Dimeric amino acid compounds and their preparation methods

Abstract

This disclosure provides a method for preparing a dimer amino acid compound, comprising the following steps: providing L-serine, an indole derivative, and Tm9D8* protein, PmLAAD protein, and a dimer synthase, and culturing them; wherein the dimer synthase comprises an amino acid sequence as shown in any one of SEQ ID NO: 1-4 or having at least 90% identity with it. This disclosure also provides a cell division inhibitor comprising the dimer amino acid compound, and a composition comprising the dimer amino acid compound or the cell division inhibitor.

Description

Technical Field

[0001] This disclosure pertains to the field of biomedicine, and particularly relates to dimer amino acid compounds. Background Technology

[0002] In recent years, dimeric amino acid bioactive natural products have received widespread attention in the pharmaceutical field due to their generally good biological activities.

[0003] Through tireless research, the inventors discovered that diamino acid compounds derived from tryptophan exhibit outstanding activity in inhibiting the G2 phase checkpoint of cell division, and also possess certain inhibitory effects on topoisomerase I and protein kinase C. These biological activities make them ideal molecular models and drug precursors for anticancer, antimalarial, and leukemia treatments, and their synthesis methods have long attracted the attention of pharmaceutical companies. Currently, no naturally occurring tryptophan dimer derivatives have been found in nature, and the reaction route for the de novo preparation of this analogue using chemical reactions is only found in a research paper published at the end of the last century. This method has low synthesis efficiency and small scale, and uses toxic raw materials and byproducts with unknown effects, raising concerns about biosafety. However, with the significant advancements in molecular biology and synthetic biology techniques, a preliminary understanding of the biosynthetic pathways of natural diamino acid products has been gained.

[0004] Several synthetic schemes for chromopyrrolic acid (CPA, chemically named 3,4-bis(1H-indol-3-yl)-1H-pyrrole-2,5-dicarboxylic acid), an analog derived from tryptophan dimers, have been reported. The chemical structure of CPA is as follows:

[0005]

[0006] The dimeric amino acid compound of this application exhibits significantly more significant PKA and PKC pathway inhibitory activity compared to chromopyrrolidone (CPA), and thus has greater drug development potential. Existing CPA synthesis methods (e.g., Zheng et al., Biomimetic Total Synthesis of the Spiroindimicin Family of Natural Products) Angew. Chem. Int. Ed. 2022 , 61,The key synthetic protein used in doi.org / 10.1002 / anie.202208802 can only synthesize CPA or its specific derivatives, and has no catalytic effect on the synthesis of other CPA derivatives, such as the dimeric amino acid compounds of this application. At the same time, there is a need to provide novel CPA derivatives with improved biological activity to facilitate their application in drugs and disease treatment. Summary of the Invention

[0007] Through the inventors' painstaking research, a method for preparing the dimeric amino acid compound of this application using enzymatic methods based on synthetic biology was finally developed, and a dimeric amino acid compound with superior biological inhibitory activity was also provided.

[0008] The first aspect of this disclosure provides a method for preparing a dimer amino acid compound, comprising the following steps: 1) providing L-serine, an indole derivative, Tm9D8* protein, PmLAAD protein and a dimer synthase and culturing them.

[0009] In some specific embodiments, the Tm9D8* protein comprises an amino acid sequence as shown in SEQ ID NO: 5 or having at least 90% identity with it.

[0010] In some specific embodiments, the Tm9D8* protein comprises an amino acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with SEQ ID NO: 5.

[0011] In some specific embodiments, the Tm9D8* protein comprises the amino acid sequence shown in SEQ ID NO: 5.

[0012] In some specific embodiments, the nucleic acid encoding the Tm9D8* protein comprises a nucleic acid sequence as shown in SEQ ID NO: 15 or having at least 90% identity with it.

[0013] In some preferred embodiments, the nucleic acid encoding the Tm9D8* protein comprises a nucleic acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with SEQ ID NO: 15.

[0014] In some preferred embodiments, the nucleic acid encoding the Tm9D8* protein has the nucleic acid sequence shown in SEQ ID NO: 15.

[0015] In some specific embodiments, the dimer synthase comprises an amino acid sequence as shown in any one of SEQ ID NO: 1-4 or having at least 90% identity with it.

[0016] In some preferred embodiments, the dimerase comprises an amino acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with any one of SEQ ID NO: 1-4.

[0017] In some preferred embodiments, the dimerase has an amino acid sequence shown in any one of SEQ ID NO: 1-4.

[0018] In some specific embodiments, the nucleic acid encoding the dimer synthase comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 11-14 or having at least 90% identity with it.

[0019] In some preferred embodiments, the nucleic acid encoding the dimerase comprises a nucleic acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with any one of SEQ ID NO: 11-14.

[0020] In some preferred embodiments, the nucleic acid encoding the dimerase has a nucleic acid sequence shown in any one of SEQ ID NO: 11-14.

[0021] In some specific implementations, step 1) includes the following reaction:

[0022] .

[0023] In some specific implementations, the reaction in step 1) is carried out in the same reaction system and / or reaction vessel.

[0024] In some specific embodiments, the indole derivative is selected from 5-cyanoindole, 7-cyanoindole, 5-methoxyindole, and / or 6-methoxyindole.

[0025] In some specific implementations, the reaction temperature in step 1) is 28 ℃-37 ℃.

[0026] In some specific implementations, the pH in step 1) is 7-9.

[0027] In some specific embodiments, the pH of the reaction system is adjusted by using a NaH2PO4-Na2HPO4 buffer solution and an aqueous solution of (NH4)2SO4.

[0028] In some specific embodiments, the dimer amino acid compound is a dimer amino acid compound derived from tryptophan (ditryptophan derivative).

[0029] In some preferred embodiments, the dimer amino acid compound is selected from 5',5''-dicyano-chromopyrrolic acid (bis-5cn-M), 7',7''-dicyano-chromopyrrolic acid (bis-7cn-M), 5',5''-dimethoxy-chromopyrrolic acid (bis-5mo-M) and / or 6',6''-dimethoxy-chromopyrrolic acid (bis-6mo-M), each having the following chemical structure:

[0030]

[0031] In some specific embodiments, the Tm9D8* protein, PmLAAD protein, and the dimer synthase are provided in whole-cell form of E. coli expressing the vector pRSFduet-Tm9D8*-PmLAAD-dimer synthase.

[0032] In some specific embodiments, the Tm9D8* protein, PmLAAD protein, and the dimer synthase are provided in the form of a culture lysate of the expression vector pRSFduet-Tm9D8*-PmLAAD-dimer synthase.

[0033] In some specific embodiments, the Tm9D8* protein, PmLAAD protein, and the dimer synthase are provided in the form of whole E. coli cells and culture lysates expressing the vector pRSFduet-Tm9D8*-PmLAAD-dimer synthase.

[0034] In some specific embodiments, the final concentration of the Tm9D8* protein is approximately 20-30 μM.

[0035] In some specific embodiments, the final concentration of the PmLAAD protein is approximately 15-20 μM.

[0036] In some specific embodiments, the final concentration of the dimer synthase is about 30-50 μM.

[0037] In some specific embodiments, the reaction in step 1) can also be carried out in steps. For example, a method for preparing a dimer amino acid compound includes the following steps: 1-A) providing L-serine, an indole derivative, and Tm9D8* protein and culturing them; 1-B) subsequently providing PmLAAD protein and dimer synthase and culturing them, wherein the Tm9D8* protein, dimer synthase, reaction temperature, reaction pH, etc., are as described above.

[0038] When the reaction in step 1) is carried out stepwise, the final concentration of the Tm9D8* protein can be about 20-30 μM; the final concentration of the PmLAAD protein can be about 4-8 μM; and the final concentration of the dimer synthase can be about 40-60 μM.

[0039] In some specific embodiments, the aforementioned method further includes the following step: 2) separating the dimer amino acid compound from the reaction system.

[0040] Another aspect of this disclosure provides a synthesizing enzyme for dimer amino acid compounds comprising an amino acid sequence as shown in any one of SEQ ID NO: 1-4 or having at least 90% identity with it.

[0041] In some preferred embodiments, the synthase comprises an amino acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with any one of SEQ ID NO: 1-4.

[0042] In some preferred embodiments, the synthase has an amino acid sequence shown in any one of SEQ ID NO: 1-4.

[0043] Another aspect of this disclosure provides a nucleic acid encoding a synthase of a dimer amino acid compound, comprising or having at least 90% identity with any of the nucleic acid sequences shown in or having at least 90% identity with any of SEQ ID NO: 11-14.

[0044] In some preferred embodiments, the nucleic acid of the synthase encoding the dimer amino acid compound comprises a nucleic acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with any one of SEQ ID NO: 11-14.

[0045] In some preferred embodiments, the nucleic acid of the synthase encoding the dimer amino acid compound has the nucleic acid sequence shown in any one of SEQ ID NO: 11-14.

[0046] Another aspect of this disclosure provides a Tm9D8* protein for the synthesis of dimeric amino acid compounds, comprising an amino acid sequence as shown in SEQ ID NO: 5 or having at least 90% identity with it.

[0047] In some preferred embodiments, the Tm9D8* protein comprises an amino acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with SEQ ID NO: 5.

[0048] In some preferred embodiments, the Tm9D8* protein has the amino acid sequence shown in SEQ ID NO: 5.

[0049] Another aspect of this disclosure provides a nucleic acid encoding the Tm9D8* protein, comprising a nucleic acid sequence as shown in SEQ ID NO: 15 or having at least 90% identity with it.

[0050] In some preferred embodiments, the nucleic acid encoding the Tm9D8* protein comprises a nucleic acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with SEQ ID NO: 15.

[0051] In some preferred embodiments, the nucleic acid encoding the Tm9D8* protein has the nucleic acid sequence shown in SEQ ID NO: 15.

[0052] In some specific embodiments, the above-mentioned dimer amino acid compounds are selected from bis-5cn-M, bis-7cn-M, bis-5mo-M and / or bis-6mo-M.

[0053] Another aspect of this disclosure provides the use of the aforementioned synthase in the preparation of dimer amino acid compounds.

[0054] Another aspect of this disclosure provides the use of the aforementioned nucleic acid-encoded synthase in the preparation of dimeric amino acid compounds.

[0055] Another aspect of this disclosure provides the use of the aforementioned Tm9D8* protein in the preparation of dimeric amino acid compounds.

[0056] Another aspect of this disclosure provides the use of the aforementioned nucleic acid-encoded Tm9D8* protein in the preparation of dimeric amino acid compounds.

[0057] In some specific embodiments, the dimer amino acid compounds used in the above-mentioned applications are selected from bis-5cn-M, bis-7cn-M, bis-5mo-M and / or bis-6mo-M.

[0058] Another aspect of this disclosure provides a dimer amino acid compound prepared according to the aforementioned method.

[0059] The second aspect of this disclosure provides a method for preparing a dimer amino acid compound, comprising the following steps: i) providing L-serine, an indole derivative and Tm2F3 protein and culturing them; ii) subsequently providing PmLAAD protein and a dimer synthase and culturing them.

[0060] In some specific embodiments, the dimer synthase comprises an amino acid sequence as shown in any one of SEQ ID NO: 1-4 or having at least 90% identity with it.

[0061] In some preferred embodiments, the dimerase comprises an amino acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with any one of SEQ ID NO: 1-4.

[0062] In some preferred embodiments, the dimerase has an amino acid sequence shown in any one of SEQ ID NO: 1-4.

[0063] In some specific embodiments, the nucleic acid encoding the dimer synthase comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 11-14 or having at least 90% identity with it.

[0064] In some preferred embodiments, the nucleic acid encoding the dimerase comprises a nucleic acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with any one of SEQ ID NO: 11-14.

[0065] In some preferred embodiments, the nucleic acid encoding the dimerase has a nucleic acid sequence shown in any one of SEQ ID NO: 11-14.

