English

• 1.   Introduction

  All the time, natural products play a highly significant role in the drug discovery and development process and still held out the best options for finding novel agents/ active templates, which when worked on in conjunction with organic chemists and biologists.^[1] Thiopeptide antibiotics are a class of ribosomally synthesized and post-translationally modified peptide (RiPP) natural products, displaying nanomolar potency toward various drugresistant strains of Gram-positive pathogens.^[2] Nosiheptide (NOS), as a typical representative of thiopeptide antibiotics, is one of the oldest known thiopeptide antibiotics, which is firstly isolated from Streptomyces actuosus 40037 (NRRL 2954), and later isolated from Streptomyces antibioticus 8446-CC1^[3] and strain CNT-373.^[4] NOS has been used as a feed additive to promote growth in pigs and poultry. NOS is very active in vitro against Gram-positive bacteria (MIC 0.9 ng/mL against Staphylococcus aureus ATCC 6538P) but inactive in vivo in experimentally infected mice.^[3] Mechanically, NOS exerts its antibacterial effect by binding within a cleft located between the L11 protein and 23S rRNA of the 50S large ribosomal subunit, thereby perturbing translation factor binding and subsequent bacterial protein synthesis.^[5] The mode of action is unique and distinct from those of current chemotherapeutics targeting the bacterial ribosome.^[6] The clinical application of NOS, however, is largely hindered due to its poor water solubility and low bioavailability.

  Structurally, NOS is composed of a macrocycle with a fused side-ring structure. The core macrocycle contains a central pyridine ring, multiple thiazoles, dehydrated serine and threonine amino acids. The side-ring structure is formed by connecting the side chains of Glu6 and Cys8 on the core macrocycle via ester and thioester bonds, respectively, with a 3-methyl-4-hydroxymethyl-2-indolic acid bridge. NOS has been chemically synthesized in 2016, ^[7] however, using chemical synthesis to improve its pharmacological properties have not been reported, most likely is subjected to its highly complex chemical structure. The biosynthetic pathway for NOS formation, which relies on the extensive modification of a ribosomally synthesized precursor peptide, offers the potential for relatively facile generation of analogues via biosynthetic methods.^[8] The biosynthetic gene cluster of NOS was identified and contains 16 genes (Figure 1A), designated nosA-nosP.^[9] The formation of NOS starts from NosM, a ribosomally synthesized precursor peptide of 50 amino acids (aa). As shown in Figure 1B, the first 37 aa of the precursor peptide, termed the leader peptide, primarily provide recognition for some of the downstream enzymes, while the remaining 13 aa, called the core peptide, are post-translationally modified and incorporated into the final product. The biosynthesis of core macrocycle is initiated by thiazole formation, catalyzed by NosF, NosG and NosH. However, Cys8 is left intact during thiazole formation and is immediately acylated with 3-methyl- 2-indolic acid (MIA) by NosI, NosJ and NosK.^{[10, 13-14]} MIA is derived from L-Trp via the action of NosL.^[11-12] And then NosN transfered a C₁ unit from SAM to C(4) of MIA with concomitant formation of a bond between the carboxylate of Glu6 of the core peptide and the nascent C₁ unit.^[15-17] Dehydroalanine (Dha) and dehydrobutyrine (Dhb) are likely to be formed via the hydroxyl groups on the side chains of Ser1, Ser10, Ser12, Ser13, and Thr3 activization and dehydration, catalyzed by NosD and NosE.^[18] The Dha derived from Ser1 and Ser10 are then cyclized by NosO via a [4+2] azacycloaddition to remove the leader peptide and form the core pyridine motif.^[19-20] NosA, NosB and NosC are tailoring enzymes in the later stages of the maturation of NOS (Scheme 1).^[21-22]

  Figure 1

  

  Figure 1.  (A) Biosynthetic gene cluster of NOS and (B) amino acid sequence of NosM

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  Scheme 1

  

