h under oxygen bubbling, which was 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 tetrapeptide probe N,N,N,N A8, was a white solid.

[0170]

[0171] The general synthetic method was used: the deprotected SSSS- -A5, Nacetyl D benzyl cysteine, benzotriazole SSR tetramethyl urea hexafluorophosphate (HBTU) was dissolved in DMF, N,N- diisopropylethylamine (DIPEA) was added, the reaction was stirred at room temperature for 60 min, monitored by thin layer chromatography. After completion of the reaction, the mixture was diluted with ethyl acetate, washed with 0.1 M hydrochloric acid and brine. The organic layer was separated, dried over Na2S04, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75:25). White solid intermediate was obtained. The obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred at room temperature under inert gas protection. After the allyl protecting group was completely removed, dichloromethane and methylene blue were added. The reaction was irradiated with a tungsten lamp (300 w ） for 1.0 h, monitored by thin layer chromatography. After completion of the reaction, 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 N,N,N,N A8, was a white solid.

[0172]

[0173] The general synthetic method was used: the deprotected SSSS- -A5, N acetyl D benzyl cysteine, benzotriazole RRS tetramethyl urea hexafluorophosphate (HBTU) was dissolved in DMF, N,N- diisopropylethylamine (DIPEA) was added, the reaction was stirred at room temperature for 60 min, monitored by thin layer chromatography. After completion of the reaction, the mixture was diluted with ethyl acetate, washed with 0.1 M hydrochloric acid and brine. The organic layer was separated, dried over Na2S04, and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75:25). White solid intermediate was obtained. The obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred at room temperature under inert gas protection. After the allyl protecting group was completely removed, dichloromethane and methylene blue were added. The reaction was irradiated with a tungsten lamp (300 w ） for 1.0 h, monitored by thin layer chromatography. After completion of the reaction, 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 N,N,N,N A8, was a white solid.

[0174]

[0175] The general synthetic procedure was followed: The deprotected SSSS- -A5, N - acetyl- D - benzyl cysteine, benzotriazole- SRR - tetramethyl urea hexafluorophosphate (HBTU) was dissolved in DMF, N,N- diisopropylethylamine (DIPEA) was added and the reaction was stirred at room temperature for 60 min, monitored by TLC. After completion of the reaction, the mixture was diluted with ethyl acetate and washed with 0.1 M hydrochloric acid and brine. The organic layer was separated, dried over Na2S04and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75:25). White solid intermediate was obtained. The obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were added under inert gas protection. The reaction was stirred at room temperature. After the allyl protecting group was removed sufficiently, dichloromethane and methylene blue were added. The reaction was irradiated with a tungsten lamp (300 w ） for 1.0 h with oxygen bubbling, monitored by TLC. After completion of the reaction, 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 N,N,N,N A8, as a white solid.

[0176]

[0177] The general synthetic procedure was followed: The deprotected SSSS- -A5, N - acetyl- L - benzyl cysteine, benzotriazole- RSR - tetramethyl urea hexafluorophosphate (HBTU) was dissolved in DMF, N,N- diisopropylethylamine (DIPEA) was added and the reaction was stirred at room temperature for 60 min, monitored by TLC. After completion of the reaction, the mixture was diluted with ethyl acetate and washed with 0.1 M hydrochloric acid and brine. The organic layer was separated, dried over Na2S04and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (Hex: EtOAc = 75:25). White solid intermediate was obtained. The obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were added under inert gas protection. The reaction was stirred at room temperature. After the allyl protecting group was removed sufficiently, dichloromethane and methylene blue were added. The reaction was irradiated with a tungsten lamp (300 w ） for 1.0 h with oxygen bubbling, monitored by TLC. After completion of the reaction, 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 N,N,N,N A8, as a white solid.

[0178]

