CN117512038A - Sansansamycin derivatives and application thereof - Google Patents
Sansansamycin derivatives and application thereof Download PDFInfo
- Publication number
- CN117512038A CN117512038A CN202311335796.1A CN202311335796A CN117512038A CN 117512038 A CN117512038 A CN 117512038A CN 202311335796 A CN202311335796 A CN 202311335796A CN 117512038 A CN117512038 A CN 117512038A
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- derivative
- tyr
- aminouridine
- follows
- substituent
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Classifications
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- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
- C12P21/00—Preparation of peptides or proteins
- C12P21/02—Preparation of peptides or proteins having a known sequence of two or more amino acids, e.g. glutathione
-
- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- A61P31/00—Antiinfectives, i.e. antibiotics, antiseptics, chemotherapeutics
- A61P31/04—Antibacterial agents
- A61P31/06—Antibacterial agents for tuberculosis
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K5/00—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof
- C07K5/04—Peptides containing up to four amino acids in a fully defined sequence; Derivatives thereof containing only normal peptide links
- C07K5/10—Tetrapeptides
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- A—HUMAN NECESSITIES
- A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
- A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
- A61K38/00—Medicinal preparations containing peptides
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
- C12R2001/00—Microorganisms ; Processes using microorganisms
- C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
- C12R2001/465—Streptomyces
Landscapes
- Health & Medical Sciences (AREA)
- Chemical & Material Sciences (AREA)
- Life Sciences & Earth Sciences (AREA)
- Organic Chemistry (AREA)
- General Health & Medical Sciences (AREA)
- Medicinal Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- General Chemical & Material Sciences (AREA)
- Genetics & Genomics (AREA)
- Molecular Biology (AREA)
- Wood Science & Technology (AREA)
- Zoology (AREA)
- Biochemistry (AREA)
- Proteomics, Peptides & Aminoacids (AREA)
- Bioinformatics & Cheminformatics (AREA)
- General Engineering & Computer Science (AREA)
- Microbiology (AREA)
- Biophysics (AREA)
- Biotechnology (AREA)
- Pulmonology (AREA)
- Communicable Diseases (AREA)
- Oncology (AREA)
- Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
- Pharmacology & Pharmacy (AREA)
- Animal Behavior & Ethology (AREA)
- Public Health (AREA)
- Veterinary Medicine (AREA)
- Preparation Of Compounds By Using Micro-Organisms (AREA)
Abstract
The invention relates to a group of sansanmycin derivatives and application thereof, wherein the sanmycin derivatives have the structure as followsWherein R is H or CH 3 AA1 is Tyr, m-Tyr, dicyclohexyl a/b/c/d, N-Acetyl- (m-) Tyr, N-Gly- (m-) Tyr; AA2 is 2,3-diaminobutyric acid (2, 3-diaminobutyric acid, DABA); AA3 is Met, met SO Leu; AA4 is Trp, (m-) Tyr, phe. The invention also relates to application of the sansamycin derivative in preparation of antibacterial drugs.
Description
Technical Field
The invention belongs to the technical field of medical biology, and particularly relates to a group of sansandamycin derivatives and application thereof.
Background
Tuberculosis (TB) is an infectious disease caused by mycobacterium tuberculosis and is still one of the most serious global health threats. Recently published global tuberculosis report 2022 (world health organization, 2022) indicated that tuberculosis is the second largest deadly infectious disease following covd-19. The number of deaths from global tuberculosis increases from 140 ten thousand in 2019 to 150 ten thousand in 2020, and further increases from 2021 to 160 ten thousand. Current methods of treating tuberculosis include four-way therapies for six months, including Rifampicin (RIF), isoniazid (INH), ethambutol and pyrazinamide (Zumla et al, 2013). Although this treatment is effective in controlling drug-sensitive tuberculosis, it is ineffective against drug-resistant tuberculosis infection, which is becoming more and more common worldwide. In view of the severity of the current tuberculosis situation and the propensity for drug resistance, there is a great need to develop new drugs for drug resistant tuberculosis.
Sansammycins (Xie et al, 2007) produced by Streptomyces SS belong to the family of Uridine Peptide Antibiotics (UPA), including pacidamycin (Karwowski et al, 1989), napsamycins (Chatterjee et al, 1994) and mureidomycins (Inukai et al, 1989). They all have a core backbone, i.e., a 3' -deoxyuridine unit linked to a pseudo-tetra/pentapeptide by an unusual enamide linkage. UPAs blocks bacterial cell wall assembly by inhibiting the synthesis of UDP-N-acetylmuramic acid-pentapeptidyl translocase (MraY, translocase I), with significant antibacterial activity against mycobacterium tuberculosis (including drug resistant mycobacterium tuberculosis) (Shi et al, 2016) and pseudomonas aeruginosa (Pseudomonas aeruginosa) (win et al, 2010). Because this unique mode of action has not been used clinically, UPA has become the lead compound for the search for new antitubercular drugs.
Structural modification of UPA has been focused mainly on substitution of amino acid residues in peptide chains, such as C-terminal modified pacidamycin derivatives (Gruschow et al 2009; and sansanansamycin derivatives (Zhang et al 2016) obtained by precursor directed biosynthesis. A series of novel sanansamycins analogues modified at the N-terminal amino acid were obtained by mutant biosynthesis (Shi et al, 2016). However, few analogues have significantly improved antibacterial activity.