[0066] In some specific implementations, the reaction formula for step i) is:

[0067] .

[0068] In some specific implementations, the reaction formula for step ii) is:

[0069] .

[0070] In some specific embodiments, the indole derivative is selected from 5-cyanoindole, 7-cyanoindole, 5-methoxyindole, and / or 6-methoxyindole.

[0071] In some specific implementations, the reaction temperature in step i) is 50 ℃-65 ℃.

[0072] In some specific implementations, the pH in step i) is 7-9.

[0073] In some specific implementations, the reaction temperature in step ii) is 35 ℃-40 ℃.

[0074] In some specific implementations, the pH in step ii) is 7-9.

[0075] In some specific embodiments, the pH of the reaction system is adjusted by using a NaH2PO4-Na2HPO4 buffer solution and an aqueous solution of (NH4)2SO4.

[0076] In some specific embodiments, the dimer amino acid compound is a dimer amino acid compound derived from tryptophan (ditryptophan derivative).

[0077] In some preferred embodiments, the dimer amino acid compound is selected from 5',5''-dicyano-chromopyrrolic acid (bis-5cn-M), 7',7''-dicyano-chromopyrrolic acid (bis-7cn-M), 5',5''-dimethoxy-chromopyrrolic acid (bis-5mo-M) and / or 6',6''-dimethoxy-chromopyrrolic acid (bis-6mo-M), each having the following chemical structure:

[0078]

[0079] In some specific embodiments, the Tm2F3 protein and the PmLAAD protein are provided in the form of culture lysates of expression vectors pET28a(+)-Tm2F3 and pET28a(+)-PmLAAD, respectively.

[0080] In some specific embodiments, the dimer synthase is provided in the form of a culture lysate of the expression vector pMCSG10-dimer synthase.

[0081] In some specific embodiments, the final concentration of the Tm2F3 protein is approximately 20-30 μM.

[0082] In some specific embodiments, the final concentration of the PmLAAD protein is approximately 4-8 μM.

[0083] In some specific embodiments, the final concentration of the dimer synthase is approximately 40-60 μM.

[0084] In some specific embodiments, the aforementioned method further includes the following step: iii) separating the dimer amino acid compound from the reaction system.

[0085] Another aspect of this disclosure provides a synthesizing enzyme for dimer amino acid compounds comprising an amino acid sequence as shown in any one of SEQ ID NO: 1-4 or having at least 90% identity with it.

[0086] In some preferred embodiments, the synthase comprises an amino acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with any one of SEQ ID NO: 1-4.

[0087] In some preferred embodiments, the synthase has an amino acid sequence shown in any one of SEQ ID NO: 1-4.

[0088] Another aspect of this disclosure provides a nucleic acid encoding a synthase of a dimer amino acid compound, comprising or having at least 90% identity with any of the nucleic acid sequences shown in or having at least 90% identity with any of SEQ ID NO: 11-14.

[0089] In some preferred embodiments, the nucleic acid comprises a nucleic acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with any one of SEQ ID NO: 11-14.

[0090] In some preferred embodiments, the nucleic acid has a nucleic acid sequence shown in any one of SEQ ID NO: 11-14.

[0091] In some specific embodiments, the above-mentioned dimer amino acid compounds are selected from bis-5cn-M, bis-7cn-M, bis-5mo-M and / or bis-6mo-M.

[0092] Another aspect of this disclosure provides the use of the aforementioned synthase in the preparation of dimer amino acid compounds.

[0093] Another aspect of this disclosure provides the use of the aforementioned nucleic acid-encoded synthase in the preparation of dimeric amino acid compounds.

[0094] In some embodiments of this disclosure, the dimer amino acid compounds used in the above-described applications are selected from bis-5cn-M, bis-7cn-M, bis-5mo-M, and / or bis-6mo-M.

[0095] A third aspect of this disclosure also provides a dimer amino acid compound having the structure of formula (I):

[0096] ,

[0097] Among them, R 1 and R 2 Each time it appears, it is independently selected from either cyano or methoxy.

[0098] Or its pharmaceutically acceptable salt.

[0099] In some specific implementations, R 1 and R 2 They are the same substituents.

[0100] In some specific embodiments, the aforementioned dimer amino acid compounds having the structure of formula (I) are selected from:

[0101]

[0102] The fourth aspect of this disclosure also provides a cell division inhibitor comprising a dimer amino acid compound prepared by the aforementioned method or the aforementioned dimer amino acid compound.

[0103] The fifth aspect of this disclosure also provides a composition comprising a dimer amino acid compound prepared by the aforementioned method, the aforementioned dimer amino acid compound, or the aforementioned cell division inhibitor.

[0104] The sixth aspect of this disclosure also provides the use of the dimer amino acid compounds prepared by the aforementioned methods, the aforementioned dimer amino acid compounds, the aforementioned cell division inhibitors, or the aforementioned compositions in the preparation of medicaments for inhibiting the G2 phase checkpoint of cell division, inhibiting topoisomerase I, and / or inhibiting protein kinase C.

[0105] The seventh aspect of this disclosure also provides the use of the dimer amino acid compounds prepared by the aforementioned methods, the aforementioned dimer amino acid compounds, the aforementioned cell division inhibitors, or the aforementioned compositions in the preparation of a medicament for treating a disease, disorder, or condition in a subject.

[0106] In some specific implementations, the disease, obstacle, or ailment is cancer or malaria.

[0107] In some specific implementations, the cancer is melanoma.

[0108] The eighth aspect of this disclosure also provides a method for inhibiting the G2 phase checkpoint of cell division, inhibiting topoisomerase I, and / or inhibiting protein kinase C, comprising administering to a subject a dimeric amino acid compound prepared by the aforementioned method, the aforementioned dimeric amino acid compound, the aforementioned cell division inhibitor, or the aforementioned composition.

[0109] The ninth aspect of this disclosure also provides a method for treating a disease, disorder, or symptom in a subject, comprising administering to the subject a dimer amino acid compound prepared by the aforementioned method, the aforementioned dimer amino acid compound, the aforementioned cell division inhibitor, or the aforementioned composition.

[0110] In some specific implementations, the disease, obstacle, or ailment is cancer or malaria.

[0111] In some specific implementations, the cancer is melanoma.

[0112] Beneficial effects

[0113] This disclosure provides a method for preparing dimer amino acid compounds, filling the gap in de novo synthetic reaction routes, and maximizing the reaction scale and yield while taking into account good economy and low toxicity. Attached Figure Description

[0114] This disclosure can be more fully understood with reference to the following figures.

[0115] Figure 1 A plasmid map of the Tm9D8*-PmLAAD-AaStaD* gene recombination of one embodiment of this disclosure is shown.

[0116] Figure 2 A plasmid map of the Tm9D8*-PmLAAD-CpStaD* gene recombination of one embodiment of this disclosure is shown.

[0117] Figure 3 A plasmid map of the Tm9D8*-PmLAAD-AbeD* gene recombination of one embodiment of this disclosure is shown.

[0118] Figure 4 A plasmid map of the Tm9D8*-PmLAAD-BorD* gene recombination of one embodiment of the present disclosure is shown.

[0119] Figure 5 The HPLC chromatogram of the product of one embodiment of this disclosure is shown.

[0120] Figure 6 This disclosure illustrates a bis-5cn-M product according to one embodiment. 1 H NMR spectrum (600 MHz, DMSO).

[0121] Figure 7 This disclosure illustrates a bis-7cn-M product according to one embodiment. 1 H NMR spectrum (600 MHz, DMSO).

[0122] Figure 8 This disclosure illustrates a bis-5 mo-M product according to one embodiment. 1 H NMR spectrum (600 MHz, DMSO).

[0123] Figure 9 This disclosure illustrates a bis-6mo-M product according to one embodiment. 1 H NMR spectrum (600 MHz, DMSO).

[0124] Figure 10 This disclosure illustrates a bis-5cn-M product according to one embodiment. 13 C NMR spectrum (600 MHz, DMSO).

[0125] Figure 11 This disclosure illustrates a bis-7cn-M product according to one embodiment. 13 C NMR spectrum (600 MHz, DMSO).

[0126] Figure 12 This disclosure illustrates a bis-5 mo-M product according to one embodiment. 13 C NMR spectrum (600 MHz, DMSO).

[0127] Figure 13 This disclosure illustrates a bis-6mo-M product according to one embodiment. 13 C NMR spectrum (600 MHz, DMSO).

[0128] Figure 14 The catalytic synthesis capabilities of existing synthesis schemes are shown in comparison with those of one embodiment of this disclosure.

[0129] Figure 15 The yield comparison of different dimeric amino acid synthases based on one embodiment of this disclosure is shown.

[0130] Figure 16 The yield comparison of products generated by different amino acid oxidases is shown.

[0131] Figure 17 A plasmid map of the AaStaD* gene recombination of another embodiment of this disclosure is shown.

[0132] Figure 18 A plasmid map of the CpStaD* gene recombination of another embodiment of this disclosure is shown.

[0133] Figure 19 A plasmid map of the AbeD* gene recombination of another embodiment of this disclosure is shown.

[0134] Figure 20 A plasmid map of the BorD* gene recombination of another embodiment of this disclosure is shown.

[0135] Figure 21 A plasmid map of the Tm2F3 gene recombination according to another embodiment of this disclosure is shown.

[0136] Figure 22 A plasmid map of the PmLAAD gene recombination of another embodiment of this disclosure is shown.

[0137] Figure 23 The catalytic synthesis capabilities of existing synthesis schemes are shown in comparison with those of another embodiment of this disclosure.

[0138] Figure 24 A comparison of the yields of different protein-generating products from another embodiment of this disclosure is shown.

[0139] Figure 25 The yield comparison of products generated by different amino acid synthases is shown. Detailed Implementation

[0140] The following description of this disclosure is merely intended to illustrate various embodiments of the disclosure. Therefore, the specific modifications discussed should not be construed as limiting the scope of this disclosure. It will be apparent to those skilled in the art that various equivalents, changes, and modifications can be made without departing from the scope of this disclosure, and it should be understood that these equivalent embodiments are included herein. All references cited herein, including publications, patents, and patent applications, are incorporated herein by reference in their entirety.

[0141] The following are definitions of terms used in this specification. Unless otherwise stated, the initial definitions provided herein apply to the use of a term individually throughout the specification or as part of another set of definitions. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art.

[0142] As used herein, the term "dimeric amino acid compound" refers to a dimer compound derived from amino acids, and is used interchangeably with "amino acid dimer" and "dimeric amino acid". When used herein, "tryptophan dimer derivative" is used interchangeably with "dimeric amino acid compound derived from tryptophan" and "analyte derived from tryptophan dimer". Exemplary dimeric amino acid compounds used in this application may be, for example, bis-5cn-M, bis-7cn-M, bis-5mo-M, or bis-6mo-M.

[0143] The term "indole derivative" refers to indole substituted with more than one substituent, for example, by more than one group on more than one carbon atom selected from the 4, 5, 6, and 7 positions of indole, and / or a group on the nitrogen atom of indole. The more than one group substituting for one or more carbon atoms can be, but is not limited to, halogen groups, alkyl groups, alkoxy groups, aryl groups, aryloxy groups, amino groups, nitro groups, and carbonyl groups. The group substituting for the nitrogen atom can be, but is not limited to, alkyl groups, aryl groups, and acyl groups. Therefore, the indole derivatives can be, for example, 5-cyanoindole, 7-cyanoindole, 5-methoxyindole, 6-methoxyindole, 4-chloroindole, 5-chloroindole, 6-chloroindole, 7-chloroindole, 5-bromoindole, 6-bromoindole, 5-nitroindole, 5-hydroxyindole, 5-methylindole, 5-methoxyindole, 6-methylindole, 7-methylindole, 5-aminoindole, 1-methylindole, and indole-6-carboxaldehyde. Any other indole derivatives can be used in the process of this invention, provided that these indole derivatives can be used to prepare the corresponding dimer amino acid compounds by the methods disclosed herein.