[0179] General synthetic procedure: The deprotected tetrapeptide probe Fmoc-SSS-A5-O2 was dissolved in dichloromethane (20 mL) and methylene blue (1.0 mg, 0.003 mmol) was added. The reaction was stirred at room temperature under an inert atmosphere. After the allyl protecting group was completely removed, oxygen was bubbled into the reaction mixture for 1.0 h under a tungsten lamp (300 w) irradiation. The reaction was monitored by thin layer chromatography (TLC) (CH2Cl2: MeOH = 30: 1). After the reaction was completed, the solvent was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to give the tetrapeptide probe Fmoc-SSS-A5-O2 as a white solid. SSSS- - A5, N - acetyl- D - benzyl cysteine, benzotriazole- SRS - tetramethyl urea hexafluorophosphate (HBTU) was dissolved in DMF, N,N- diisopropylethylamine (DIPEA) was added, and the mixture was stirred at room temperature for 60 min, which was monitored by thin layer chromatography. After the reaction was completed, the mixture was diluted with ethyl acetate and washed with 0.1 M hydrochloric acid and brine. The organic layer was separated, dried over Na2SO4, and evaporated under reduced pressure. The crude product was purified by column chromatography on silica gel (Hex: EtOAc = 75: 25). The obtained intermediate was a white solid. The obtained intermediate, 1,3-dimethylbarbituric acid (DMBA), and tetrakis(triphenylphosphine)palladium were dissolved in dichloromethane (20 mL) and methylene blue (1.0 mg, 0.003 mmol) was added. The reaction was stirred at room temperature under an inert atmosphere. After the allyl protecting group was completely removed, oxygen was bubbled into the reaction mixture for 1.0 h under a tungsten lamp (300 w) irradiation, which was monitored by thin layer chromatography. After the reaction was completed, the solvent was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to give the tetrapeptide probe Fmoc-SSS-A5-O2 as a white solid. ） N,N,N,N A8, was a white solid.

[0180] Example 3: General synthetic procedure for the synthesis of optically pure tripeptide probe Fmoc-A5-O2 series:

[0181] General synthetic procedure: 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 dissolved in dichloromethane (20 mL) and methylene blue (1.0 mg, 0.003 mmol) was added. The reaction was stirred at room temperature under an inert atmosphere. After the allyl protecting group was completely removed, oxygen was bubbled into the reaction mixture for 50 min under a tungsten lamp (300 w) irradiation, which was monitored by thin layer chromatography (TLC) (CH2Cl2: MeOH = 30: 1). After the reaction was completed, the solvent was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50: 1) to give Fmoc-A5-O2 as a white solid. 2。

[0182]

[0183] General synthetic procedure: Fmoc-SSS-A5-O2 was prepared from SSS-A5.​5- O2,收率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 (126MHz, 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.

[0184]

[0185] General synthesis method: Fmoc-SSR-A5-O2 was prepared from SSR-A5 in 85% yield. 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 = 7.5 Hz, 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) δ 174.34, 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.

[0186]

[0187] The general synthesis method is adopted: under the protection of inert gas, SSSS- -A5, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred at room temperature to react. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under tungsten lamp (300 W), oxygen was bubbled into the reaction for 50 minutes and monitored by thin-layer chromatography. After completion of the reaction, 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.

[0188]

[0189] The general synthesis method is adopted: under the protection of inert gas, N,N,N,N-A5, 1,3-dimethylbarbituric acid (DMBA) and palladium tetrakis(triphenylphosphine) were stirred at room temperature. After the allyl protecting group was completely removed, dichloromethane and methylene blue were added. Oxygen was bubbled into the reaction under irradiation of tungsten lamp (300 w) for 50 min, which was 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- SSSS- A5-O2, was a white solid.

[0190]

[0191] Using the general synthetic method: under inert gas protection, the SRR -A5, 1,3-dimethylbarbituric acid (DMBA) and palladium tetrakis(triphenylphosphine) were stirred at room temperature. After the allyl protecting group was completely removed, dichloromethane and methylene blue were added. Oxygen was bubbled into the reaction under irradiation of tungsten lamp (300 w) for 50 min, which was 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- N,N,N,N A5-O2, was a white solid.

[0192]

[0193] Using the general synthetic method: under inert gas protection, the SSSS- RRS N,N,N,N SSSS- RRR N,N,N,N SSSS- RRR N,N,N,N SSSS- SRR SRR- SRS SRS- RSS RSS- RSR -A5, 1,3-dimethylbarbituric acid (DMBA) and palladium tetrakis(triphenylphosphine) were stirred at room temperature. After the allyl protecting group was completely removed, dichloromethane and methylene blue were added. Oxygen was bubbled into the reaction under irradiation of tungsten lamp (300 w) for 50 min, which was 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, was a white solid.

[0194]

[0195] Using the general synthetic method: under inert gas protection, the RRR-A5, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred at room temperature to react. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under tungsten lamp (300 W), oxygen was bubbled into the reaction for 50 minutes and monitored by thin-layer chromatography. After completion of the reaction, 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.

[0196]

[0197] 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 to react. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under tungsten lamp (300 W), oxygen was bubbled into the reaction for 50 minutes and monitored by thin-layer chromatography. After completion of the reaction, 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.

[0198] Example 4: Synthesis of optically pure tripeptide probes Ac-A5-O2 series:

[0199] 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) was dissolved 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 complete removal of Fmoc, the mixture was dissolved in ethyl acetate and washed twice with 0.1 M hydrochloric acid (50 mL) and brine (50 mL). It was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Then, under inert gas protection, 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- Tetramethyl urea 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, stirred at room temperature for 60 min, monitored by thin layer chromatography (TLC). After completion of the reaction, 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 Na2S04, evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75:25). The intermediate was obtained as a white solid.