The 5'-aminouridine (5' -aminouridine) moiety in UPAs is believed to be necessary to compete for active site binding with the natural substrate UDP-N-acetylmuramyl (UDP-MurNAc) of Mray (Walsh and Zhang, 2011). Recently, the structural data of the MraY-inhibitor complex show that the uridine binding pocket of natural nucleoside inhibitors, including UPAs, share a common feature, and that the residues forming the uridine binding pocket are likely involved in the binding of the MraY natural substrate (mashis et al, 2019).
The modified 5' -aminouridine analogs reported so far are almost entirely obtained by chemical synthesis (fig. 1). Dihydropacidamycin D is the first 5'-aminouridine modified UPA derivative obtained by hydrogenation of the C-4' exocyclic olefin of pacidamycin D and has an inhibitory activity against P.aeruginosa comparable to that of pacidamycin D (FIGS. 1A-C, F), indicating that the reduction of the enamide bond does not affect UPAs activity. In recent years, certain peptide chain modified dihydrosan mycin analogs have been reported to have improved activity against Mycobacterium tuberculosis (FIGS. 1A-C, F). Notably, the stereochemical configuration of the ribose moiety C-4' in dihydroasansamycin and dihydroopadamycin is believed to be a critical factor in maintaining antimicrobial activity, and analogues of the S-configuration generally result in reduced or complete loss of activity. The antibacterial activity of 3'-hydroxypacidamycin D against P.aeruginosa was comparable to that of pacidamycin D, and the inhibitory activity of 3' -hydroxymureidomycin A against MraY was also comparable to that of mureidamycin A (FIGS. 1A, D, F). These findings indicate that the introduction of a hydroxyl group at the 3 'position of 5' -aminouridine is well tolerated for both antibacterial activity and inhibitory activity of MraY. In 2020, niro et al reported a novel hybrid structure consisting of the peptide chain of sansantomycin B and the uridine portion of 5' -deoxymuraymycin C (FIGS. 1A, E-F). Like UPA, 5' -deoxymuraymycin C4 is also a uridine-derived nucleoside antibiotic that contains (6'S) -5' -deoxyglycyluridine and exhibits inhibitory activity against Mray. The Muraymycin-sansanmycin B hybrid structure has inhibitory activity against Mrays, but no apparent antibacterial activity against P.aeromonas. This suggests that hybrid structures formed by incorporating uridine building blocks of other nucleoside antibiotics into uridine peptide compounds may still have inhibitory activity against MraY. The chemical synthesis of UPAs is difficult and has low efficiency, but provides some insight for the structure-activity relationship of UPAs. The above studies suggest that suitable modification of 5' -aminouridine can be used to obtain UPA new structural derivatives to increase their activity and/or patentability. In the sansansansamycin biosynthetic gene cluster SsaM, ssaK and SsaE have a high degree of homology to PacM, pacK and PacE, respectively, which have been shown to be responsible for the biosynthesis of 5' -aminouridine of pacidamycin by in vitro experiments (CN 201610219358.2). However, 5' -aminouridine has not been reported as an important component of pharmacology to be structurally modified by biosynthesis.
Based on this, the present invention has been proposed.
Disclosure of Invention
The invention firstly relates to a method for biosynthesis of sansanmycin derivatives, which is characterized by comprising the following steps
(1) Adding a 5' -aminouridine analogue, preferably a 5' -aminouridine analogue with C-4' chirality of R-configuration, preferably 2' -deoxy-5 ' -aminouridine or 5-methyl-2 ' -deoxy-5 ' -aminouridine, to a culture medium of ssaM, ssaK-disrupted strain of Streptomyces sp.SS;
the chemical structure of the 2 '-deoxy-5' -aminouridine is shown as the following formula:
the chemical structure of the 5-methyl-2 '-deoxy-5' -aminouridine is shown as the following formula:
the ssaM and ssaK blocking strains of the Streptomyces sp.SS are as follows: streptomyces sp.SS, with China general microbiological culture Collection center (CGMCC) with the collection number of CGMCC No.1764, is taken as a host, and a gene engineering means is used to knock out ssaM genes or ssaK genes of the Streptomyces sp.SS, so as to obtain a blocking strain;
the 5' -aminouridine analogue is added into a fermentation medium when transferring strains after shaking flask fermentation, wherein the final concentration of the substrate is 3mM and the substrate is dissolved in water.
(2) Collecting fermentation liquor, and separating and purifying to obtain the sansanansamycin derivative.