[0144] As used herein, the term "dimer synthase" refers to a protease that catalyzes the dimerization of tryptophan derivatives, for example, comprising an amino acid sequence as shown in or having at least 90% identity with any of SEQ ID NO: 1-4; in some preferred embodiments, the "dimer synthase" comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or more than 99% sequence identity with any of SEQ ID NO: 1-4. In some more preferred embodiments, the "dimer synthase" has the amino acid sequence described in any one of SEQ ID NO: 1-4. In some specific embodiments, the nucleic acid encoding the "dimer synthase" comprises, for example, a nucleic acid sequence as shown in any one of SEQ ID NO: 11-14 or having at least 90% identity with it; in some preferred embodiments, the nucleic acid encoding the "dimer synthase" comprises a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with any one of SEQ ID NO: 11-14; in some more preferred embodiments, the nucleic acid encoding the "dimer synthase" has the nucleic acid sequence as described in any one of SEQ ID NO: 11-14.

[0145] As used herein, the term "whole Escherichia coli" refers to an Escherichia coli cell entity that retains the integrity of its cell wall and cell membrane. The term encompasses both live, proliferating cells and cells that have been inactivated and are no longer proliferating but retain their cellular structure and immunogenicity, optionally after washing, concentration, and / or lyophilization.

[0146] In this invention, "culture lysis buffer" refers to the product obtained after lysing cells by physical (e.g., high-pressure homogenization, ultrasonic disruption, repeated freeze-thaw cycles, glass bead grinding, etc.), chemical (e.g., using denaturing agents (e.g., urea, guanidine hydrochloride), detergents (e.g., SDS, Triton X-100), alkalis or enzymes (e.g., lysozyme)), and / or a combination of both methods). This lysis buffer contains intracellular contents (e.g., cytoplasm, ribosomes, nucleic acids (DNA, RNA), expressed recombinant proteins, enzymes, metabolites, etc.) and cell membrane debris (e.g., cell wall and cell membrane debris), and may optionally be centrifuged or filtered to remove cell debris, yielding a clear supernatant portion.

[0147] As used herein, when the reaction proceeds in one step, the final concentration of the protein is obtained by the following method: First, a purified protein mixture containing three components—Tm9D8*, PmLAAD, and dimer synthase—with a 6 × His tag is separated from the total protein of *E. coli* using affinity chromatography. Subsequently, the sample is further separated using size exclusion chromatography to obtain purified proteins of Tm9D8*, PmLAAD, and dimer synthase, respectively. The content of each purified protein is determined, and the original final concentration is then calculated. For the two-step method, the final protein concentration is obtained directly by obtaining purified proteins of Tm2F3, PmLAAD, and dimer synthase separately using affinity chromatography, and then determining their concentrations to infer the final protein concentration in the protein lysate. Similarly, a similar determination method is used for the two-step method involving Tm9D8*, PmLAAD, and dimer synthase.

[0148] The term "cell division inhibitor" generally refers to substances that can inhibit cell division. Cell division generally refers to the process during cell proliferation in which the number of cells increases from one to two or more through division.

[0149] "Cell cycle checkpoints" are a set of checks during the cell cycle to ensure the quality of DNA replication and chromosome segregation. The "cell cycle" refers to the entire process a cell undergoes from the completion of one division to the end of the next, divided into two phases: interphase and mitotic phase. Interphase is further divided into three phases: pre-DNA synthesis (G1 phase), DNA synthesis (S phase), and post-DNA synthesis (G2 phase). In this article, "cell division G2 phase checkpoint" refers to the checkpoint mechanism involved in the G2 phase.

[0150] "Topoisomerase I" is an important intracellular enzyme in living organisms, participating in all key intracellular processes such as DNA replication, transcription, recombination and repair.

[0151] The term "protein kinase C (PKC)" can refer to a protein kinase that regulates protein function by phosphorylating the hydroxyl groups of serine and threonine residues in proteins.

[0152] The term “inhibition” means to slow down, stop or reverse the relevant process, including any level of inhibition, such as at least partial inhibition, such as inhibition of at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85%, at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99%.

[0153] As used herein, the term "subject" includes, but is not limited to, humans, i.e., men or women of any age group, such as pediatric subjects (e.g., infants, children, adolescents) or adult subjects (e.g., young adults, middle-aged adults, or elderly adults) and / or non-human animals, such as mammals, including primates (e.g., cynomolgus monkeys, rhesus monkeys), cattle, pigs, horses, sheep, goats, rodents, cats, and / or dogs. In some embodiments, the subject is a human. In some embodiments, the subject is a non-human animal. The terms "human," "patient," "subject," and "individual" are used interchangeably herein. None of these terms require active supervision by a medical professional.

[0154] Disease, obstacle, and symptom are used interchangeably in this article.

[0155] As used herein, unless otherwise stated, the term “treatment” refers to an effect that occurs when a subject has a particular disease, disorder, or condition, which reduces the severity of the disease, disorder, or condition, or reverses or slows the progression of the disease, disorder, or condition (also known as “therapeutic treatment”).

[0156] The first aspect of this disclosure provides a method for preparing a dimer amino acid compound, comprising the following steps: 1) providing L-serine, an indole derivative, Tm9D8* protein, PmLAAD protein and a dimer synthase and culturing them.

[0157] In some embodiments of this disclosure, the Tm9D8* protein comprises an amino acid sequence as shown in SEQ ID NO: 5 or having at least 90% identity with it.

[0158] In some preferred embodiments of this disclosure, the Tm9D8* protein comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with SEQ ID NO: 5. In some more preferred embodiments, the Tm9D8* protein comprises the amino acid sequence shown in SEQ ID NO: 5.

[0159] In some embodiments of this disclosure, the nucleic acid encoding the Tm9D8* protein comprises a nucleic acid sequence as shown in SEQ ID NO: 15 or having at least 90% identity with it.

[0160] In some preferred embodiments of this disclosure, the nucleic acid encoding the Tm9D8* protein comprises a nucleic acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with SEQ ID NO: 15. In some more preferred embodiments, the nucleic acid encoding the Tm9D8* protein has the nucleic acid sequence shown in SEQ ID NO: 15.

[0161] In some embodiments of this disclosure, the dimer synthase comprises an amino acid sequence as shown in any one of SEQ ID NO: 1-4 or having at least 90% identity with it.

[0162] In some preferred embodiments of this disclosure, the dimerase comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with any one of SEQ ID NO: 1-4. In some more preferred embodiments, the dimerase has an amino acid sequence shown in any one of SEQ ID NO: 1-4.

[0163] In some embodiments of this disclosure, the nucleic acid encoding the dimer synthase comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 11-14 or having at least 90% identity with it.

[0164] In some preferred embodiments of this disclosure, the nucleic acid encoding the dimerase comprises a nucleic acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with any one of SEQ ID NO: 11-14. In some more preferred embodiments, the nucleic acid encoding the dimerase has a nucleic acid sequence shown in any one of SEQ ID NO: 11-14.

[0165] In some specific implementations, step 1) includes the following reaction:

[0166] .

[0167] In some specific embodiments, the reaction in step 1) is carried out in the same reaction system and / or reaction vessel. That is, according to the preparation method of this disclosure, dimer amino acid compounds can be prepared by a "one-pot" process.

[0168] In some embodiments of this disclosure, the indole derivative is selected from 5-cyanoindole, 7-cyanoindole, 5-methoxyindole, and / or 6-methoxyindole.

[0169] In some embodiments of this disclosure, the reaction temperature in step 1) is 28 ℃-37 ℃. The reaction temperature can be any range or any temperature within the above range, for example, the reaction temperature can be 28 ℃-37 ℃, 50 ℃-30 ℃, 30 ℃-37 ℃, 32 ℃-35 ℃, or 35 ℃-37 ℃; for example, the reaction temperature can be 28 ℃, 29 ℃, 30 ℃, 31 ℃, 32 ℃, 33 ℃, 34 ℃, 35 ℃, 36 ℃, or 37 ℃.

[0170] In some embodiments of this disclosure, the pH in step 1) is 7-9. The pH can be any range or any value within the above range, for example, pH can be 7.1-9, 7.2-9, 7.3-9, 7.4-9, 7.5-9, 7.6-9, 7.7-9, 7.8-9, 7.9-9, 8-9, 8.1-9, 8.2-9, 8.3-9, 8.4-9, 8.5-9, 8.6-9, 8.7-9, 8.8-9 or 8.9-9; for example, pH can be 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.

[0171] In some embodiments of this disclosure, the pH of the reaction system is adjusted by using a NaH2PO4-Na2HPO4 buffer solution and an aqueous (NH4)2SO4 solution.

[0172] In some embodiments of this disclosure, the dimer amino acid compound is a dimer amino acid compound derived from tryptophan (ditryptophan derivative).

[0173] In some preferred embodiments of this disclosure, the dimer amino acid compound is selected from 5',5''-dicyano-chromopyrrolic acid (bis-5cn-M), 7',7''-dicyano-chromopyrrolic acid (bis-7cn-M), 5',5''-dimethoxy-chromopyrrolic acid (bis-5mo-M) and / or 6',6''-dimethoxy-chromopyrrolic acid (bis-6mo-M), and each has the following chemical structure:

[0174]

[0175] In some embodiments of this disclosure, the Tm9D8* protein, PmLAAD protein, and the dimer synthase are provided in whole-cell form of E. coli expressing the vector pRSFduet-Tm9D8*-PmLAAD-dimer synthase.

[0176] In some embodiments of this disclosure, the Tm9D8* protein, PmLAAD protein, and the dimer synthase are provided in the form of a culture lysate of the expression vector pRSFduet-Tm9D8*-PmLAAD-dimer synthase.

[0177] In some embodiments of this disclosure, the Tm9D8* protein, PmLAAD protein, and the dimer synthase are provided in the form of whole E. coli cells and culture lysates expressing the vector pRSFduet-Tm9D8*-PmLAAD-dimer synthase.

[0178] In some embodiments of this disclosure, the final concentration of the Tm9D8* protein is about 20-30 μM. For example, the concentration of the Tm9D8* protein is approximately 20-30 μM, 21-30 μM, 22-30 μM, 23-30 μM, 24-30 μM, 25-30 μM, 26-30 μM, 27-30 μM, 28-30 μM, 29-30 μM, 20-29 μM, 20-28 μM, 20-27 μM, 20-26 μM, 20-25 μM, 20-24 μM, 20-23 μM, 20-22 μM, or 20-21 μM; for example, the concentration of the Tm9D8* protein is approximately 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, or 30 μM.

[0179] In some embodiments of this disclosure, the final concentration of the PmLAAD protein is about 15-20 μM. For example, the concentration of the PmLAAD protein is about 15-20 μM, 16-20 μM, 17-20 μM, 18-20 μM, 19-20 μM, 15-19 μM, 15-18 μM, 15-17 μM, or 15-16 μM; the concentration of the PmLAAD protein is about 15 μM, 16 μM, 17 μM, 18 μM, 19 μM, or 20 μM.

[0180] In some embodiments of this disclosure, the final concentration of the dimer synthase is about 30-50 μM. For example, the concentration of the dimer synthase is about 30-50 μM, 31-50 μM, 32-50 μM, 33-50 μM, 34-50 μM, 35-50 μM, 36-50 μM, 37-50 μM, 38-50 μM, 39-50 μM, 40-50 μM, 41-50 μM, 42-50 μM, 43-50 μM, 44-50 μM, 45-50 μM, 46-50 μM, 47-50 μM, 48-50 μM, 49-50 μM, 30-49 μM, 30-48 μM, 30-47 μM, 30-46 μM, 30-45 μM, 30-44 μM, 30-43 μM、30-42 μM、30-41 μM, 30-40 μM, 30-39 μM, 30-38 μM, 30-37 μM, 30-36 μM, 30-35 μM, 30-34 μM, 30-33 μM, 30-32 μM, or 30-31 μM. For example, the concentration of the dimer synthase is about 30 μM, 31 μM, 32 μM, 33 μM, 34 μM, 35 μM, 36 μM, 37 μM, 38 μM, 39 μM, 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, or 50 μM.