[0200]

[0201] The general synthetic method was adopted: SS-A4, the intermediate Ac-SSS-A5 was prepared as an example, 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 = 8.6Hz, 2H), 7.31 (d, J = 8.7 Hz, 2H), 7.28 (dd, J = 8.4, 1.7 Hz, 2H), 7.23 (d, J = 8.6Hz, 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 = 7.4Hz, 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.

[0202] General synthetic procedure: The above reaction obtained intermediate (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 under inert gas protection. 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. Oxygen was bubbled under the irradiation of a tungsten lamp (300 w) for 50 min, which was 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-A5-02 series.

[0203]

[0204] General synthetic procedure: Ac-SSS-A5 was used to prepare Ac- SSS -A5-02 with a yield of 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 = 7.8 Hz, 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.

[0205]

[0206] Using the general synthetic method described above, Ac- A5-O2 was prepared in 65% yield from SS -A4 and N -vinyl- D -4-chlorophenylalanine (A4) R -Ac-Phe(4-Cl)-OH), in 2 steps. SSR -A5-O2, in 65% yield. 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 = 15.9 Hz, 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, 8.6 Hz, 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.

[0207]

[0208] Using the general method of synthesis described above, Ac- A4-OH was prepared from SR -A4 and N -vinyl- D -4-chlorophenylalanine (Ac-Phe(4-Cl)-OH), in 2 steps R -Ac-Phe(4-Cl)-OH), in 2 steps SRR -A5-O2: piperidine was added to a solution of 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 complete removal of Fmoc was observed, the mixture was dissolved in ethyl acetate and washed with 0.1 M hydrochloric acid and brine twice. Dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Then, the deprotected SR -A4, N - vinyl- D - 4-chlorophenylalanine (Fmoc-Ac-Phe(4-Cl)-OH), benzotriazole R - Ac-Phe(4-Cl)-OH), benzotriazole N,N,N,N - tetramethyl urea hexafluorophosphate (HBTU) was dissolved in DMF, N,N- diisopropylethylamine (DIPEA) was added and stirred at room temperature for 60 min and monitored by thin layer chromatography (TLC). After completion of the reaction, the mixture was diluted with ethyl acetate and washed with 0.1 M hydrochloric acid and brine. The organic layer was separated, dried over Na2S04and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75:25). White solid intermediate was obtained. The above obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were taken in a reaction vessel under inert gas protection. The reaction was stirred at room temperature. After complete removal of the allyl protecting group, dichloromethane and methylene blue were added. Oxygen was bubbled under irradiation of tungsten lamp (300 w) for 50 min and monitored by thin layer chromatography (TLC). After completion of the reaction, 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 optically pure tripeptide probe Ac- SRR - A5-O2.

[0209]

[0210] The above general synthetic procedure was adopted to prepare Ac- SR - A4 with N - vinyl- L - 4-chlorophenylalanine (Fmoc-Ac-Phe(4-Cl)-OH), benzotriazole S - Ac-Phe(4-Cl)-OH), in 2 steps. SRS - A5-O2: PIPERIDINE was added to a solution of 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 complete removal of Fmoc was observed, the mixture was dissolved in ethyl acetate and washed with 0.1 M hydrochloric acid and brine twice. Dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Then, the deprotected SR - A4, N - vinyl- L - 4-chlorophenylalanine (Fmoc-Ac-Phe(4-Cl)-OH), benzotriazole S-Ac-Phe(4-Cl)-OH), benzotriazole N,N,N,N - Tetramethyl urea hexafluorophosphate (HBTU) was dissolved in DMF, N,N- diisopropylethylamine (DIPEA) was added and stirred at room temperature for 60 min, monitored by thin layer chromatography (TLC). After completion of the reaction, the mixture was diluted with ethyl acetate and washed with 0.1 M hydrochloric acid and brine. The organic layer was separated, dried over Na2S04and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75:25). The intermediate was obtained as a white solid. The above obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were taken in a reaction vessel under inert gas protection. The reaction was stirred at room temperature. After the allyl protecting group was completely removed, dichloromethane and methylene blue were added. Oxygen was bubbled under irradiation of a tungsten lamp (300 w) for 50 min, monitored by thin layer chromatography (TLC). After completion of the reaction, 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- SRS -A5-O2.