  Scheme 1.  proposed NOS biosynthetic pathway

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  In our previous work, by feeding 5-F-Trp into wild type NOS strain, we obtained 5'-F-NOS whose antibacterial activity is improved, suggesting that the side-ring structure of NOS is important for its antibiotic properties.^[12] However, due to the competition of L-Trp in the native biosynthetic machinery, a mixture of NOS and 5'-F-NOS were co-produced, leading to complex compound purification burden. Based on precursor-directed biosynthesis, mutational biosynthesis can utilize mutant strains deficient in a key aspect of the biosynthetic pathway and directionally produce natural product analogues via supplementation of particular precursors.^[23] Recently, we established an efficient mutational biosynthesis strategy for the structural expansion of the side-ring system of NOS. By feeding 6-F-MIA into a nosL-deficient mutant strain, ΔnosL, 6'-F-NOS with significantly improved activities against drug-resistant pathogens were successfully obtained. What's more, an unexpected product, named as 6'-F-NOSint, with an open side ring and a bis-dehydro-alanine (Dha) tail, was also trapped. The fact gives us a clue that the size and electrical property of the substituent group will affect the enzymatic activity of related enzymes involved in NOS biosynthesis pathway.^[24] However, the relationship between the size and electrical property of the substituent group in MIA and the substrate tolerance of enzymes involved in NOS biosynthesis pathway remains undefined. Herein, we provide experimental evidence that substrate tolerance of enzymes involved in NOS biosynthesis pathway by using MIA analogues with different size and chemical property substituent group, such as F, Cl, CH₃, NO₂ and CF₃.

  2.   Results and discussion

  For MIA analogues preparation, an efficient two-step chemical synthesis route was developed.^[24] Briefly, 2-, 3-, 4-substituted 1-(2-bromophenyl)ethan-1-ones were reacted with ethyl isocyanoacetate in the presence of Cs₂CO₃ and CuI, resulting in substituted indole-2-carboxylic acid esters in 85%~90% yield. The substituted-MIA esters were further hydrolyzed by NaOH followed by acidification, furnishing substituted-MIA in 80%~88% yield (Scheme 2, A). The substituted-MIA was exogenously fed into the fermentation broth with a NosL-deficient mutant strain, ΔnosL, in which the biosynthetic route to MIA was blocked. The fermentation products were analyzed by high pressure liquid chromatography (HPLC) (Scheme 2, B).

  Scheme 2

  

  Scheme 2.  (A) Chemical synthesis of the substituted-MIA and (B) enzymes involved in NOS biosynthesis pathway to generate NOS analogues or NOSint in NosL mutant, by chemically feeding substituted-MIA

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  5-F-MIA was firstly exogenously fed into the fermentation broth with ΔnosL mutant strain (Figure 2, Ⅲ). The target NOS analogue 5'-F-NOS and an unexpected compound 5'-F-NOSint were co-produced. HR-ESI-MS data established the molecular formula as C₅₁H₄₃FN₁₃O₁₂S₆ ([M+H]⁺, calcd 1240.1457, found 1240.1436) and C₅₃H₄₅FN₁₃O₁₂S₆ ([M+H]⁺, calcd 1266.1613, found 1266.1578) respectively. As shown in Figure 2 (Ⅲ), the output of 5'-F-NOSint is more than that of 5'-F-NOS. Regarding to the fact that electronegativity of F (4.0) is higher than H (2.1) while atomic radius of F (0.071 nm) is similar to H (0.079 nm), the introduction of a fluorine atom at C(5) of MIA will reduce the electron cloud density of MIA and hence affect losing an electron to generate the resulting electrophilic aryl cation intermediate (Scheme 3), and thus partly side-ring-opening intermediate was accumulated. Fortunately, the side-ring-opening intermediate can be tolerated by NosO and then be enzymatically transformed to form 5'-F-NOSint. Since the substrates of tailoring enzymes NosA, NosB and NosC feature the closure of the side ring, 5'-F-NOSint can't be enzymatically transformed by these three enzymes.^[22]

  Figure 2

  

  Figure 2.  HPLC analysis of the fermentation products from the wild-type NOS-producing strain streptomyces actuosus ATCC25421 (Ⅱ), the nosL-deficient mutant strain ΔnosL (Ⅰ) and ΔnosL complemented by chemically feeding substituted-MIA (Ⅲ~X).