[0181] In some specific embodiments, the reaction in step 1) can also be carried out in steps. For example, a method for preparing a dimer amino acid compound includes the following steps: 1-A) providing L-serine, an indole derivative, and Tm9D8* protein and culturing them; 1-B) subsequently providing PmLAAD protein and dimer synthase and culturing them, wherein the Tm9D8* protein and dimer synthase are as described above.

[0182] When the reaction in step 1) is carried out in steps, the final concentration of the Tm9D8* protein can be approximately 20-30 μM. For example, the concentration of the Tm9D8* protein is approximately 20-30 μM, 21-30 μM, 22-30 μM, 23-30 μM, 24-30 μM, 25-30 μM, 26-30 μM, 27-30 μM, 28-30 μM, 29-30 μM, 20-29 μM, 20-28 μM, 20-27 μM, 20-26 μM, 20-25 μM, 20-24 μM, 20-23 μM, 20-22 μM, or 20-21 μM; for example, the concentration of the Tm9D8* protein is approximately 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, or 30 μM. The final concentration of the PmLAAD protein can be approximately 4-8 μM. For example, the concentration of the PmLAAD protein can be approximately 4-8 μM, 5-8 μM, 6-8 μM, 7-8 μM, 4-7 μM, 4-6 μM, or 4-5 μM; the concentration of the PmLAAD protein can be approximately 4 μM, 5 μM, 6 μM, 7 μM, or 8 μM. The final concentration of the dimer synthase can be approximately 40-60 μM. For example, the concentration of the dimer synthase is about 40-60 μM, 41-60 μM, 42-60 μM, 43-60 μM, 44-60 μM, 45-60 μM, 46-60 μM, 47-60 μM, 48-60 μM, 49-60 μM, 50-60 μM, 51-60 μM, 52-60 μM, 53-60 μM, 54-60 μM, 55-60 μM, 56-60 μM, 57-60 μM, 58-60 μM, 59-60 μM, 40-59 μM, 40-58 μM, 40-57 μM, 40-56 μM, 40-55 μM, 40-54 μM, 40-53 μM、40-52 μM、40-51 μM, 40-50 μM, 40-49 μM, 40-48 μM, 40-47 μM, 40-46 μM, 40-45 μM, 40-44 μM, 40-43 μM, 40-42 μM, or 40-41 μM. For example, the concentration of the dimer synthase is about 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 50 μM, 51 μM, 52 μM, 53 μM, 54 μM, 55 μM, 56 μM, 57 μM, 58 μM, 59 μM, or 60 μM.

[0183] In some embodiments of this disclosure, the aforementioned method further includes the following step: 2) separating the dimer amino acid compound from the reaction system.

[0184] Another aspect of this disclosure provides a synthesizing enzyme for dimer amino acid compounds comprising an amino acid sequence as shown in any one of SEQ ID NO: 1-4 or having at least 90% identity with it.

[0185] In some preferred embodiments of this disclosure, the synthase comprises an amino acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with any one of SEQ ID NO: 1-4.

[0186] In some preferred embodiments of this disclosure, the synthase has an amino acid sequence shown in any one of SEQ ID NO: 1-4.

[0187] Another aspect of this disclosure provides a nucleic acid encoding a synthase of a dimer amino acid compound, comprising or having at least 90% identity with any of the nucleic acid sequences shown in or having at least 90% identity with any of SEQ ID NO: 11-14.

[0188] In some preferred embodiments of this disclosure, the nucleic acid of the synthase encoding the dimer amino acid compound comprises a nucleic acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with any one of SEQ ID NO: 11-14.

[0189] In some preferred embodiments of this disclosure, the nucleic acid of the synthase encoding the dimer amino acid compound has a nucleic acid sequence shown in any one of SEQ ID NO: 11-14.

[0190] Another aspect of this disclosure provides a Tm9D8* protein for the synthesis of dimeric amino acid compounds, comprising an amino acid sequence as shown in SEQ ID NO: 5 or having at least 90% identity with it.

[0191] In some preferred embodiments of this disclosure, the Tm9D8* protein comprises an amino acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with SEQ ID NO: 5.

[0192] In some preferred embodiments of this disclosure, the Tm9D8* protein has the amino acid sequence shown in SEQ ID NO: 5.

[0193] Another aspect of this disclosure provides a nucleic acid encoding the Tm9D8* protein, comprising a nucleic acid sequence as shown in SEQ ID NO: 15 or having at least 90% identity with it.

[0194] In some preferred embodiments of this disclosure, the nucleic acid encoding the Tm9D8* protein comprises a nucleic acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with SEQ ID NO: 15.

[0195] In some preferred embodiments of this disclosure, the nucleic acid encoding the Tm9D8* protein has the nucleic acid sequence shown in SEQ ID NO: 15.

[0196] In some embodiments of this disclosure, the dimer amino acid compounds are selected from bis-5cn-M, bis-7cn-M, bis-5mo-M and / or bis-6mo-M.

[0197] Another aspect of this disclosure provides the use of the aforementioned synthase in the preparation of dimer amino acid compounds.

[0198] Another aspect of this disclosure provides the use of the aforementioned nucleic acid-encoded synthase in the preparation of dimeric amino acid compounds.

[0199] Another aspect of this disclosure provides the use of the aforementioned Tm9D8* protein in the preparation of dimeric amino acid compounds.

[0200] Another aspect of this disclosure provides the use of the aforementioned nucleic acid-encoded Tm9D8* protein in the preparation of dimeric amino acid compounds.

[0201] In some embodiments of this disclosure, the dimer amino acid compounds used in the above-described applications are selected from bis-5cn-M, bis-7cn-M, bis-5mo-M, and / or bis-6mo-M.

[0202] Another aspect of this disclosure provides a dimer amino acid compound prepared according to the aforementioned method.

[0203] The second aspect of this disclosure provides a method for preparing a dimer amino acid compound, comprising the following steps: i) providing L-serine, an indole derivative and Tm2F3 protein and culturing them; ii) subsequently providing PmLAAD protein and a dimer synthase and culturing them.

[0204] In some embodiments of this disclosure, the dimer synthase comprises an amino acid sequence as shown in any one of SEQ ID NO: 1-4 or having at least 90% identity with it.

[0205] In some preferred embodiments of this disclosure, the dimerase comprises an amino acid sequence having 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more sequence identity with any one of SEQ ID NO: 1-4. In some more preferred embodiments, the dimerase has an amino acid sequence shown in any one of SEQ ID NO: 1-4.

[0206] In some embodiments of this disclosure, the nucleic acid encoding the dimer synthase comprises a nucleic acid sequence as shown in any one of SEQ ID NO: 11-14 or having at least 90% identity with it.

[0207] In some preferred embodiments of this disclosure, the nucleic acid encoding the dimerase comprises a nucleic acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with any one of SEQ ID NO: 11-14. In some more preferred embodiments, the nucleic acid encoding the dimerase has a nucleic acid sequence shown in any one of SEQ ID NO: 11-14.

[0208] In some specific implementations, the reaction formula for step i) is:

[0209] .

[0210] In some specific implementations, the reaction formula for step ii) is:

[0211] .

[0212] In some embodiments of this disclosure, the indole derivative is selected from 5-cyanoindole, 7-cyanoindole, 5-methoxyindole, and / or 6-methoxyindole.

[0213] In some embodiments of this disclosure, the reaction temperature in step i) is 50 ℃-65 ℃. The reaction temperature can be any range or any temperature within the above range, for example, the reaction temperature can be 50 ℃-65 ℃, 50 ℃-60 ℃, 50 ℃-55 ℃, 55 ℃-60 ℃, or 55 ℃-65 ℃; for example, the reaction temperature can be 50 ℃, 51 ℃, 52 ℃, 53 ℃, 54 ℃, 55 ℃, 56 ℃, 57 ℃, 58 ℃, 59 ℃, 60 ℃, 61 ℃, 62 ℃, 63 ℃, 64 ℃, or 65 ℃.

[0214] In some embodiments of this disclosure, the pH in step i) is 7-9. The pH can be any range or any value within the above range, for example, pH can be 7.1-9, 7.2-9, 7.3-9, 7.4-9, 7.5-9, 7.6-9, 7.7-9, 7.8-9, 7.9-9, 8-9, 8.1-9, 8.2-9, 8.3-9, 8.4-9, 8.5-9, 8.6-9, 8.7-9, 8.8-9 or 8.9-9; for example, pH can be 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.

[0215] In some embodiments of this disclosure, the reaction temperature in step ii) is 35°C-40°C. For example, 35°C-40°C, 36°C-40°C, 37°C-40°C, 38°C-40°C, or 39°C-40°C; for example, 35°C, 36°C, 37°C, 38°C, 39°C, or 40°C.

[0216] In some embodiments of this disclosure, the pH in step ii) is 7-9. The pH can be any range or any value within the above range, for example, pH can be 7.1-9, 7.2-9, 7.3-9, 7.4-9, 7.5-9, 7.6-9, 7.7-9, 7.8-9, 7.9-9, 8-9, 8.1-9, 8.2-9, 8.3-9, 8.4-9, 8.5-9, 8.6-9, 8.7-9, 8.8-9 or 8.9-9; for example, pH can be 7, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, 8, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9 or 9.

[0217] In some embodiments of this disclosure, the pH of the reaction system is adjusted by using a NaH2PO4-Na2HPO4 buffer solution and an aqueous (NH4)2SO4 solution.

[0218] In some embodiments of this disclosure, the dimer amino acid compound is a dimer amino acid compound derived from tryptophan (ditryptophan derivative).

[0219] In some preferred embodiments of this disclosure, the dimer amino acid compound is selected from 5',5''-dicyano-chromopyrrolic acid (bis-5cn-M), 7',7''-dicyano-chromopyrrolic acid (bis-7cn-M), 5',5''-dimethoxy-chromopyrrolic acid (bis-5mo-M) and / or 6',6''-dimethoxy-chromopyrrolic acid (bis-6mo-M), and each has the following chemical structure:

[0220]

[0221] In some embodiments of this disclosure, the Tm2F3 protein and the PmLAAD protein are provided in the form of culture lysates of expression vectors pET28a(+)-Tm2F3 and pET28a(+)-PmLAAD, respectively.

[0222] In some embodiments of this disclosure, the dimer synthase is provided in the form of a culture lysate of the expression vector pMCSG10-dimer synthase.

[0223] In some embodiments of this disclosure, the final concentration of the Tm2F3 protein is about 20-30 μM. For example, the concentration of the Tm2F3 protein is approximately 20-30 μM, 21-30 μM, 22-30 μM, 23-30 μM, 24-30 μM, 25-30 μM, 26-30 μM, 27-30 μM, 28-30 μM, 29-30 μM, 20-29 μM, 20-28 μM, 20-27 μM, 20-26 μM, 20-25 μM, 20-24 μM, 20-23 μM, 20-22 μM, or 20-21 μM; for example, the concentration of the Tm2F3 protein is approximately 20 μM, 21 μM, 22 μM, 23 μM, 24 μM, 25 μM, 26 μM, 27 μM, 28 μM, 29 μM, or 30 μM.

[0224] In some embodiments of this disclosure, the final concentration of the PmLAAD protein is about 4-8 μM. For example, the concentration of the PmLAAD protein is about 4-8 μM, 5-8 μM, 6-8 μM, 7-8 μM, 4-7 μM, 4-6 μM, or 4-5 μM; the concentration of the PmLAAD protein is about 4 μM, 5 μM, 6 μM, 7 μM, or 8 μM.