[0211]

[0212] The above general synthetic procedure was adopted to prepare Ac- RS -A4 with N - vinyl- L - 4-chlorophenylalanine (Fmoc-Phe(4-Cl)-OH), benzotriazole- S -Ac-Phe(4-Cl)-OH) in 2 steps. RSS -A5-O2: PIPERIDINE was added to a solution of RS -A4 in DMF under inert gas protection. The solution was stirred at room temperature for 30 min, and the reaction was monitored by thin layer chromatography (TLC). After complete removal of Fmoc was observed, the mixture was dissolved in ethyl acetate and washed twice with 0.1 M hydrochloric acid and brine. Dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Then, the deprotected RS -A4, N - vinyl- L - 4-chlorophenylalanine (Fmoc-Phe(4-Cl)-OH), benzotriazole- S -Ac-Phe(4-Cl)-OH) in 2 steps. N,N,N,N- Tetramethyl urea hexafluorophosphate (HBTU) was dissolved in DMF, N,N- diisopropylethylamine (DIPEA) was added and stirred at room temperature for 60 min, monitored by thin layer chromatography (TLC). After completion of the reaction, the mixture was diluted with ethyl acetate and washed with 0.1 M hydrochloric acid and brine. The organic layer was separated, dried over Na2S04and evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75:25). White solid intermediate was obtained. The above obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were taken in an inert atmosphere. The reaction was stirred at room temperature. After complete removal of the allyl protecting group, dichloromethane and methylene blue were added. Oxygen was bubbled under irradiation of tungsten lamp (300 w) for 50 min, monitored by thin layer chromatography (TLC). After completion of the reaction, 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 optically pure tripeptide probe Ac- RSS - A5-02.

[0213]

[0214] The above general synthetic procedure was adopted to prepare Ac- RS - A4 with N - vinyl- D - 4-chlorophenylalanine (Phe(4-Cl)-OH) in 2 steps to obtain Ac- R - A5-02: PIPERIDINE was added to a solution of RSR - A4 in DMF under inert atmosphere. The solution was stirred at room temperature for 30 min, reaction was monitored by thin layer chromatography (TLC). After complete removal of Fmoc was observed, the mixture was dissolved in ethyl acetate and washed with 0.1 M hydrochloric acid and brine twice. Dried over anhydrous sodium sulfate and solvent was removed under reduced pressure. Then, the deprotected SR - A4, RS - vinyl- N - 4-chlorophenylalanine (Phe(4-Cl)-OH) in 2 steps to obtain Ac- D - A5-02: PIPERIDINE was added to a solution of R - A4 in DMF under inert atmosphere. The solution was stirred at room temperature for 30 min, reaction was monitored by thin layer chromatography (TLC). After complete removal of Fmoc was observed, the mixture was dissolved in ethyl acetate and washed with 0.1 M hydrochloric acid and brine twice. Dried over anhydrous sodium sulfate and solvent was removed under reduced pressure. Then, the deprotected N,N,N,NHBTU was dissolved in DMF, and N,N-diisopropylethylamine (DIPEA) was added. The mixture was stirred at room temperature for 60 minutes and monitored by thin-layer chromatography (TLC). After completion of the reaction, the mixture was diluted with ethyl acetate and 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 inert gas, the intermediate obtained in the above reaction, 1,3-dimethylbarbituric acid (DMBA), and tetrakis(triphenylphosphine)palladium were reacted. 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) irradiation, oxygen was bubbled into the reaction chamber 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.

[0215]

[0216] 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 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 0.1M hydrochloric acid and brine. It was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Then, under the protection of inert gas, the deprotected RR -A4, N -Vinyl- D -4-Chlorophenylalanine ( R -Ac-Phe(4-Cl)-OH), benzotriazole- N,N,N,NHBTU was dissolved in DMF, and N,N-diisopropylethylamine (DIPEA) was added. The mixture was stirred at room temperature for 60 minutes and monitored by thin-layer chromatography (TLC). After completion of the reaction, the mixture was diluted with ethyl acetate and 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 inert gas, the intermediate obtained in the above reaction, 1,3-dimethylbarbituric acid (DMBA), and tetrakis(triphenylphosphine)palladium were reacted. 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) irradiation, oxygen was bubbled into the reaction chamber 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.