Specifically, the fermentation broth obtained in the step (2) is collected, separated and purified to obtain the sansanmycin derivative, which comprises the following steps of
1) Centrifuging the fermentation product at 4 ℃ and 4,500rpm for 20min, discarding the precipitate, and collecting the supernatant of the fermentation broth;
2) Adsorbing the crude product by using macroporous resin, preferably, loading the fermentation supernatant on a macroporous resin D4006 column at a speed of 50mL/min, sequentially eluting by using pure water without acetone, 10% acetone aqueous solution, 20% acetone aqueous solution and 30% acetone aqueous solution, performing gradient elution for 5-6 column volumes of each elution, collecting elution fractions of 20% acetone aqueous solution and 30% acetone aqueous solution, merging the fractions, concentrating and freeze-drying to obtain a sansandamycin derivative mixed crude product;
3) Performing preliminary separation of crude products of the derivatives by using anion exchange resin, preferably, the anion filler is DEAE sephadex A-25, completely dissolving and loading the crude product powder by using 0.02M Tris buffer solution with pH value of 9, eluting by using 0.02M Tris buffer solution added with NaCl after loading, taking 0.01M NaCl as an initial gradient, performing HPLC-MS detection on effluent after eluting 10-12 column volumes, if no sansamycin derivative signal exists, increasing the concentration of NaCl, continuously eluting, increasing the concentration of 0.01M each time, eluting 10-12 column volumes until no sansamycin derivative signal exists in the effluent; combining effluent liquid containing single same HPLC-MS detection signals, and desalting by a solid-phase extraction method after rotary evaporation concentration to obtain crude products of each sansamycin derivative;
4) The preparation method comprises purifying with high performance liquid chromatography, preferably with Shimadzu LC-20AD high performance liquid chromatography, and introducing 100 μl of sample each time, using chromatographic column asprep C18column (5 μm, 250X 10 mm), flow rate 3mL/min, detection wavelength 254nm, column temperature 40 ℃, mobile phase 0.1% (w/v) CH 3 COONH 4 Aqueous solution-MeOH; after the peaks of each compound are collected, the purity is detected by HPLC, so that the purity is ensured to be more than 90 percent, and the preparation is needed again if the purity is insufficient; desalting the effluent with qualified purity with a solid phase extraction column, concentrating with a rotary evaporator, and lyophilizing to obtain pure product of each sansanmycin derivative.
The invention also relates to a group of sansanmycin derivatives obtained by the method through biosynthesis, and the structures of the sansanmycin derivatives are shown as the following formula
In the method, in the process of the invention,
r is H or CH 3 ，
AA1 is Tyr, m-Tyr, dicyclohexyl a/b/c/d, N-Acetyl- (m-) Tyr, N-Gly- (m-) Tyr;
AA2 is 2,3-diaminobutyric acid (2, 3-diaminobutyric acid, DABA);
AA3 is Met, met SO 、Leu；
AA4 is Trp, (m-) Tyr, phe.
Wherein,
the structural formula of the (m-) Tyr substituent is as follows:
Met so the structural formula of the substituent is as follows:the structural formula of the m-Tyr substituent is as follows: />The structural formula of the dicyclohe a substituent is as follows: />The structural formula of the dicyclohe b substituent is as follows:the structural formula of the dicyclohec substituent is as follows: />The structural formula of the dicyclohed substituent is as follows: />The structural formula of the N-Acetyl- (m-) Tyr substituent is as follows: />
The structural formula of the N-Gly- (m-) Tyr substituent is as follows:
preferably, the sansamycin derivatives are SS-KK-1 to 13 and SS-KK-A to I, and the structures of the sansamycin derivatives are shown in the following table
The invention also relates to application of the sansamycin derivative in preparing medicines, wherein the medicines are medicines for treating bacterial infection, preferably medicines for inhibiting mycobacterium tuberculosis, most preferably medicines for treating diseases caused by drug-resistant mycobacterium tuberculosis infection.
The invention also relates to a pharmaceutical composition containing the sansamycin derivative, and the pharmaceutical composition comprises a therapeutically effective amount of the sansamycin derivative and necessary pharmaceutical excipients.
The invention has the advantages that,
we extended the structural diversity of Sansanmycins by mutant biosynthesis and explored the relationship between the structure and activity of 5' -aminouridine molecules. In the sansamycin biosynthesis gene cluster SsaM, ssaK and SsaE have a high degree of homology to PacM, pacK and PacE, respectively, which have been shown to be responsible for the biosynthesis of 5' -aminouridine of pacidamycin by in vitro experiments.
In this study, we verified by in vivo experiments that SsaM and SsaK are responsible for the biosynthesis of 5' -aminouridine in the sansanmycin biosynthetic pathway, and by detection of a new hydrated derivative, the role of SsaM as dehydratase was demonstrated.
On this basis, two 5' -aminouridine analogs were fed to ssaK and ssaM gene knockout strains, respectively, and as a result, it was found that the ssaK gene knockout strain had higher derivative yields. 22 new derivatives were found by molecular network analysis of LC-MS/MS data. Wherein four monomers were purified, one of which was further confirmed by nuclear magnetic resonance. The antibacterial activity of SS-KK-2 on Escherichia coli DeltatolC is improved. SS-KK-3 has the same activity to mycobacterium tuberculosis (including clinically isolated drug-resistant strains) and better structural stability.
Drawings
FIG. 1, chemical structures of native UPAs (black formula, X1 substituent) and chemically synthesized 5' -aminouridine modified UPA derivatives (blue formula, X2-X4 substituent) and their corresponding antibacterial activities (Pae, pseudomonas aeruginosa; mtb, mycobacterium tuberculosis; B.subtilis, bacillus subtilis; S.aureus, staphylococcus aureus; A.aeolicus, a thermophilic bacterium),
1A, UPAs;
1B, the chemical structure of 5' -aminouridine of native UPAs;
1C-1E, modified 5' -aminouridine chemical structure of chemically synthesized UPA derivative;
detailed information on the structure and bacteriostatic activity of 1F native UPAs and synthetic UPA derivatives.