[0225] In some embodiments of this disclosure, the final concentration of the dimer synthase is about 40-60 μM. For example, the concentration of the dimer synthase is about 40-60 μM, 41-60 μM, 42-60 μM, 43-60 μM, 44-60 μM, 45-60 μM, 46-60 μM, 47-60 μM, 48-60 μM, 49-60 μM, 50-60 μM, 51-60 μM, 52-60 μM, 53-60 μM, 54-60 μM, 55-60 μM, 56-60 μM, 57-60 μM, 58-60 μM, 59-60 μM, 40-59 μM, 40-58 μM, 40-57 μM, 40-56 μM, 40-55 μM, 40-54 μM, 40-53 μM、40-52 μM、40-51 μM, 40-50 μM, 40-49 μM, 40-48 μM, 40-47 μM, 40-46 μM, 40-45 μM, 40-44 μM, 40-43 μM, 40-42 μM, or 40-41 μM. For example, the concentration of the dimer synthase is about 40 μM, 41 μM, 42 μM, 43 μM, 44 μM, 45 μM, 46 μM, 47 μM, 48 μM, 49 μM, 50 μM, 51 μM, 52 μM, 53 μM, 54 μM, 55 μM, 56 μM, 57 μM, 58 μM, 59 μM, or 60 μM.

[0226] In some embodiments of this disclosure, the aforementioned method further includes the step of: iii) separating the dimer amino acid compound from the reaction system.

[0227] Another aspect of this disclosure provides a synthesizing enzyme for dimer amino acid compounds comprising an amino acid sequence as shown in any one of SEQ ID NO: 1-4 or having at least 90% identity with it.

[0228] In some preferred embodiments of this disclosure, the synthase comprises an amino acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with any one of SEQ ID NO: 1-4.

[0229] In some preferred embodiments of this disclosure, the synthase has an amino acid sequence shown in any one of SEQ ID NO: 1-4.

[0230] Another aspect of this disclosure provides a nucleic acid encoding a synthase of a dimeric amino acid compound, comprising or having at least 90% identity with any one of SEQ ID NO: 1-4.

[0231] In some preferred embodiments of this disclosure, the nucleic acid comprises a nucleic acid sequence having a sequence identity of 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% or more with any one of SEQ ID NO: 1-4.

[0232] In some preferred embodiments of this disclosure, the nucleic acid has a nucleic acid sequence shown in any one of SEQ ID NO: 1-4.

[0233] In some embodiments of this disclosure, the dimer amino acid compounds are selected from bis-5cn-M, bis-7cn-M, bis-5mo-M and / or bis-6mo-M.

[0234] Another aspect of this disclosure provides the use of the aforementioned synthase in the preparation of dimer amino acid compounds.

[0235] Another aspect of this disclosure provides the use of the aforementioned nucleic acid-encoded synthase in the preparation of dimeric amino acid compounds.

[0236] In some embodiments of this disclosure, the dimer amino acid compounds used in the above-described applications are selected from bis-5cn-M, bis-7cn-M, bis-5mo-M, and / or bis-6mo-M.

[0237] Another aspect of this disclosure provides a dimer amino acid compound prepared according to the aforementioned method.

[0238] A third aspect of this disclosure also provides a dimer amino acid compound having the structure of formula (I):

[0239] ,

[0240] Among them, R 1 and R 2 Each time it appears, it is independently selected from either cyano or methoxy.

[0241] Or its pharmaceutically acceptable salt.

[0242] In some embodiments of this disclosure, R 1 and R 2 They are the same substituents.

[0243] In some embodiments of this disclosure, the aforementioned dimer amino acid compounds having the structure of formula (I) are selected from:

[0244]

[0245] The fourth aspect of this disclosure also provides a cell division inhibitor comprising a dimer amino acid compound prepared by the aforementioned method or the aforementioned dimer amino acid compound.

[0246] The fifth aspect of this disclosure also provides a composition comprising a dimer amino acid compound prepared by the aforementioned method, the aforementioned dimer amino acid compound, or the aforementioned cell division inhibitor.

[0247] The sixth aspect of this disclosure also provides the use of the dimer amino acid compounds prepared by the aforementioned methods, the aforementioned dimer amino acid compounds, the aforementioned cell division inhibitors, or the aforementioned compositions in the preparation of medicaments for inhibiting the G2 phase checkpoint of cell division, inhibiting topoisomerase I, and / or inhibiting protein kinase C.

[0248] The seventh aspect of this disclosure also provides the use of the dimer amino acid compounds prepared by the aforementioned methods, the aforementioned dimer amino acid compounds, the aforementioned cell division inhibitors, or the aforementioned compositions in the preparation of a medicament for treating a disease, disorder, or condition in a subject.

[0249] In some embodiments of this disclosure, the disease, obstacle, or ailment is cancer or malaria.

[0250] In some embodiments of this disclosure, the cancer is melanoma.

[0251] The eighth aspect of this disclosure also provides a method for inhibiting the G2 phase checkpoint of cell division, inhibiting topoisomerase I, and / or inhibiting protein kinase C, comprising administering to a subject a dimeric amino acid compound prepared by the aforementioned method, the aforementioned dimeric amino acid compound, the aforementioned cell division inhibitor, or the aforementioned composition.

[0252] The ninth aspect of this disclosure also provides a method for treating a disease, disorder, or symptom in a subject, comprising administering to the subject a dimer amino acid compound prepared by the aforementioned method, the aforementioned dimer amino acid compound, the aforementioned cell division inhibitor, or the aforementioned composition.

[0253] In some embodiments of this disclosure, the disease, obstacle, or ailment is cancer or malaria.

[0254] In some embodiments of this disclosure, the cancer is melanoma.

[0255] Example

[0256] To enable those skilled in the art to better understand the present disclosure, the technical solutions in the embodiments of the present disclosure will be clearly and completely described below. Obviously, the described embodiments are only some embodiments of the present disclosure, and not all embodiments.

[0257] Reagents, Instruments and Experimental Methods

[0258] I. Reagents and consumables used

[0259] The starting materials used in the examples are commercially available and / or can be prepared using various methods well known to those skilled in the art of organic synthesis. Those skilled in the art of organic synthesis will appropriately select the reaction conditions (including solvent, reaction atmosphere, reaction temperature, duration of the experiment, and post-treatment) from the synthetic methods described below. Those skilled in the art of organic synthesis will understand that the functional groups present on the various parts of the molecule should be compatible with the proposed reagents and reactions.

[0260] All reagents and compounds synthesized are available through general commercial channels. Please see Table A below for details.

[0261] Table A. Reagent Information

[0262]

[0263]

[0264] II. Instruments Used

[0265] Table B. Instrument Information

[0266]

[0267] Example 1: An embodiment based on the technical solution of the first aspect of this disclosure

[0268] Example 1.1: Modification, amino acid sequence, and expression vector of dimeric synthase

[0269] 1. Bis-5 CN-M synthesis-related enzymes

[0270] The bis-5cn-M dimer synthase was designed and prepared by the inventors from Anabaena. Anabaena azotica A structurally modified variant of the heme-dependent oxidase WP_190478047.1 (SEQ ID NO: 7) was named AaStaD* in this paper (amino acid sequence shown in SEQ ID NO: 1, and nucleic acid sequence encoding this synthase shown in SEQ ID NO: 11). To construct the expression vector, the AaStaD* gene fragment was cloned into the vector pRSFduet using Gibson splicing technology. The recombinant plasmid map is shown below. Figure 1 As shown.

[0271] In addition, the reaction utilizes the commonly used amino acid synthase Tm9D8* (amino acid sequence shown in SEQ ID NO: 5, nucleic acid sequence encoding this synthase shown in SEQ ID NO: 15) and L-amino acid dehydrogenase PmLAAD (amino acid sequence shown in SEQ ID NO: 6, nucleic acid sequence encoding this synthase shown in SEQ ID NO: 16) to assist in the production of bis-5cn-M. The expression vector was obtained by cloning the Tm9D8* and PmLAAD gene fragments into the vector pRSFduet using Gibson splicing technology. The recombinant plasmid maps are shown below. Figure 1 As shown.

[0272] The constructed expression vector pRSFduet-Tm9D8*-PmLAAD-AaStaD* was transformed into competent BL21(DE3) cells to obtain a bacterial culture solution, which was stored at -80 ℃.

[0273] 2. bis-7cn-M synthesis-related enzymes

[0274] The bis-7cn-M dimer synthase was designed and prepared by the inventors from Myxococcus bacteria. Calothrix parasitica A structurally modified variant of the heme-dependent oxidase BAY81287.1 (SEQ ID NO: 8) was named CpStaD* in this paper (the amino acid sequence is shown in SEQ ID NO: 2, and the nucleic acid sequence encoding this synthase is shown in SEQ ID NO: 12).

[0275] In addition, the synthesis of bis-7cn-M also requires the assistance of the amino acid synthase Tm9D8* (amino acid sequence as shown in SEQ ID NO: 5, nucleic acid sequence encoding this synthase as shown in SEQ ID NO: 15) and the L-amino acid dehydrogenase PmLAAD (amino acid sequence as shown in SEQ ID NO: 6, nucleic acid sequence encoding this synthase as shown in SEQ ID NO: 16). The expression vector was obtained in the same manner as described above. The recombinant plasmid map is shown below. Figure 2 As shown.

[0276] The constructed expression vector pRSFduet-Tm9D8*-PmLAAD-CpStaD* was transformed into competent BL21(DE3) cells to obtain a bacterial culture solution, which was stored at -80 ℃.

[0277] 3. Bis-5 mo-M synthesis-related enzymes

[0278] The key synthase of bis-5mo-M is an original enzyme derived from actinomycetes, developed by the applicant. Lentzea waywayandensis A structurally modified variant of the heme-dependent oxidase AEF32092.1 (SEQ ID NO: 9) was named AbeD* (amino acid sequence as shown in SEQ ID NO: 3, nucleic acid sequence encoding this synthase as shown in SEQ ID NO: 13). Furthermore, the synthesis of bis-5mo-M also requires the amino acid synthase Tm9D8* (amino acid sequence as shown in SEQ ID NO: 5, nucleic acid sequence encoding this synthase as shown in SEQ ID NO: 15) and the L-amino acid dehydrogenase PmLAAD (amino acid sequence as shown in SEQ ID NO: 6, nucleic acid sequence encoding this synthase as shown in SEQ ID NO: 16) for assistance. The expression vector was obtained in the same manner as described above. The recombinant plasmid map is shown below. Figure 3 As shown.

[0279] The constructed expression vector pRSFduet-Tm9D8*-PmLAAD-AbeD* was transformed into competent BL21(DE3) cells to obtain a bacterial culture solution, which was stored at -80 ℃.

[0280] 4. Bis-6 mo-M synthesis-related enzymes

[0281] The key synthase for bis-6mo-M is a structurally modified variant of the heme-dependent oxidase WP_073788971.1 (SEQ ID NO: 10) from Streptomyces uncialis environmental samples in the Global Protein Database, originally developed by the applicant. We named it BorD* (amino acid sequence as shown in SEQ ID NO: 4, nucleic acid sequence encoding this synthase as shown in SEQ ID NO: 14). The synthesis of bis-6mo-M also requires the amino acid synthase Tm9D8* (amino acid sequence as shown in SEQ ID NO: 5, nucleic acid sequence encoding this synthase as shown in SEQ ID NO: 15) and the L-amino acid dehydrogenase PmLAAD (amino acid sequence as shown in SEQ ID NO: 6, nucleic acid sequence encoding this synthase as shown in SEQ ID NO: 16) for assistance. The expression vector was obtained in the same manner as described above. The recombinant plasmid map is shown below. Figure 4 As shown.

[0282] The constructed expression vector pRSFduet-Tm9D8*-PmLAAD-BorD* was transformed into competent BL21(DE3) cells to obtain a bacterial culture solution, which was stored at -80 ℃.