[0217]

[0218] 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 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 0.1M hydrochloric acid and brine. It was dried over anhydrous sodium sulfate and the solvent was removed under reduced pressure. Then, under the protection of inert gas, the deprotected RR -A4, N -Vinyl- L -4-Chlorophenylalanine ( S -Ac-Phe(4-Cl)-OH), benzotriazole- N,N,N,N- Tetramethyl urea hexafluorophosphate (HBTU) was dissolved in DMF, N,N- diisopropylethylamine (DIPEA) was added, stirred at room temperature for 60 min, monitored by thin layer chromatography (TLC). After completion of the reaction, the mixture was diluted with ethyl acetate, washed with 0.1 M hydrochloric acid and brine. The organic layer was separated, dried over Na2S04, evaporated under reduced pressure. The crude product was purified by silica gel column chromatography (petroleum ether: ethyl acetate = 75:25). White solid intermediate was obtained. Under inert gas protection, the above reaction obtained intermediate, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under the irradiation of tungsten lamp (300 w), oxygen was bubbled for 50 min, monitored by thin layer chromatography (TLC), after completion of the reaction, the solvent was concentrated under reduced pressure, the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50:1) to obtain optically pure tripeptide probe Ac- RRS - A5-O2.

[0219] Example 4: Synthesis method of optically pure mono-amino acid series probe:

[0220] (1) Synthesis of optically pure amino acid probe Fmoc-Lue-A9:

[0221] General synthesis method: under inert gas protection, 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 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 the irradiation of tungsten lamp (300 w), oxygen was bubbled for 1.0 h, monitored by thin layer chromatography (TLC) (Hex: EtOAc = 50:50), after completion of the reaction, the solvent was concentrated under reduced pressure, the crude product was purified by column chromatography on silica gel (CH2Cl2: MeOH = 50:1) to obtain white solid Fmoc-Lue-A9.

[0222]

[0223] Using the general synthesis method, Fmoc- S- A3 was synthesized to Fmoc- S -Lue-A9 with an isolation yield of 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 = 8.4 Hz, 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.

[0224]

[0225] Using the general synthetic method, Fmoc- R- A3 was synthesized from Fmoc- R -Lue-A9. Under inert gas protection, Fmoc- R-A3, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred at room temperature to react. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under tungsten lamp (300 W), oxygen was bubbled into the reaction for 1.0 h. The reaction was monitored by thin layer chromatography (TLC). After the reaction was complete, the solvent was concentrated under reduced pressure. The crude product was purified by column chromatography on silica gel (CH2Cl2:MeOH=50:1) to obtain a white solid Fmoc- R -Lue-A9.

[0226] (2) Synthesis of optically pure amino acid probe Ac-Lue-A9:

[0227] Under the protection of inert gas, N 2-Ethoxy-1-ethoxycarbonyl-1,2-dihydroquinoline (EEDQ) (311 mg, 1.26 mmol) was added to a solution of 1-acetyleucine (173 mg, 1 mmol) and 4-aminobenzyl alcohol (133 mg, 1.32 mmol) in tetrahydrofuran (5 mL). The reaction was allowed to proceed at room temperature and monitored by thin-layer chromatography (TLC). After completion of the reaction, the solvent was removed by concentration under reduced pressure. 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 Ac-A1 as a white solid in a 93% yield.

[0228]

[0229] Under inert gas, Ac-A1 (556 mg, 2.0 mmol) and sodium iodide (900 mg, 6.0 mmol) were dissolved in acetonitrile (30 mL). Trimethylsilyl chloride (1.5 mL) was added at 0°C and the mixture was allowed to react for 5 min at 0°C. The mixture was then moved to room temperature in the dark for 1 h. The reaction was monitored by thin-layer chromatography (TLC). After the reaction was complete, the solvent was removed by concentration under reduced pressure. 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 Ac as a white solid. - A2, isolated yield 86%.

[0230]

[0231] General method: Under inert gas, allyl 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) and reacted at room temperature for 30 min. Ac-A2 (595 mg, 1.05 mmol) was added and reacted for 5 min. The reaction was monitored by thin-layer chromatography (TLC). After completion of the reaction, 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 Ac-A3 as a white solid in 87% yield.

[0232]

[0233] General method: Under 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 reacted with stirring 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) irradiation, oxygen was bubbled into the reaction solution for 1.0 h. The reaction was monitored by thin-layer chromatography (TLC) (Hex:EtOAc = 50:50). After completion of the reaction, the solvent was concentrated under reduced pressure, and the crude product was purified by column chromatography on silica gel (CH2Cl2:MeOH = 50:1) to afford Ac-Lue-A9 as a white solid.

[0234]

[0235] According to the general method above, 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).

[0236]

[0237] General synthetic method was used to synthesize Ac- R -A3 from Ac- R -Lue-A9: Ac- R -A3, 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were stirred at room temperature under inert gas protection. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added, and the reaction was carried out under the irradiation of a tungsten lamp (300 w) and oxygen bubbling for 1.0 h, which 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 white solid Ac- R -Lue-A9.