FIG. 2 production of a new structural analogue of 5' -aminouridine modified sansanansamycin by mutant biosynthesis,
structural formulas of 2A, 2 '-deoxy-5' -aminouridine (substrate 1) and 5-methyl-2 '-deoxy-5' -aminouridine (substrate 2);
2B ESI-MS analysis of the fermentation products after feeding substrate 1 and substrate 2 with different blocking strains.
FIG. 3, molecular network directed analysis of novel sansamycin class compounds,
3A, molecular networks composed of all parent ions in SS/KKO strains fed 1 or 2 were examined by LC-MS. A potential subset of sanansamycin was identified from the entire molecular network using SS-KK-1/2 and SS-KK-A/B (indicated in red) as probes, and an enlarged display was made showing that the derivative produced by feeding substrate 1 with SS/KKO was labeled blue and the derivative produced by feeding substrate 2 with SS/KKO was labeled yellow. All nodes for which an analog of sansandamycin has been identified are represented by diamond symbols;
3B, structures of analyzed SS-KK-1 through SS-KK-13 and SS-KK-A through SS-KK-I;
3C, MS/MS analysis of the corresponding compound, feature fragments are identified.
FIG. 4, HPLC-DAD-MS purity determination of SS-KK-1,
DAD chromatogram and ultraviolet absorption spectrum extracted from 4A, SS-KK-1 at 254 nm;
total ion flow chromatogram of 4B, SS-KK-1.
FIG. 5, HPLC-DAD-MS purity determination of SS-KK-2,
a DAD chromatogram and an ultraviolet absorption spectrum extracted from 5A, SS-KK-2 at 254 nm;
total ion flow chromatogram of 5B, SS-KK-2.
FIG. 6, HPLC-DAD-MS purity determination of SS-KK-3,
DAD chromatogram and ultraviolet absorption spectrum extracted from 6A, SS-KK-3 at 254 nm;
total ion flow chromatogram of 6B, SS-KK-3.
FIG. 7, HPLC-DAD-MS purity determination of SS-KK-C,
DAD chromatogram and ultraviolet absorption spectrum extracted from 6A, SS-KK-C at 254 nm;
total ion flow chromatogram of 6B, SS-KK-C.
FIG. 8, two-dimensional NMR correlation signal of SS-KK-1.
FIG. 9, stability of SS-A, SS-KK-2 and SS-KK-3 (all samples were analyzed by HPLC and quantified in terms of peak area, each sample was repeated three times in parallel)
8A-8C, in use KH 2 PO 4 HPLC analysis was performed on SS-A, SS-KK-2 and SS-KK-3 in buffer (pH 6.0) at the indicated times; residual amounts of 8D, SS-A, SS-KK-2 and SS-KK-3 over time.
Detailed Description
Strains, materials
The step of constructing ssaM, ssaK blocking strains using Streptomyces sp.SS as a host, china general microbiological culture Collection center with a collection number of CGMCC No.1764, was found in the prior work of the subject group (Fan Jiahui. Initial research of the synthetic biology of Sansansanmycin. Beijing institute of coordination, medical institute of medical biotechnology, 2018.);
2 '-deoxy-5' -aminouridine (feeding substrate 1), CAS number 35959-38-7, product number drug transition longitude and latitude information technology (Beijing) Co., ltd., YD0001468;
5-methyl-2 '-deoxy-5' -aminouridine (fed substrate 2), CAS number 25152-20-9, product number drug transition latitude and longitude information technology (Beijing) Co., ltd., YD0001469.
EXAMPLE 1 MS analysis of feeding uridine analogs and fermentation broth products to Streptomyces sp.SS ssaM, ssaK blocking strains
Streptomyces strains were grown at 28℃for 7d on corresponding solid culture plates, 1X 4cm pieces were shoveled off with a sterile spatula in an ultra-clean bench, crushed and added to 100mL of seed medium, and cultured at 28℃with shaking at 220rpm for 2d. After 2d, the seed medium was transferred to the fermentation medium at an inoculum size of 5%, and the shaking culture was continued at 28℃and 220rpm for 6d.
In the feeding experiment, shake flask fermentation is adopted, the final concentration of the fed substrate is 3mM, and 1mL ddH is used for feeding the substrate 2 The O-sonications were complete, filtered using a 0.22 μm sterile filter, and added to the fermentation medium at the time of transfer.
(1) A series of 5' -OH containing uridine analogs were first added to ssaK blocking strain SS/KKO, however, no new derivatives were detected in the LC-MS/MS analysis;
(2) We used 5'-aminouridine analogs, 2' -deoxy-5 '-aminouridine (substrate 1) and 5-methyl-2' -deoxy-5 '-aminouridine (substrate 2), respectively, which have both C-4' chiralities in the R-configuration (FIG. 2A). Feeding the above two 5' -aminouridine analogs to SS/MKO (ssaM-disrupted strain) and SS/KKO (ssaK-disrupted strain) mutants, respectively, at a concentration of 3 mM;
(3) Analysis of the fermentation product by extraction of the ion chromatogram of the target compound derived from parent compound SS-se:Sup>A revealed that two peaks corresponding to the new structural derivatives appeared simultaneously in both fermentation broths (fig. 2B), and that two peaks in the same broth had the same MS/MS fragments.