[0283] Table 1. Sequence Information

[0284]

[0285] Example 1.2: Expression of key synthases

[0286] 1. Preparation of whole-cell fermentation broth

[0287] Preparation of Tm9D8*-PmLAAD-AaStaD* whole-cell fermentation broth:

[0288] (1) Take 10 μL of pRSFduet-Tm9D8*-PmLAAD-AaStaD* bacterial culture solution and add it to 5 mL of LB liquid medium. Incubate overnight at 37 ℃ and 200 rpm to obtain pRSFduet-Tm9D8*-PmLAAD-AaStaD* seed culture, wherein: the LB liquid medium contains kanamycin at a concentration of 50 μg / mL;

[0289] (2) The pRSFduet-Tm9D8*-PmLAAD-AaStaD* seed culture obtained in step (1) was added to an experimental container containing 1 L of TB liquid culture medium and cultured in a shaker at 37 ℃ and 200 rpm until the expression vector containing Tm9D8*-PmLAAD-AaStaD* was OD 600 =0.8-1.2, then place the test container in an ice bath for 30 min, wherein: TB liquid culture medium contains kanamycin at a concentration of 50 μg / mL;

[0290] (3) Add appropriate amounts of ferrous ammonium sulfate, 5-aminolevulinic acid and IPTG to the bacterial solution treated in step (2) so that the concentration of ferrous ammonium sulfate in the bacterial solution is 40 μM, the concentration of 5-aminolevulinic acid in the bacterial solution is 0.25 mM and the concentration of IPTG in the bacterial solution is 0.1 mM. Then mix well and place in a shaker to induce culture at 18 ℃ and 200 rpm for 16 h.

[0291] (4) Pour the bacterial culture induced in step (3) into a centrifuge cup, centrifuge at 4000 g for 15 min, discard the supernatant, wash the surface of the precipitate with ddH2O, and then add 100 mL of reaction buffer to resuspend the precipitated bacterial culture until there are no obvious bacterial clumps to obtain the bacterial culture resuspension. The pH value of the reaction buffer is 8.0, and the reaction buffer includes: 100 mM NaH2PO4-Na2HPO4, 2 mM ammonium sulfate, and the remainder is water.

[0292] Tm9D8*-PmLAAD-CpStaD* whole-cell fermentation broth, Tm9D8*-PmLAAD-AbeD* whole-cell fermentation broth, and Tm9D8*-PmLAAD-BorD* whole-cell fermentation broth can be prepared using the same method.

[0293] Example 1.3: Synthesis Reaction

[0294] 1. Reaction route

[0295] (1) De novo synthesis route of bis-5cn-M

[0296]

[0297] (2) De novo synthesis route of bis-7cn-M

[0298]

[0299] (3) De novo synthesis route of bis-5mo-M

[0300]

[0301] (4) De novo synthesis route of bis-6mo-M

[0302]

[0303] 2. Construction of the enzymatic reaction system (fermentation system)

[0304] Add the corresponding substances to reaction vessel 4 L fermenter 1-4 one by one according to Table 2-5. Specifically, the indole derivative is added in four batches, each batch containing 1 / 4 of the total amount, with a 3-hour interval between each addition, for a total reaction time of 12 hours. The reaction is carried out at 37℃ with the reaction vessel kept open throughout (i.e., the oxygen concentration at the top of the reaction vessel is the same as the oxygen concentration in the air). After the final addition, the reaction is shaken for 3 hours to obtain the final reaction solution.

[0305] Table 2. Material information in fermenter 1 (preparation of bis-5cn-M)

[0306]

[0307] Table 3. Material information in fermenter 2 (preparation of bis-7cn-M)

[0308]

[0309] Table 4. Material information in fermenter 3 (preparation of bis-5 mo-M)

[0310]

[0311] Table 5. Material information in fermenter 4 (preparation of bis-6 mo-M)

[0312]

[0313] The final concentrations of Tm9D8* protein were 20-30 μM (fermenter 1'), 20-30 μM (fermenter 2'), 20-30 μM (fermenter 3'), and 20-30 μM (fermenter 4').

[0314] The final concentrations of PmLAAD protein were 15-20 μM (fermenter 1'), 15-20 μM (fermenter 2'), 15-20 μM (fermenter 3'), and 15-20 μM (fermenter 4').

[0315] The final concentrations of the dimer synthase were 30-40 μM (fermenter 1'), 30-40 μM (fermenter 2'), 30-40 μM (fermenter 3'), and 30-40 μM (fermenter 4').

[0316] Example 1.4: Isolation, Identification and Bioactivity of the Product

[0317] The reaction solution obtained in fermenter 1 was adjusted to pH=1 with 1 N dilute hydrochloric acid, and then 1 L ethyl acetate was added for extraction at least three times. The emulsion layer was filtered with diatomaceous earth and separated. The organic phases were combined and dried with anhydrous sodium sulfate. The crude product was concentrated under reduced pressure and then purified by silica gel column chromatography. The yellow product obtained by elution under DCM / MeOH = 16:1 was bis-5cn-M (product mass was 8.359 g, yield was 96.1%).

[0318] The reaction solution obtained in fermenter 2 was adjusted to pH=1 with 1 N dilute hydrochloric acid, and then 1 L ethyl acetate was added for extraction at least three times. The emulsion layer was filtered with diatomaceous earth and separated. The organic phases were combined and dried with anhydrous sodium sulfate. The crude product was concentrated under reduced pressure and then purified by silica gel column chromatography. The yellow product obtained by elution under DCM / MeOH = 16:1 was bis-7cn-M (product mass was 8.229 g, yield was 94.5%).

[0319] The reaction solution obtained in fermenter 3 was adjusted to pH=1 with 1 N dilute hydrochloric acid, and then 1 L ethyl acetate was added for extraction at least three times. The emulsion layer was filtered with diatomaceous earth and separated. The organic phases were combined and dried with anhydrous sodium sulfate. The crude product was concentrated under reduced pressure and then purified by silica gel column chromatography. The pale yellow product obtained by elution under DCM / MeOH = 18:1 was bis-5mo-M (product mass was 8.694 g, yield was 97.6%).

[0320] The reaction solution obtained in fermenter 4 was adjusted to pH=1 with 1 N dilute hydrochloric acid, and then 1 L ethyl acetate was added for extraction at least three times. The emulsion layer was filtered with diatomaceous earth and separated. The organic phases were combined and dried with anhydrous sodium sulfate. The crude product was concentrated under reduced pressure and then purified by silica gel column chromatography. The pale yellow product obtained by elution under DCM / MeOH = 18:1 was bis-6mo-M (product mass was 8.480 g, yield was 95.2%).

[0321] Figures 5-13 The HPLC and NMR spectra of the product are shown respectively.

[0322] Example 1.5: IC 50 test

[0323] The IC50 values ​​of the bis-5cn-M, bis-7cn-M, bis-5mo-M, and bis-6mo-M products prepared using this method against PKC and PKA were determined using a protein kinase C (PKC) activity assay kit (PKC Kinase Activity Assay Kit, Abcam, catalog number ab139437) and a protein kinase A (PKA) activity assay kit (PKA Kinase Activity Assay Kit, Abcam, catalog number ab139435), respectively. 50 Values ​​were also tested; simultaneously, the bis-5cn-M, bis-7cn-M, bis-5mo-M, and bis-6mo-M products prepared by this method were examined for their effectiveness against *Plasmodium falciparum*. Plasmodium falciparum and the inhibitory activity of B16 in mouse melanoma cells (IC50). 50 Simultaneously testing existing similar products CPA and 5',5''-di-Cl-CPA ICs. 50The values, in which CPA and 5',5''-di-Cl-CPA in this test were derived from the enzymatic synthesis described in the literature (Zheng et al., Biomimetic Total Synthesis of the Spiroindimicin Family of Natural Products, Angew. Chem. Int. Ed. 2022 , 61, doi.org / 10.1002 / anie.202208802).

[0324] The structures of CPA and 5',5''-di-Cl-CPA are as follows:

[0325] , .

[0326] Inhibitory activity assay for protein kinase C (PKC): A double-antibody sandwich method was used. Purified human PKC antibody was coated onto microplates to prepare a solid-phase antibody. PKC was added sequentially to the wells coated with monoclonal antibody, followed by binding with HRP-labeled PKC antibody to form an antibody-antigen-enzyme-labeled antibody complex. After thorough washing, the substrate TMB was added for color development. TMB was converted to blue under the catalysis of HRP enzyme, and then to yellow under acidic conditions. The absorbance was measured at 450 nm using a microplate reader, and it showed a positive correlation with the PKC content in the sample. The PKC concentration in the sample was calculated using a standard curve. The substrate concentrations used were 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, and 200 μM, with a reaction time of 15 min and a reaction temperature of 37℃.

[0327] Inhibitory activity against protein kinase A (PKA) was determined using an ELISA method. The analyte was simultaneously incubated with biotin-labeled TXA2 antibody. After washing, avidin-labeled HRP was added, followed by washing again and then the substrate TMB for color development. The absorbance was measured at 450 nm using a microplate reader, and it showed a positive correlation with the PKC content in the sample. The PKA concentration in the sample was calculated using a standard curve. The substrate concentrations used were 5 μM, 10 μM, 20 μM, 50 μM, 100 μM, and 200 μM, with a reaction time of 10 min and a reaction temperature of 37 °C.

[0328] against Plasmodium falciparum Plasmodium falciparum Inhibitory activity assay: The test substrate was dissolved in DMSO and serially diluted using assay medium (RPMI 1640 medium containing 5% Albumax II, 0.2% w / v glucose, and 0.03% L-glutamine) and added to each well of a microtiter plate. Subsequently, cultured *Plasmodium falciparum* were... Plasmodium falciparum(Source: Artemisinin Research Center, China Academy of Chinese Medical Sciences) Add to each well, with a final volume of 100 μL (final DMSO concentration ≤0.25%). Incubate the plates at 37°C for 48 hours, and add 0.1 μCi to each well. 3 H-hypoxanthine. Mix the plates thoroughly and incubate for 24 hours. Terminate the reaction by placing the plates at -80°C. Thaw the cells using a 96-well plate and transfer them to a glass fiber filter pad. Count the bound radioactivity using a liquid Betalux scintillation counter and calculate the IC50. 50 The substrate concentrations used were 1 μM, 2 μM, 5 μM, 10 μM, 20 μM, and 50 μM.

[0329] Inhibitory activity against mouse melanoma cells B16 was determined by adding mouse melanoma cells B16 and serially diluted DMSO solution to a 200 μL reaction system. The final substrate concentrations were 0.1 μM, 0.2 μM, 0.5 μM, 1 μM, 2 μM, and 5 μM. After incubation at 37°C with shaking for 1 h, B16 cell viability was determined using the MTT assay, and the IC50 was calculated. 50 value.

[0330] The obtained detection results are shown in Table 6. The table shows that the compounds prepared by the method of this disclosure are effective against PKC, PKA, and... Plasmodium falciparum The inhibitory activity against B16 in mouse melanoma cells was superior to that of CPA and 5',5''-di-Cl-CPA.

[0331] Table 6. IC 50 Test Results

[0332]

[0333] Comparative Example 1: The role of key synthetic proteins in the existing CPA synthesis protocol in the synthesis of bis-5cn-M, bis-7cn-M, bis-5mo-M, and bis-6mo-M.