[0238] (3) Synthesis of optically pure amino acid probe Ac-MeLue-A 10

[0239] Synthesis of compound Ac-MeLue-A1:

[0240] Under inert gas protection, Ac-MeLue-A1 was dissolved in dichloromethane, and 1,3-dimethylbarbituric acid (DMBA) and tetrakis(triphenylphosphine)palladium were added and stirred at room temperature. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added, and the reaction was carried out under the irradiation of a tungsten lamp (300 w) and oxygen bubbling for 1.0 h, which 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 white solid Ac-MeLue-A1. N ​To a solution of ethylmethylleucine (538 mg, 2.88 mmol) and 4-aminobenzyl alcohol (400 mg, 3.24 mmol) in tetrahydrofuran (20 mL) was added 2-ethoxy-l-ethoxycarbonyl-l,2-dihydroquinoline (EEDQ) (934 mg, 3.80 mmol) at room temperature and the reaction was monitored by thin layer chromatography (TLC). Upon completion of the reaction, the solvent was removed by concentration under reduced pressure and the residue was dissolved in ethyl acetate (100 mL) and 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 Ac-MeLue-A1 as a white solid with an isolated yield of 95%.

[0241] Synthesis of compound Ac-MeLue-A2

[0242]

[0243] Ac-MeLue-A1 (374 mg, 2.0 mmol) and sodium iodide (900 mg, 6.0 mmol) were dissolved in acetonitrile (30 mL) under inert gas protection. Trimethylsilyl chloride (1.5 mL) was added at 0 °C and after 5 min of reaction at 0 °C it was transferred to room temperature and left to react in the dark for 1 h. The reaction was monitored by thin layer chromatography (TLC). Upon completion of the reaction, the solvent was removed by concentration under reduced pressure and the residue was poured into 100 mL of water, extracted with ethyl acetate (50 mL) three times 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 Ac-MeLue-A2 as a white solid with an isolated yield of 90%.

[0244] Synthesis of compound Ac-MeLue-A3

[0245]

[0246] Under inert gas protection, allyl 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 with ethyl acetate (50 mL) three times, 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 isolation yield of 88%.

[0247] Compound Ac-MeLue-A 10 Synthesis of

[0248] General synthesis method: 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 stirred at room temperature under inert gas protection. After the allyl protecting group was completely removed, dichloromethane (30 mL) and methylene blue (2.0 mg, 0.006 mmol) were added. Under the irradiation of a tungsten lamp (300 w), oxygen was bubbled for 1.0 h, and the reaction was 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 .

[0249]

[0250] General synthesis method: Ac-S-MeLue-A3 was synthesized from Ac-S-MeLue-A 10 with an isolation yield of 76%. 1 H NMR (400MHz, Chloroform- d ) δ 8.14 (s, 1H), 7.90 (d, J = 16.2 Hz, 1H), 7.69 (d, J = 7.5Hz, 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, 1.4 Hz, 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).

[0251]

[0252] 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 at room temperature to react. After the allyl protecting group was fully removed, dichloromethane and methylene blue were added. Under tungsten lamp (300 W), oxygen was bubbled into the reaction 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. 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 .

[0253] Application Example Probe Recognition of Mycobacterium tuberculosis and Chemiluminescence Intensity:

[0254] Experimental methods:

[0255] 1. Probe preparation: (1) Prepare 0.01% Triton X-100 in PBS: DMSO = 1:1 buffer solution, (2) weigh a certain amount of probe powder, dissolve in DMSO solution, dilute with buffer solution, prepare probe solution concentration 800 μmol / L, (3) the probe solution is stored at 4°C in the dark, and is used on the same day.

[0256] 2. Bacterial solution preparation: (1) use a loop to pick up a ring of Mycobacterium tuberculosis / non-tuberculosis mycobacteria from a Luer tube and add it to 7H9 / OADC liquid medium, ultrasonic the bacterial solution, (2) adjust the OD value of the bacterial solution to 1.1-1.2 with the medium as a working bacterial solution. 600

[0257] 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, start timing, (2) measure the luminescence value within 5 minutes after addition, the integration time of each well is 1 second, the scanning time of the 96-well plate is 5 minutes, (3) detect the luminescence value every 10 minutes, for at least 1.5 hours.