(4) The novel structural derivatives produced by feeding substrate 1 are designated SS-KK-1 and SS-KK-2, while the novel structural derivatives produced by feeding substrate 2 are designated SS-KK-A and SS-KK-B.
Example 2 Structure analysis of novel Structure derivatives
(1) The feeding of substrate 1 produced SS-KK-1 and SS-KK-2 with se:Sup>A molecular weight 2 masses greater than that of SS-A, while substrate 1 had se:Sup>A molecular weight 2 masses greater than that of the natural 5'-aminouridine moiety, initially indicating that the 5' -aminouridine moiety in SS-KK-1 and SS-KK-2 was replaced by substrate 1. The MS/MS datse:Sup>A also supports the assumption (FIG. 3C) that characteristic fragment ions, such as F5, F6, F12, F13, comprising se:Sup>A 5' -aminouracil moiety in SS-KK-1 and SS-KK-2 are each 2 mass numbers greater than SS-A, while other characteristic fragment ions are consistent with SS-A. Fragment ion F1 (m/z 136) is a characteristic fragment ion corresponding to m-Tyr or Tyr, and fragment ion F2 (m/z 703) corresponds to a characteristic fragment ion missing one m-Tyr or Tyr residue at the N-terminus. As can be derived from the presence of fragments of the two features F1 and F2, SS-KK-1 and SS-KK-2 contain an m-Tyr or Tyr at the AA1 position, as previously described in SS-A [1] And SS MX-2 [2] As found in the above.
(2) The molecular weight of the substrate 2 is 14 mass numbers greater than that of the substrate 1, and similarly, the molecular ion peak of the SS-KK-A/B is 14 mass numbers greater than that of the SS-KK-1/2, so that the substrate 2 fed is preliminarily proved to replace natural 5' -aminouridine to be doped into the structure of the SS-KK-A/B. The corresponding MS/MS datA also supports this conclusion, with the characteristic fragment ions (including F2, F5, F9, F10) comprising the 5' -aminouridine moiety in SS-KK-A/B each being 14 mass numbers greater than the corresponding characteristic fragment ions of SS-KK-1/2, while the other characteristic fragment ions are identical to SS-KK-1/2 (FIG. 3C). Thus, SS-KK-A/B differs from SS-KK-1/2 in the structure of only the 5' -aminouridine moiety.
(3) The production of SS-KK-1/2 and SS-KK-A/B indicated that feeding 5' -aminouridine analogs into blocking strains SS/MKO and SS/KKO could produce new structural derivatives. Among them, the yield of new structural derivatives produced by feeding two 5'-aminouridine analogs in SS/KKO was higher (fig. 2B), indicating that SS/KKO (ssaM-disrupted strain) is a good host for expanding the structural diversity of 5' -aminouridine by mutant biosynthesis.
Example 3 structural identification of novel sansamycin derivatives
1. Analysis and identification of the Structure of novel sansamycin derivatives produced on feeding substrate 1 (FIG. 3)
(1) The presence of fragment ions F5 and F9 of the same character indicates that SS-KK-3 has the same AA1 and AA3 as SS-KK-1/2, but that the total molecular weight of SS-KK-3 is 23 masses lower than that of SS-KK-1, indicating that AA4 of SS-KK-3 is 23 masses lower than Trp of (m-) Tyr.
(2) Because of the presence of the same characteristic fragment ions F2, F5, F7 and F8, it is presumed that SS-KK-4, SS-KK-5 and SS-KK-8 differ only in AA1 from SS-KK-1/2.
(3) AA1 of SS-KK-4 was 12 masses greater than that of m-Tyr, indicating that AA1 of SS-KK-4 is a bicyclic a/b that was found in sansaminomycin K and sansaminomycin J [3] 。
(4) In SS-KK-5, AA1 is 42 masses greater than m-Tyr, probably due to AA1 being acetylated, becoming N-Acetyl- (m-) Tyr. AA1 is 57 mass numbers greater than AA1 of m-Tyr, presumably with an additional Gly substitution on AA1 of SS-KK-8, resulting in AA1 becoming N-Gly- (m-) Tyr.
(5) SS-KK-6 has a molecular weight 16 masses greater than that of SS-KK-1/2, indicating that SS-KK-6 may have one more oxygen atom, presumably AA3 of SS-KK-1 is sulfoxylated by one more oxygen atom than methionine (Met) (Met SO ) Instead, this assumption is also supported by the two characteristic fragment ions M/z 818 (M-64) and M/z 614 (F10-64) that lose methylsulfinyl groups (64 mass numbers).