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  Scheme 3

  

  Scheme 3.  Proposed mechanism of NosN

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  When 5-Cl-MIA was fed into the fermentation broth with ΔnosL mutant strain, the target NOS analogue, 5'-Cl-NOS was produced, and HR-ESI-MS data established the molecular formula as C₅₁H₄₃ClN₁₃O₁₂S₆ ([M+H]⁺, calcd 1256.1161, found 1256.1154). 5'-Cl-NOSint, however, was not found in the fermentation products (Figure 2, Ⅳ). The electronegativity of Cl (3.2) is a little weaker than that of F (4.0) while atomic radius of Cl (0.099 nm) is similar to F (0.071 nm), reveals that the electron cloud density adjacent to C(4) have the key effect on the NosN enzymatic activity. When 5-CH₃-MIA was exogenously fed into the fermentation broth with ΔnosL mutant strain, the target NOS analogue, 5'-CH₃-NOS, was produced and HR-ESI-MS data established the molecular formula as C₅₂H₄₆N₁₃O₁₂S₆ ([M+H]⁺, calcd 1236.1708, found 1236.1626). The CH₃ is an electron donor group which will facilitate losing an electron to form the resulting electrophilic aryl cation intermediate (Scheme 3). The above experiments show that the chemical property of substituent group at C(5) is the main barrier to affect NosN enzymatic fuction.

  For 5-CF₃-MIA and 5-NO₂-MIA, the target NOS analogues or NOSint analogues, were not found in the fermentation products. The electronegativity of CF₃ (3.6) or NO₂ (3.4) is a little weaker than the electronegativity of F (4.0) while atomic radius of CF₃ or NO₂ is much larger than that of F.^[25] On the one hand, their steric hindrance might hinder methylene radical addition to C(4) of MIA to give an aryl radical intermediate (Scheme 3), and thus might hinder the following dehydratase (NosD and NosE) and azacyclase (NosO). On the other hand, MIA is activated and uploaded by NosI, NosJ, and NosK, and the above results can't deny the possibility that introduction CF₃ or NO₂ into C(5) of MIA might affect enzymatic activities of NosI, NosJ, and NosK.

  When 6-Cl-MIA was exogenously fed into the fermentation broth with ΔnosL mutant strain, 6'-chloro-NOS and 6'-Cl-NOSint, were co-produced. HR-ESI-MS data estab-lished the molecular formula as C₅₁H₄₃ClN₁₃O₁₂S₆ ([M+H]⁺, calcd 1256.1161, found 1256.1141) and C₅₃H₄₅Cl-N₁₃O₁₂S₆ ([M+H]⁺, calcd 1282.1318, found 1282.1290) respectively. As shown in Figure 2 (Ⅵ), the output of 6'-Cl-NOSint is more than 6'-Cl-NOS, revealing that introduction of a chlorine atom at C(6) of MIA, perhaps interferes the enzymatic function of NosN via the steric hindrance.

  When 7-F-MIA or 7-Cl-MIA was exogenously fed into the fermentation broth with ΔnosL mutant strain, 7'-F-NOSint or 7'-Cl-NOSint was respectively produced. HR-ESI-MS data established the molecular formula as C₅₃H₄₅FN₁₃O₁₂S₆ ([M+H]⁺, calcd 1266.1613, found 1266.1591) or C₅₃H₄₅ClN₁₃O₁₂S₆ ([M+H]⁺, calcd 1282.1318, found 1282.1274) respectively. Figure 2 shows that 7'-F-NOSint and 7'-Cl-NOSint are the main product while the output of 7'-Cl-NOSint is somewhat lower. The above result reveals that the steric hindrance at C(7) of MIA may have the key effect on NosN enzymatic activity. Furthermore, we got no product when larger substituent group on MIA, such as 7-CF₃-MIA or 7-NO₂-MIA, was fed into the fermentation broth. The possible mechanism of activation and upload of 7-CF₃-MIA or 7-NO₂-MIA is similar to that of 5-NO₂-MIA.

  For disubstituted MIA, When 5, 6-F₂-MIA or 5-F-6- Cl-MIA was exogenously fed into the fermentation broth with ΔnosL mutant strain, 5', 6'-F₂-NOSint or 5'-F-6'-Cl- NOSint was respectively produced. HR-ESI-MS data established the molecular formula as C₅₃H₄₄F₂N₁₃O₁₂S₆ ([M+H]⁺, calcd 1284.1519, found 1284.1490) or C₅₃H₄₄ClFN₁₃O₁₂S₆ ([M+H]⁺, calcd 1300.1224, found 1300.1166) respectively. Figure 2 (Ⅸ and Ⅹ) shows that the output of 5'-F-6'-Cl-NOSint is much more than that of 5', 6'-F₂-NOSint. The above result reveals that the dual strong negative inductive effect at C(5) or strong negative inductive effect at C(5) plus the steric hindrance at C(6) of MIA may have the key effect on NosN enzymatic function to generate an aryl radical intermediate (Scheme 3).