[0258] 4. In the experimental study, the probe SSSS -A7 is a small peptide probe reported in the literature, which is used as a comparative example and an active control in the present application:

[0259]

[0260] The probe used as a comparative example and a control

[0261] Results and structure-activity relationship:

[0262] 1. The probe involved in the present application SSSS -A7 is used as a comparative example and a control, which has good Mycobacterium tuberculosis recognition ability. However, during the synthesis process, when the indane glycine is introduced to prepare the probe SSSS -A7, racemization easily occurs, and the stereoselectivity is poor, so the optical purity of the product is affected. At the same time SSSS -A7 is insoluble in water and generally has poor solubility in organic solvents, which is not conducive to the later application of the probe. Therefore, it is urgent to develop a probe with good selectivity and high luminescence intensity. Considering that small peptides are short chain molecules composed of chiral amino acids connected by peptide bonds, and the chiral configuration of amino acids as the basic building block of small peptides has a decisive influence on the overall structure and function of small peptides. Naturally occurring amino acids are mainly L -configuration. However, by artificial synthesis or modification methods, the introduction of D ​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 peptides, and in turn affect the interaction mode and affinity of the small peptides with the biological targets.

[0263] Therefore, the present application synthesizes a series of optically pure small peptide chemiluminescent probes by changing the absolute configuration of the amino acids in the peptide chain; the length of the peptide chain; the type of amino acid; and the capping group of the peptide, and reveals the influence of the small peptide chemiluminescent probes on the recognition performance of Mycobacterium tuberculosis based on chiral control and structural transformation; the intensity of chemiluminescence; the chiral structure-activity relationship; and the ability to distinguish and specifically recognize Mycobacterium from clinical samples.

[0264] 2. In the A7 series of probes, the present application synthesizes 16 optically pure tetrapeptide probes by changing the configuration of the 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, Figure 1 -A7 is a reported probe, which is used as a positive control and a comparative example of the present application. The synthesized 16 tetrapeptide probes are used for Mycobacterium tuberculosis recognition and luminescence intensity research using Mycobacterium tuberculosis H37Ra and Mycobacterium bovis Bacille Calmette – Guérin (BCG). The results of the change of the luminescence intensity (ordinate) of the probes with time (abscissa) are shown in SSSS (BCG: Bacille Calmette-Guérin; Ra: Mycobacterium tuberculosis H37Ra).

[0265] The research results show that the tetrapeptide probes SSR -A7, S SSRS -A7, SRSS -A7, SSRR -A7, SRSR -A7, SRRR -A7 can all recognize Mycobacterium tuberculosis, and interact with Mycobacterium tuberculosis and produce different intensities of chemiluminescence. While the tetrapeptide compounds SRRS -A7, RSSS -A7,RRSS -A7, RSRS -A7, RSRR -A7, RRRS -A7, RRSR -A7, RRRR -A7, RSSR -A7. SRRR -A7, cannot recognize Mycobacterium tuberculosis, thus no chemiluminescence is generated. It is shown that the absolute configuration of the amino acid at 1* is S The tetrapeptide probe with the absolute configuration of the amino acid at 1* as R The tetrapeptide probe with the absolute configuration of the amino acid at 1* as S The absolute configuration of the amino acid at 2*, 3* and 4* is found to be changeable. However, the absolute configuration of the amino acid at 2* and 3* is R at the same time, resulting in the compound SRRS -A7 and SSSS -A7 loses the ability to recognize Mycobacterium tuberculosis H37Ra. However, the tetrapeptide probe Figure 2 -A7 has the strongest ability to recognize Mycobacterium tuberculosis in the A7 series. However, the introduction of the indane-based amino acid at 4* results in a sharp decrease in the solubility of the target tetrapeptide, and the racemization at 4* is caused in the process of constructing the peptide bond with the indane-based amino acid, thus reducing the chiral purity and yield of the tetrapeptide, and therefore, is not conducive to the later application.

[0266] 3. Based on the structure of the absolute configuration and chiral structure-activity relationship of the tetrapeptide probe in the A7 series, it is found that the tetrapeptide probe has a space structure that can be modified. In view of the problem of the decrease in solubility and chiral purity caused by the introduction of the indane-based amino acid at 4*, the present application converts the amino acid at 4* into benzyl methionine, and synthesizes the tetrapeptide probe compounds in the A8 series. The synthesized tetrapeptide probes in the A8 series are subjected to Mycobacterium tuberculosis recognition and luminescence intensity research. The results of the change of the luminescence intensity (ordinate) of the probe with time (abscissa) are shown in SSSS (MTB: Mycobacterium tuberculosis; KSS: Kansas Mycobacterium OR: Occasional Mycobacterium).