(6) (+) -ESI-MS of SS-KK-7 shows molecular ion peaks [ M+H ]]+m/z 894, 16 more mass numbers than SS-KK-4, and it was found by analysis that all of the characteristic fragment ions (F2, F5 and F10) containing AA3 in SS-KK-7 were 16 more mass numbers than SS-KK-4, indicating that it had one more oxygen atom at the AA3 position than SS-KK-1, and that AA3 was Met SO 。
(7) The molecular ion peak [ M+H ] + of SS-KK-9 is M/z 839, 39 mass numbers lower than that of SS-KK-4. MS/MS data analysis demonstrated the presence of a uniform characteristic fragment ion at m/z674 (F10), indicating that AA4 of SS-KK-9 changed from Trp in SS-KK-4 to Phe 39 mass numbers lower than Trp.
(8) SS-KK-10 has a molecular weight 18 mass numbers lower than that of SS-KK-1. By analysis, it was found that both of fragment ions F7 and F10 containing AA3 characteristic of SS-KK-10 were lower by 18 mass numbers than SS-KK-1, and therefore, AA3 of SS-KK-10 was presumed to be Leu, which was lower by 18 mass numbers than Met.
(9) SS-KK-11 has a molecular weight 854, which is 12 mass numbers greater than that of SS-KK-3. Both feature fragment ions F7 at m/z 339 are identical, but feature fragment ion F3 (m/z 207) of SS-KK-11 is 12 mass numbers higher than F3 (m/z 195) of SS-KK-3, from which it can be inferred that AA1 of SS-KK-11 is dicyclohec a/b.
(10) MS/MS data for SS-KK-12 were significantly different from those identified for sansamycin. Due to the presence of the characteristic fragment M/z 835 (M-64), we speculate that AA3 of SS-KK-12 is Met SO . The characteristic fragment ion F1 (m/z 176) corresponding to the AA1 residue was 28 masses higher than the dicyclohexyl a/b, suggesting that AA1 of SS-KK-12 is a dicyclohexyl d with two more methyl groups than the dicyclohexyl a/b. Based on the above presumption, AA4 is presumed to be m-Tyr/Tyr based on the molecular weight of SS-KK-12. The remaining characteristic fragment ions also support this inference.
(11) The molecular weight of SS-KK-13 is 16 mass numbers higher than that of SS-KK-5. Binding characteristic fragment ion m/z 655 (F2-64), it was inferred that AA3 of SS-KK-13 was Met SO 。
2. The structure of the novel sansaminomycin derivatives produced by feeding substrate 2 was analyzed and identified (FIG. 3).
(1) Substrate 2 has a molecular weight greater than substrate 1 by 14 mass numbers, and similarly, SS-KK-C, SS-KK-F and SS-KK-H have molecular weights greater than SS-KK-4, SS-KK-6 and SS-KK-12 by 14 mass numbers, respectively. It is therefore assumed that SS-KK-C, SS-KK-F and SS-KK-H have the same amino acids as SS-KK-4, SS-KK-6 and SS-KK-12, respectively, with only 5' -aminouridine being replaced by substrate 2. The corresponding MS/MS data also supports this inference.
(2) SS-KK-D has a molecular weight 16 masses higher than SS-KK-C, suggesting that AA3 of SS-KK-D may be Met 16 masses greater than Met SO . This assumption is also confirmed by the presence of characteristic fragment ions M/z 844 (M-64) and M/z 640 (F10-64).
(3) The molecular weight of SS-KK-E was 28 mass numbers higher than that of SS-KK-C, probably due to the two more methyl substitutions on AA1, suggesting that AA1 is a dicyclohec d. The presence of the same characteristic fragment ion m/z 717 (F2) also indicates that this change only occurred in AA1, while the characteristic fragment ion m/z 176 (F1) of SS-KK-E corresponds to the residue of dicyclohec d, further supporting this conclusion.
(4) The higher molecular weight of SS-KK-G than SS-KK-C by 14 mass numbers is likely due to one more methyl substitution. Both compounds had the same characteristic fragment ion F7 at m/z 362, indicating that they had the same AA3 and AA4, further inferring that AA1 in SS-KK-G may be a methyl substituted dicyclohec more than dicyclohec a/b.
(5) SS-KK-I has a molecular weight 18 masses lower than that of SS-KK-E, since both have the same characteristic fragment ions m/z 176 (F1) and m/z235 (F3), it is inferred that they contain the same AA1. The characteristic fragment ion F10 (m/z 698) in SS-KK-I was 18 mass numbers lower than F10 (m/z 716) of SS-KK-E, suggesting that AA3 of SS-KK-I should be Leu instead of Met.
EXAMPLE 4 isolation and Structure identification of novel structural derivatives of sansandamycin
The extended fermentations were performed on SS/KKO fed substrates 1 and 2, respectively. Further purification by semi-preparative high performance liquid chromatography gave SS-KK-1 (2.1 mg), SS-KK-2 (0.8 mg), SS-KK-3 (0.9 mg) and SS-KK-C (0.4 mg), see the summary of the invention. The purity of these compound monomers was determined by HPLC (fig. 4-7).