[0334] Referring to the synthetic route in Example 1.3, the AaStaD* protein, CpStaD* protein, AbeD* protein, and BorD* protein were replaced with existing CPA synthetic protocols (Zheng et al., Biomimetic Total Synthesis of the Spiroindimicin Family of Natural Products). Angew. Chem. Int. Ed. 2022 , 61,The key synthetic protein VioB (SEQ ID NO: 17) used in the reaction (doi.org / 10.1002 / anie.202208802) was reacted, and the AaStaD* protein, CpStaD* protein, AbeD* protein, and BorD* protein were replaced with their respective wild-type proteins AaStaD. WT Protein (WP_190478047.1, SEQ ID NO: 7), CpStaD WT Protein (BAY81287.1, SEQ ID NO: 8), AbeD WT Protein (AEF32092.1, SEQ ID NO: 9), BorD WT The protein (WP_073788971.1, SEQ ID NO: 10) was reacted, and the results are as follows: Figures 14-15 As shown, the bis-5cn-M reference standard, bis-7cn-M reference standard, bis-5mo-M reference standard, and bis-6mo-M reference standard were synthesized using the enzymatic method described in Example 1.1 of this disclosure.

[0335] The results showed that VioB had no catalytic effect on the synthesis of the dimeric amino acid compounds in this application, and wild-type AaStaD... WT The protein (WP_190478047.1, SEQ ID NO: 7) has a limited substrate range and low catalytic ability to synthesize the product bis-5cn-M involved in this invention; wild-type CpStaD WT The protein (BAY81287.1, SEQ ID NO: 8) has a limited substrate range and low catalytic ability to synthesize the product bis-7cn-M involved in this invention; wild-type AbeD WT The protein (AEF32092.1, SEQ ID NO: 9) has a limited substrate range and low catalytic ability to synthesize the product bis-5mo-M involved in this invention; wild-type BorD WT The protein (WP_073788971.1, SEQ ID NO: 10) has a limited substrate range and no catalytic ability to synthesize the product bis-6mo-M involved in this invention.

[0336] Comparative Example 2: Effects of common amino acid oxidases on the synthesis of bis-5cn-M, bis-7cn-M, bis-5mo-M, and bis-6mo-M

[0337] Referring to the synthetic route in Example 1.3, the PmLAAD protein was replaced with (Zheng et al., Biomimetic Total Synthesis of the Spiroindimicin Family of Natural Products, Angew. Chem. Int. Ed. 2022 , 61, The reaction was performed using the amino acid oxidase StaO (SEQ ID NO: 18) or VioA (SEQ ID NO: 19) used in doi.org / 10.1002 / anie.202208802, and the Tm9D8* protein was replaced with its wild-type Tm9D8. WT The reaction was carried out using (SEQ ID NO: 20), and the results were as follows: Figure 16 and Figure 25 As shown in the figure. The results show that the technical solution disclosed herein demonstrates excellent technical effects.

[0338] Example 2: An embodiment based on the technical solution of the second aspect of this disclosure

[0339] Example 2.1: Modification of the dimer synthase, amino acid sequence and expression vector

[0340] 1. Bis-5 CN-M synthesis-related enzymes

[0341] The bis-5cn-M dimer synthase is as described in Example 1.1. To construct the expression vector, the AaStaD* gene fragment was cloned into the vector pET26b using Gibson splicing technology. The recombinant plasmid map is shown below. Figure 17 As shown.

[0342] In addition, the commonly used amino acid synthase Tm2F3 (amino acid sequence shown in SEQ ID NO: 21, nucleic acid sequence encoding this synthase shown in SEQ ID NO: 22) and L-amino acid dehydrogenase PmLAAD (amino acid sequence shown in SEQ ID NO: 6, nucleic acid sequence encoding this synthase shown in SEQ ID NO: 16) were used to assist in the production of bis-5cn-M. The expression vector was obtained by cloning the Tm2F3 and PmLAAD gene fragments into the vector pET28a(+) using Gibson splicing technology. The recombinant plasmid maps are shown below. Figures 21-22 As shown.

[0343] The constructed expression vectors pET28a(+)-Tm2F3, pET28a(+)-PmLAAD, and pET26b-AaStaD* were transformed into competent BL21(DE3) cells to obtain bacterial strain solutions, which were then stored at -80 °C.

[0344] 2. bis-7cn-M synthesis-related enzymes

[0345] The bis-7cn-M dimer synthase is as described in Example 1.1.

[0346] In addition, the synthesis of bis-7cn-M also requires the assistance of the amino acid synthase Tm2F3 (amino acid sequence as shown in SEQ ID NO: 21, nucleic acid sequence encoding this synthase as shown in SEQ ID NO: 22) and the L-amino acid dehydrogenase PmLAAD (amino acid sequence as shown in SEQ ID NO: 6, nucleic acid sequence encoding this synthase as shown in SEQ ID NO: 16). The expression vector was obtained in the same manner as described above. The recombinant plasmid maps are shown below. Figure 18 and Figures 21-22 As shown.

[0347] The constructed expression vectors pET28a(+)-Tm2F3, pET28a(+)-PmLAAD, and pET26b-CpStaD* were transformed into competent BL21(DE3) cells to obtain bacterial strain solutions, which were then stored at -80 °C.

[0348] 3. Bis-5 mo-M synthesis-related enzymes

[0349] The key synthase for bis-5mo-M is as described in Example 1.1. Additionally, the synthesis of bis-5mo-M also requires the assistance of the amino acid synthase Tm2F3 (amino acid sequence as shown in SEQ ID NO: 21, nucleic acid sequence encoding this synthase as shown in SEQ ID NO: 22) and the L-amino acid dehydrogenase PmLAAD (amino acid sequence as shown in SEQ ID NO: 6, nucleic acid sequence encoding this synthase as shown in SEQ ID NO: 16). The expression vector was obtained in the same manner as described above. The recombinant plasmid map is shown below. Figure 19 and Figures 21-22 As shown.

[0350] The constructed expression vectors pET28a(+)-Tm2F3, pET28a(+)-PmLAAD, and pET26b-AbeD* were transformed into competent BL21(DE3) cells to obtain bacterial strain solutions, which were then stored at -80 °C.

[0351] 4. Bis-6 mo-M synthesis-related enzymes

[0352] The key synthase for bis-6mo-M is as described in Example 1.1. Additionally, the synthesis of bis-6mo-M also requires the assistance of the amino acid synthase Tm2F3 (amino acid sequence as shown in SEQ ID NO: 21, nucleic acid sequence encoding this synthase as shown in SEQ ID NO: 22) and the L-amino acid dehydrogenase PmLAAD (amino acid sequence as shown in SEQ ID NO: 6, nucleic acid sequence encoding this synthase as shown in SEQ ID NO: 16). The expression vector was obtained in the same manner as described above. The recombinant plasmid map is shown below. Figures 20-22 As shown.

[0353] The constructed expression vectors pET28a(+)-Tm2F3, pET28a(+)-PmLAAD, and pET26b-BorD* were transformed into competent BL21(DE3) cells to obtain bacterial strain solutions, which were then stored at -80 °C.

[0354] Table 7. Sequence Information

[0355]

[0356] Example 2.2: Expression of key synthases

[0357] 1. Preparation of crude lysate of dimerase protein

[0358] Preparation of crude lysis buffer for AaStaD* protein:

[0359] (1) Take 10 μL of pET26b-AaStaD* bacterial solution and add it to 5 mL of LB liquid medium. Incubate overnight at 37 ℃ and 200 rpm to obtain pET26b-AaStaD* seed culture. The LB liquid medium contains ampicillin at a concentration of 50 μg / mL.

[0360] (2) Add the pET26b-AaStaD* seed culture obtained in step (1) to a test container containing 1 L of LB liquid medium, and culture in a shaker at 37 ℃ and 200 rpm until the AaStaD* expression vector reaches OD. 600 = up to 0.7-0.9, then place the test container in an ice bath for 30 min, wherein: LB liquid medium contains ampicillin at a concentration of 50 μg / mL;

[0361] (3) Add appropriate amounts of ferrous ammonium sulfate, 5-aminolevulinic acid and IPTG to the bacterial solution treated in step (2) so that the concentration of ferrous ammonium sulfate in the bacterial solution is 40 μM, the concentration of 5-aminolevulinic acid in the bacterial solution is 0.25 mM and the concentration of IPTG in the bacterial solution is 0.1 mM. Then mix well and place in a shaker to induce culture at 18 ℃ and 200 rpm for 16 h.

[0362] (4) Pour the bacterial culture induced in step (3) into a centrifuge cup, centrifuge at 4000 g for 15 min, discard the supernatant, wash the surface of the precipitate with ddH2O, and then add 30 mL of loading buffer to resuspend the precipitated bacterial culture until there are no obvious bacterial clumps to obtain the bacterial culture resuspension. The pH value of the loading buffer is 8.0, and the loading buffer includes: 100 mM Tris-HCl, 300 mM NaCl, 15 mM imidazole, 10% v / v glycerol, and the remainder is water.

[0363] (5) The bacterial suspension obtained in step (4) was broken up by an ultrasonic disruptor. The diameter of the amplitude rod was 6 mm. The disruption program was: 40% energy, ultrasonic cycle working time 2 s, pause time 4 s, and total working time 30 min.

[0364] (6) Centrifuge the broken bacterial solution at 10000 g for 15 min at low temperature, and take the supernatant to obtain the crude lysate of AaStaD* protein.

[0365] Crude lysates of CpStaD*, AbeD*, and BorD* proteins can be prepared using the same method.

[0366] 2. Preparation of crude lysis buffer for Tm2F3 protein

[0367] (1) Take 10 μL of pET28a(+)-Tm2F3 bacterial culture solution and add it to 5 mL of LB liquid medium. Incubate overnight at 37 ℃ and 200 rpm to obtain pET28a(+)-Tm2F3 seed culture. The LB liquid medium contains kanamycin at a concentration of 50 μg / mL.

[0368] (2) The pET28a(+)-Tm2F3 seed culture obtained in step (1) was added to a test container containing 1 L of LB liquid medium and cultured in a shaker at 37 °C and 200 rpm until the bacterial culture containing the Tm2F3 expression vector reached the concentration OD. 600 =0.9-1.1, then the test container was removed and placed in an ice bath for 30 min, wherein: LB liquid medium contained kanamycin at a concentration of 50 μg / mL;

[0369] (3) Add IPTG to the bacterial solution treated in step (2) so that the concentration of IPTG in the bacterial solution is 0.1mM. After mixing, place it in a shaker and induce culture at 18 ℃ and 200 rpm for 16 h.

[0370] (4) Pour the bacterial culture induced in step (3) into a centrifuge cup, centrifuge at 4000 g for 15 min, discard the supernatant, wash the surface of the precipitate with ddH2O, and then add 30 mL of loading buffer to resuspend the precipitated bacterial culture until there are no obvious bacterial clumps to obtain the bacterial culture resuspension. The pH value of the loading buffer is 8.0, and the loading buffer includes: 100 mM Tris-HCl, 300 mM NaCl, 15 mM imidazole, 10% v / v glycerol, and the remainder is water.

[0371] (5) The bacterial weight suspension was broken up using an ultrasonic disruptor, wherein: the diameter of the amplitude rod was 6 mm, the disruption program was: energy 40%, ultrasonic cycle working time 2 s, pause time 4 s, and total working time 30 min;

[0372] (6) Centrifuge the broken bacterial solution at 10000 g for 15 min at low temperature, and take the supernatant to obtain the crude lysate of Tm2F3 protein.

[0373] 3. Preparation of crude PmLAAD protein lysis buffer

[0374] (1) Take 10 μL of pET28a(+)-PmLAAD bacterial culture solution and add it to 5 mL of LB liquid medium. Incubate overnight at 37℃ and 200 rpm to obtain pET28a(+)-PmLAAD seed culture. The LB liquid medium contains kanamycin at a concentration of 50 μg / mL.

[0375] (2) Add all of the pET28a(+)-PmLAAD seed culture obtained in step (1) to a test container containing 1L of LB liquid medium, and then culture it in a shaker at 37 ℃ and 200 rpm until the bacterial concentration of PmLAAD expression vector in the bacterial culture reaches OD. 600 =1.1-1.3, then remove the test container and place it in an ice bath for 30 min, wherein: LB liquid medium contains kanamycin at a concentration of 50 μg / mL;

[0376] (3) Add an appropriate amount of IPTG to the bacterial solution treated in step (2) so that the concentration of IPTG in the bacterial solution is 0.1 mM. After mixing, place it in a shaker and induce culture at 18 ℃ and 200 rpm for 16 h.