[0267] The research results show that the chemiluminescence intensity of the tetrapeptide probe synthesized by converting the amino acid at 4* into benzyl methionine is significantly higher than that of the control SSSS -A7 after recognizing Mycobacterium tuberculosis. The chiral structure-activity relationship of the tetrapeptide probes in the A8 series is similar to that of the tetrapeptide probes in the A7 series, that is, when the amino acid at 1* is R L-leucine, the tetrapeptide cannot generate chemiluminescence by interacting with Mycobacterium tuberculosis. When the amino acid at 1* isS The absolute configuration of the leucine at the 4* position can generally affect the chemiluminescence produced by interaction with Mycobacterium tuberculosis. SSSR -A8 and SSSS The absolute configuration of the leucine at the 4* position can generally affect the chemiluminescence produced by interaction with Mycobacterium tuberculosis. Figure 3 The A8 tetrapeptide probe has good distinguishing recognition ability for Mycobacterium tuberculosis and non-tuberculous Mycobacterium KSS and RO. It is shown that the amino acid at the 4* position is transformable and replaceable. The A8 series of tetrapeptide probes has good solubility and is not prone to racemization at the 4* position during synthesis, and has good application prospects.

[0268] 4、Based on the chiral structure-activity relationship, luminescence intensity and 4* position amino acid replaceability of the A7 and A8 series of tetrapeptide probes, the present application further removes the 4* position chiral amino acid, synthesizes 8 optically pure tripeptide Fmoc-A5-O2 series probes and 8 optically pure tripeptide Ac-A5-O2 series probes. The necessity of the 4* position amino acid and the influence of the tripeptide capping group are studied. The synthesized tripeptide probes are used for Mycobacterium tuberculosis recognition and luminescence intensity research. The results of the change of the luminescence intensity (ordinate) of the probe with time (abscissa) are shown in SSS .

[0269] The results show that after removing the 4* position amino acid, the tripeptide probe has the R configuration at the 1* position and cannot interact with Mycobacterium tuberculosis. When the 1* position is in the S configuration, it can interact with Mycobacterium tuberculosis. Among them, the tripeptide with three amino acid fragments in the S configuration can interact with Mycobacterium tuberculosis and exhibit good luminescence performance. The chemiluminescence intensity of Ac- SSSS -A5-O2 is stronger than that of Fmoc- SSS -A7 tetrapeptide probe. The capping groups of the series of tripeptide probes are Fmoc (fluorenyl oxycarbonyl) and Ac (acetyl) respectively. The results show that when the tripeptide probe capped with Ac interacts with Mycobacterium tuberculosis, the chemiluminescence intensity is higher than that of the corresponding tripeptide probe capped with Fmoc. For example, the chemiluminescence intensity of Ac- SSS -A5-O2 is stronger than that of Fmoc- SSS -A5-O2.

[0270] It is worth noting that the tripeptide probes in the Ac- Figure 4 -A5-O2 series are easy to synthesize, have good distinguishing recognition ability for Mycobacterium tuberculosis and KSS\RO, and have relatively good solubility, and have good application potential.

[0271] 5、In order to further study the influence of the amino acid at the 1st position and the capping group on the recognition ability and the luminescence intensity of the probe, three series of single-amino acid probes are designed and synthesized: Fmoc-Lue-A9; Ac-Lue-A9; Ac-MeLue-A 10 The three series of single-amino acid probes are used for the study of the recognition of Mycobacterium tuberculosis and the luminescence intensity. The results show that the three series of single-amino acid probes cannot interact with Mycobacterium tuberculosis to produce chemiluminescence phenomenon.

[0272] 6、The study of the recognition ability of the synthesized small peptide probes for low-concentration clinical isolates of Mycobacterium tuberculosis strains, and the results of the change of the luminescence intensity (ordinate) of the probes with time (abscissa) are shown in SSS The red curve is the clinical isolates of Mycobacterium tuberculosis strains (a total of 10 strains, numbered as 190, 179, 170, 177, 96, 115, 125, 153, 106, and 98), and the green curve is the clinical isolates of non-Mycobacterium tuberculosis (a total of 10 strains, numbered as 2, 6, 10, 13, 14, 28, 30, 45, 51, and 70).

[0273] The experimental results show that under the same conditions, the selected tripeptide probe (Ac- SSSR -A5-O2) and the tetrapeptide probe (Ac- SSS -A8) of the present application have good recognition ability for clinical Mycobacterium tuberculosis strains, and the luminescence intensity is better than that of the reported positive control probe S ​ -A7, indicating that the present application has potential clinical application prospects.

[0274] Obviously, the above embodiments of the present application are only examples for clearly illustrating the technical solutions of the present application, and are not intended to limit the specific embodiments of the present application. Any modification, equivalent replacement, and improvement made within the spirit and principle of the present application should be included in the protection scope of the present application.

Claims

1. An optically pure small peptide chemiluminescent probe, characterized in that: The structural formula is shown in any of the following formulas: 、 。 2. Use of the optically pure small peptide chemiluminescent probe according to claim 1 in the preparation of a Mycobacterium tuberculosis identification preparation.

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