SS-KK-1 compound monomers were dissolved in DMSO-D6 as solvent and 1D and 2D NMR spectra were collected for structural identification. Compared with the nuclear magnetic resonance spectrum of SS-A in DMSO-d6, SS-KK-1 has very similar signals except for the ribose moiety [1] . Further analysis showed that 2-deoxy-5 '-aminouridine (δH 6.05for H-sugam-1, 2.02for H-sugam-2, 3.99for H-sugam-3, 3.66for H-sugam-4, 3.21for H-sugam-5) fed in SS-KK-1 replaced 5' -aminouridine in SS-A in the 1H-1H COSY signal
This was further confirmed by the continuous correlation of H-sugar-1/H-sugar-2/H-sugar-3/H-sugar-4/H-sugar-5. Meanwhile, analysis of the 1D and 2D NMR spectra (FIG. 8 and Table 1) confirmed that the N-terminus of SS-KK-1 was m-Tyr.
Table 1, SS-KK-1 1 H NMR (600 MHz) and 13 c NMR (150 MHz) data
Spectra were recorded in DMSO-d 6. Chemical shift (δ) is in ppm.
* The structural units are abbreviated as follows: trp=tryptophan, daba=2-amino-3-methylaminobutyric acid, met=methionine
In addition, SS-KK-1 and SS-KK-2 were found to have identical secondary mass spectral fragments by MS/MS analysis, and therefore they were presumed to differ only in the N-terminal amino acid, corresponding to m-Tyr or Tyr, respectively. Since the N-terminus of SS-KK-1 was identified as m-Tyr by nuclear magnetic data, the N-terminus of SS-KK-2 was inferred to be Tyr.
EXAMPLE 5 detection of bacteriostatic Activity and stability of sansandamycin novel Structure derivatives
The isolated purified sansandamycin new structural derivatives SS-KK-1, SS-KK-2, SS-KK-3 and SS-KK-C were assayed for bacteriostatic activity (Table 2), including gram negative and gram positive bacteria, as well as M.tuberculosis H37Rv and clinically isolated M.tuberculosis S17, M6600, M3551 and M9483. Wherein M.tuberculosis M6600, M3551 and M9483 are multi-drug resistant strains with a drug resistance concentration to INH of 0.1 μg/mL and to RIF of 1 μg/mL.
The results show that the data obtained from the above-mentioned method,
(1) Both SS-KK-1 and SS-KK-C lost antibacterial activity against Pseudomonas aeruginosa, mycobacterium tuberculosis and even Escherichia coli DeltatolC.
(2) The antibacterial activity of SS-KK-3 against E.coli DeltatolC and Mycobacterium tuberculosis (including M.tuberculosis H37 Rv) was comparable to that of SS-A.
(3) Although SS-KK-2 lost inhibitory activity against Pseudomonas aeruginosse:Sup>A, it had the best inhibitory activity against E.coli DeltatolC compared to SS-KK-3 and SS-A.
TABLE 2 Activity of Sansanmycin analogues
Mycobacterium tuberculosis H37Rv, standard strain;
s17, M6600, M3551 and M9483 are clinically isolated Mycobacterium tuberculosis.
INH, isoniazid; RIF, rifampin; LZD, linezolid.
M6600, M3551 and M9483 are multi-drug resistant strains, resistant to INH at 0.1. Mu.g/ml and RIF at 1.0. Mu.g/ml, and sensitive to LZD at 2.0. Mu.g/ml.
Poor stability is one of the factors impeding the development of sansansamycin analogs into candidate drugs. Thus, according to the previously reported method [2] Stability experiments were performed on two biologically active sansamycin new structural derivatives SS-KK-2 and SS-KK-3 (fig. 9).
By taking the parent compound SS-A (sansanmycin) as se:Sup>A control, after 9 days of incubation (the incubation condition is 28 ℃), the residual amount of the SS-A is reduced to below 70%, the stability of the SS-KK-2 is slightly higher than that of the SS-A, and the SS-KK-3 is hardly degraded, so that the compound has excellent stability. Meanwhile, SS-KK-3 has an antibacterial activity equivalent to that of SS-A, so that SS-KK-3 is se:Sup>A more promising lead compound of antitubercular drugs.
[ cited documents ]
1.Winn,M.,Goss,R.J.,Kimura,K.,and Bugg,T.D.(2010).Antimicrobial nucleoside antibiotics targeting cell wall assembly:recent advances in structure-function studies and nucleoside biosynthesis.Nat.Prod.Rep.27,279-304.doi:10.1039/b816215h.
2.World Health Organization.(2022).Global tuberculosis report 2022.
https://www.who.int/teams/global-tuberculosis-programme/tb-reports/global-tuberculosis-report-2022[Accessed October 27,2022].
3.Xie,Y.,Cai,Q.,Ren,H.,Wang,L.,Xu,H.,Hong,B.,et al.(2014).NRPS substrate promiscuity leads to more potent antitubercular sansanmycin analogues.J.Nat.Prod.77,1744-1748.doi:10.1021/np5001494.
Finally, it should be noted that the above embodiments are only for helping the person skilled in the art to understand the essence of the present invention, and are not intended to limit the protection scope of the present invention.