[0377] (4) Pour the bacterial culture induced in step (3) into a centrifuge cup, centrifuge at 4000 g for 15 min, discard the supernatant, wash the surface of the precipitate with ddH2O, and then add 30 mL of loading buffer to resuspend the precipitated bacterial culture until there are no obvious bacterial clumps to obtain the bacterial culture resuspension. The pH value of the loading buffer is 8.0, and the loading buffer includes: 100 mM Tris-HCl, 300 mM NaCl, 5 mM imidazole, 10% v / v glycerol, and the remainder is water.

[0378] (5) The bacterial suspension obtained in step (4) is broken up using an ultrasonic disruptor to obtain the broken bacterial solution, wherein: the diameter of the amplitude rod is 6 mm; the disruption program is: energy 40%, ultrasonic cycle working time 2 s, pause time 4 s, and total working time 30 min;

[0379] (6) Centrifuge the broken bacterial solution at 10000 g for 15 min at low temperature, and take the supernatant to obtain the crude lysate of PmLAAD protein.

[0380] Example 2.3: Synthesis Reaction

[0381] 1. Reaction Pathway

[0382] (1) De novo synthesis route of bis-5cn-M

[0383]

[0384] (2) De novo synthesis route of bis-7cn-M

[0385]

[0386] (3) De novo synthesis route of bis-5mo-M

[0387]

[0388] (4) De novo synthesis route of bis-6mo-M

[0389]

[0390] 2. Construction of the enzymatic reaction system (fermentation system)

[0391] Add the corresponding substances to reaction vessels 4 L fermenters 1'-4' one by one according to Table 8-11. The order of addition and concentration of each substance have a decisive impact on the yield and quality of the final product. Specifically: the concentration of the NaH2PO4-Na2HPO4 buffer solution is 1 M, and the pH value is 8.0; the concentration of the (NH4)2SO4 stock solution is 2 M. After mixing thoroughly, the reaction vessel is shaken at 60℃ for 2-3 hours. Throughout the shaking reaction, the reaction vessel is kept open (i.e., the oxygen concentration at the top of the reaction vessel is the same as the oxygen concentration in the air).

[0392] Table 8. Material information in fermenter 1' (preparation of bis-5cn-M)

[0393]

[0394] Table 9. Material information in fermenter 2' (preparation bis-7cn-M)

[0395]

[0396] Table 10. Material information in fermenter 3' (preparation of bis-5 mo-M)

[0397]

[0398] Table 11. Material information in fermenter 4' (preparation of bis-6 mo-M)

[0399]

[0400] The final concentrations of Tm2F3 protein were 20-30 μM (fermenter 1'), 5-10 μM (fermenter 2'), 5-10 μM (fermenter 3'), and 5-10 μM (fermenter 4').

[0401] After the reaction was completed and each container cooled to 37 °C, 60 mL of PmLAAD crude protein lysis buffer was added to each reaction vessel, followed by 800 mL of AaStaD* crude protein lysis buffer (fermenter 1'), 800 mL of CpStaD* crude protein lysis buffer (fermenter 2'), 800 mL of AbeD* crude protein lysis buffer (fermenter 3'), and 800 mL of BorD* crude protein lysis buffer (fermenter 4'). After mixing thoroughly, each container was shaken and reacted for 4-6 h to obtain the reaction solution.

[0402] The final concentrations of PmLAAD protein were 4-8 μM (fermenter 1'), 4-8 μM (fermenter 2'), 4-8 μM (fermenter 3'), and 4-8 μM (fermenter 4').

[0403] The final concentrations of the dimer synthase were 40-60 μM (fermenter 1'), 40-60 μM (fermenter 2'), 40-60 μM (fermenter 3'), and 40-60 μM (fermenter 4').

[0404] Example 2.4: Isolation, Identification and Bioactivity of the Product

[0405] The reaction solution obtained in fermenter 1' was adjusted to pH=1 with 1 N dilute hydrochloric acid, and then 1 L ethyl acetate was added for extraction at least three times. The emulsion layer was filtered through diatomaceous earth and separated. The organic phases were combined and dried over anhydrous sodium sulfate. The crude product was concentrated under reduced pressure and then purified by silica gel column chromatography. The yellow product obtained by elution under DCM / MeOH = 16:1 was bis-5cn-M (product mass was 6.63 g, yield was 76.1%).

[0406] The reaction solution obtained in fermenter 2' was adjusted to pH=1 with 1 N dilute hydrochloric acid, and then 1 L ethyl acetate was added for extraction at least three times. The emulsion layer was filtered with diatomaceous earth and separated. The organic phases were combined and dried with anhydrous sodium sulfate. The crude product was concentrated under reduced pressure and then purified by silica gel column chromatography. The yellow product obtained by elution under DCM / MeOH = 16:1 was bis-7cn-M (product mass was 6.30 g, yield was 72.4%).

[0407] The reaction solution obtained in fermenter 3' was adjusted to pH=1 with 1 N dilute hydrochloric acid, and then 1 L ethyl acetate was added for extraction at least three times. The emulsion layer was filtered with diatomaceous earth and separated. The organic phases were combined and dried with anhydrous sodium sulfate. The crude product was concentrated under reduced pressure and then purified by silica gel column chromatography. The pale yellow product obtained by elution under DCM / MeOH = 18:1 was bis-5mo-M (product mass was 7.71 g, yield was 86.5%).

[0408] The reaction solution obtained in fermenter 4' was adjusted to pH=1 with 1 N dilute hydrochloric acid, and then 1 L ethyl acetate was added for extraction at least three times. The emulsion layer was filtered through diatomaceous earth and separated. The organic phases were combined and dried over anhydrous sodium sulfate. The crude product was concentrated under reduced pressure and then purified by silica gel column chromatography. The pale yellow product obtained by elution under DCM / MeOH = 18:1 was bis-6mo-M (product mass was 7.37 g, yield was 82.7%).

[0409] The characterization results of the dimer amino acid compound prepared in Example 2.4 were basically the same as those of the product prepared in Example 1.4, as determined by HPLC and NMR.

[0410] Example 2.5: IC 50 test

[0411] IC 50 The test method is the same as in Example 1.5, and the experimental results are basically the same as those in Example 1.5.

[0412] Comparative Example 3: The role of key synthetic proteins in the existing CPA synthesis protocol in the synthesis of bis-5cn-M, bis-7cn-M, bis-5mo-M, and bis-6mo-M

[0413] Referring to the synthetic route in Example 2.3, the AaStaD* protein, CpStaD* protein, AbeD* protein, and BorD* protein were replaced with existing CPA synthetic protocols (Zheng et al., Biomimetic Total Synthesis of the Spiroindimicin Family of Natural Products). Angew. Chem. Int. Ed. 2022 , 61, The key synthetic protein VioB (SEQ ID NO: 37) used in the reaction (doi.org / 10.1002 / anie.202208802) was used, and the AaStaD* protein, CpStaD* protein, AbeD* protein, and BorD* protein were replaced with their respective wild-type proteins AaStaD. WT Protein (WP_190478047.1, SEQ ID NO: 7), CpStaD WT Protein (BAY81287.1, SEQ ID NO: 8), AbeD WT Protein (AEF32092.1, SEQ ID NO: 9), BorD WT The protein (WP_073788971.1, SEQ ID NO: 10) was reacted, and the results are as follows: Figures 23-24 As shown, the bis-5cn-M reference standard, bis-7cn-M reference standard, bis-5mo-M reference standard, and bis-6mo-M reference standard were synthesized using the enzymatic method described in Example 2.2 of this disclosure.

[0414] The results showed that VioB had no catalytic effect on the synthesis of the dimeric amino acid compounds in this application, and wild-type AaStaD... WT The protein (WP_190478047.1, SEQ ID NO: 7) has a limited substrate range and lacks catalytic synthesis ability for the product bis-5cn-M involved in this invention; wild-type CpStaD WTThe protein (BAY81287.1, SEQ ID NO: 8) has a limited substrate range and lacks the catalytic ability to synthesize the product bis-7cn-M involved in this invention; wild-type AbeD WT The protein (AEF32092.1, SEQ ID NO: 9) has a limited substrate range and lacks the catalytic ability to synthesize the product bis-5mo-M involved in this invention; wild-type BorD WT The protein (WP_073788971.1, SEQ ID NO: 10) has a limited substrate range and no catalytic ability to synthesize the product bis-6mo-M involved in this invention.

[0415] By incorporating via reference

[0416] The full contents of every patent and scientific document mentioned in this article are incorporated herein by reference for all purposes.

[0417] Equivalence

[0418] This disclosure may be embodied in other specific ways without departing from its spirit or essential characteristics. Therefore, the above embodiments should be considered illustrative in all cases and not as limiting of the invention described herein. Consequently, the scope of this disclosure is defined by the appended claims rather than by the foregoing description and is intended to be encompassed therein by all variations within the equivalent meaning and scope of the claims.

Claims

1. A dimeric amino acid compound synthesizing enzyme, the amino acid sequence of which is shown in any one of SEQ ID NOs: 1-4.

2. A nucleic acid encoding a dimeric amino acid compound synthesizing enzyme, the nucleic acid sequence of which is shown in any one of SEQ ID NOs: 11-14.

3. Use of the synthesizing enzyme according to claim 1 or the nucleic acid encoded synthesizing enzyme according to claim 2 in the preparation of a dimeric amino acid compound.

4. Use according to claim 3, wherein, the dimeric amino acid compound is selected from ， ， , and / or .

5. A method for preparing a dimeric amino acid compound, comprising the following steps: 1) providing L-serine, an indole derivative, a Tm9D8* protein, a PmLAAD protein and a dimer synthesizing enzyme and culturing; wherein, the amino acid sequence of the Tm9D8* protein is shown in SEQ ID NO: 5; and the amino acid sequence of the dimer synthesizing enzyme is shown in any one of SEQ ID NOs: 1-4, and wherein, the indole derivative is an indole substituted with one or more substituents on one or more carbons at any position of 4, 5, 6 and 7 of the indole ring, the substituents being a halogen group, an alkyl group, an alkoxy group, a cyano group and / or a nitro group.

6. The method according to claim 5, wherein, the nucleic acid sequence of the nucleic acid encoding the Tm9D8* protein is shown in SEQ ID NO: 15; and / or the nucleic acid sequence of the nucleic acid encoding the dimer synthesizing enzyme is shown in any one of SEQ ID NOs: 11-14.

7. The method according to claim 5, wherein step 1) comprises the following reaction 。 8. The method of any one of claims 5-7, wherein, the indole derivative is selected from 5-cyanoindole, 7-cyanoindole, 5-methoxyindole and / or 6-methoxyindole.

9. The method of any one of claims 5-7, wherein, in step 1) the reaction temperature is 28-37 °C, and / or the reaction pH is 7-9.

10. The method of any one of claims 5-7, wherein, in step 1) the pH of the reaction system is adjusted by using a NaH2PO4-Na2HPO4 buffer and an aqueous (NH4)2SO4 solution.

11. The method of any one of claims 5-7, wherein, the dimeric amino acid compound is a dimeric amino acid compound derived from tryptophan.

12. The method of any one of claims 5 to 7, wherein, the dimeric amino acid compound is selected from ， ， , and / or .

13. The method according to any one of claims 5-7, wherein, the Tm9D8* protein, the PmLAAD protein and the dimer synthesizing enzyme are provided in the form of E. coli whole cells and / or culture lysate of expression vector pRSFduet-Tm9D8*-PmLAAD-dimer synthesizing enzyme.

14. The method according to any one of claims 5-7, further comprising the following step: 2) separating the dimeric amino acid compound from the reaction system.

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