Claims (7)
1. A method for biosynthesis of sansanmycin derivatives, comprising the steps of
(1) Adding a 5' -aminouridine analogue, preferably a 5' -aminouridine analogue with C-4' chirality of R-configuration, preferably 2' -deoxy-5 ' -aminouridine or 5-methyl-2 ' -deoxy-5 ' -aminouridine, to a culture medium of ssaM, ssaK-disrupted strain of Streptomyces sp.SS;
the chemical structure of the 2 '-deoxy-5' -aminouridine is shown as the following formula:
the chemical structure of the 5-methyl-2 '-deoxy-5' -aminouridine is shown as the following formula:
the ssaM and ssaK blocking strains of the Streptomyces sp.SS are as follows: taking Streptomyces sp.SS as a host, knocking out ssaM genes or ssaK genes by using a genetic engineering means, and obtaining a blocking strain;
(2) Collecting fermentation liquor, and separating and purifying to obtain the sansanansamycin derivative.
2. The method of claim 1, wherein the 5' -aminouridine analog is added by shaking the host for fermentation, the final concentration of the substrate is 3mM, and the substrate is added to the fermentation medium at the time of transferring the strain after being dissolved in water.
3. The method according to claim 1 or 2, wherein the collection of the fermentation broth of step (2) and the isolation and purification to obtain the sansamycin derivative comprises the steps of
1) Centrifuging the fermentation product at 4 ℃ and 4,500rpm for 20min, discarding the precipitate, and collecting the supernatant of the fermentation broth;
2) Using macroporous resin to adsorb crude products; preferably, the fermentation supernatant is put on a macroporous resin D4006 column at the speed of 50mL/min, pure water without acetone, 10% acetone aqueous solution, 20% acetone aqueous solution and 30% acetone aqueous solution are sequentially used for elution, gradient elution is carried out, each elution gradient is carried out for 5-6 column volumes, the elution components of the 20% acetone aqueous solution and the 30% acetone aqueous solution are collected, and the components are concentrated and freeze-dried after being combined to obtain a sansanansamycin derivative mixed crude product;
3) Performing preliminary separation of crude products of the respective derivatives by using anion exchange resin; preferably, the anionic filler is DEAE sephadex A-25, the crude powder is completely dissolved and loaded with 0.02M Tris buffer solution with pH value of 9, after loading is completed, the solution is eluted with 0.02M Tris buffer solution added with NaCl, after 10-12 column volumes are eluted with 0.01M NaCl as initial gradient, HPLC-MS detection is carried out on the effluent, if no sansamycin derivative signal exists, the concentration of NaCl is increased, the elution is continued, the concentration of 0.01M is increased each time, and 10-12 column volumes are eluted until no sanmycin derivative signal exists in the effluent; combining effluent containing single same HPLC-MS detection signals, concentrating by rotary evaporation, and desalting by adopting a solid-phase extraction method to obtain crude products of each sansamycin derivative;
4) Purifying by high performance liquid chromatography; preferably, the preparation is carried out by using a Shimadzu LC-20AD high performance liquid chromatograph, 100 mu L of sample is injected each time, and a chromatographic column is adopted asprep C18column (5 μm, 250X 10 mm), flow rate 3mL/min, detection wavelength 254nm, column temperature 40 ℃, mobile phase 0.1% (w/v) CH 3 COONH 4 Aqueous solution-MeOH; after the peaks of each compound are collected, the purity is detected by HPLC, so that the purity is ensured to be more than 90 percent, and the preparation is needed again if the purity is insufficient; desalting the effluent with qualified purity with a solid phase extraction column, concentrating with a rotary evaporator, and lyophilizing to obtain pure product of each sansanmycin derivative.
4. A sansanansamycin derivative obtainable by the process of any one of claims 1 to 3, the sanansamycin derivative having the formula
In the method, in the process of the invention,
r is H or CH 3 ，
AA1 is Tyr, m-Tyr, dicyclohexyl a/b/c/d, N-Acetyl- (m-) Tyr, N-Gly- (m-) Tyr;
AA2 is 2,3-diaminobutyric acid (2, 3-diaminobutyric acid, DABA);
AA3 is Met, met SO 、Leu；
AA4 is Trp, (m-) Tyr, phe.
Wherein,
the structural formula of the (m-) Tyr substituent is as follows:
Met so the structural formula of the substituent is as follows:
the structural formula of the m-Tyr substituent is as follows:
the structural formula of the dicyclohe a substituent is as follows:
the structural formula of the dicyclohe b substituent is as follows:
the structural formula of the dicyclohec substituent is as follows:
the structural formula of the dicyclohed substituent is as follows:
the structural formula of the N-Acetyl- (m-) Tyr substituent is as follows:
the structural formula of the N-Gly- (m-) Tyr substituent is as follows:
5. the sansamycin derivative according to claim 4, wherein the sansamycin derivative is SS-KK-1 to 13 or SS-KK-A to I, and the structure thereof is shown in the following table
6. The use of a sansandamycin derivative according to claim 4 or 5for the manufacture of a medicament for the treatment of a bacterial infection, preferably for the inhibition of mycobacterium tuberculosis, most preferably for the treatment of a disease caused by a drug-resistant mycobacterium tuberculosis infection.
7. A pharmaceutical composition comprising a sansanansamycin derivative according to claim 4 or 5, wherein the pharmaceutical composition comprises a therapeutically effective amount of the sanansamycin derivative and, if necessary, pharmaceutical excipients.
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