  The target NOS analog, 9'-CH₃-NOS, was produced when 9-CH₃-MIA was exogenously fed into the fermentation broth with ΔnosL mutant strain. HR-ESI-MS data established the molecular formula as C₅₂H₄₆N₁₃O₁₂S₆ ([M+H]⁺, calcd 1236.1708, found 1236.1695). 9'-CH₃-NOS is the main product and 9'-CH₃- NOSint was not found in the fermentation products. Considering the distance, the influence of C(9) substituent group on MIA enzymatic transformations by NosN is negligible. However, no product was got when 9-Ph-5-Cl-MIA was fed into the fermentation broth and the possible mechanism of activation and upload of 9-Ph-5-Cl-MIA is similar to that of 5-CF₃-MIA.

  3.   Conclusions

  Although MIA generation is independent biotransformation process through action of NosL, which will be recognized, activated and tolerated by at least ten enzymes before it becomes mature NOS. Although NosI, NosJ and NosK perform the activation of the MIA and upload it to the intermediate, our results show that these three enzyme's functions might intervene by the larger substituent groups, such as CF₃ or NO₂ through steric hindrance. However, the chemical property and steric hindrance of substituent group of MIA are the main aspects to affect NosN enzymatic function and the cumulative negative inductive effect of the multisubstituted groups can also affect NosN enzymatic function. The bioengineering of NosI, NosJ, NosK and NosN, based on the analysis of their protein crystal structure, will help to broaden their substrate tolerance and hence are beneficial to structural diversity bioproduction of NOS analogues.

  Under the global antibiotic resistance crisis and urgent need for antibiotics with novel mechanisms of action, our work not only explored the substrate tolerance of related enzymes involved in NOS biosynthesis pathway, but also was expected to obtain NOS analogues via mutational biosynthesis. Besides, our work provided valuable information for using directed evolution technology to improve the substrate tolerance of enzymes in NOS biosynthesis and to expand the use of NOS-producing bacteria to obtain more analogues, which is difficult to access solely by chemical synthesis.

  4.   Experimental section

  4.1   Experiment procedures and product characterization

  Analytical thin layer chromatography (TLC) was performed on 0.25 mm silica gel plates (Qingdao Haiyang Chemical China), and the compounds were visualized with a UV light at 254 nm. Flash chromatography was performed on silica gel 200~300 mesh (purchased from Qingdao Haiyang Chemical China) with commercial solvents. The ¹H NMR and ¹³C NMR spectra were recorded on a Bruker AM 500 spectrometer (500, 125, 470 MHz for ¹H NMR, ¹³C NMR and ¹⁹F NMR, respectively) and are internally referenced to residual solvent signals (note: CDCl₃ referenced at δ 7.26 and 77.00 in ¹H NMR and ¹³C NMR, respectively; DMSO-d₆ referenced at δ 2.50 and 39.52 in ¹H NMR and ¹³C NMR, respectively). High-resolution mass spectrometry (HRMS) was recorded on an Agilent Q-TOF spectrometer. Melting point was recorded on Hanon MP-420 automatic melting point meter. Commercial reagents and solvents were used as received, unless otherwise stated. 5-F-MIA, 7-F-MIA, 5-CH₃-MIA, 5-NO₂- MIA, 7-NO₂-MIA, 9-CH₃-MIA and 9-Ph-5-Cl-MIA were purchased from Cambridge, other MIA analogues were synthesized. Organic solution was concentrated under reduced pressure on an Eyela rotary evaporator.

  4.2   Chemical synthesis of ethyl substituted 3-methyl-1H-indole-2-carboxylate

  Substituted 1-(2-bromo-4-fluorophenyl)ethan-1-one (10.0 mmol), Cs₂CO₃ (20.0 mmol), CuI (1.0 mmol) and dimethyl sulfoxide (DMSO, 10 mL) were added together into a reacting tube under nitrogen atmosphere. Then ethyl isocyanoacetate (11.0 mmol) was slowly added and the mixture was stirred at 50 ℃. Once the reaction finished, ethyl acetate (100 mL) and water (40 mL) were added. The organic phase was separated, dried over sodium sulphate and evaporated in vacuum. The residue was loaded on silica gel column and purified to get ethyl substituted 3-methyl-1H-indole-2-carboxylate in 80%~90% yield.

  Ethyl 5-chloro-3-methyl-1H-indole-2-carboxylate (b-1):^[26] white solid, 2.13 g, 90% yield. m.p. 153~154 ℃(lit.^[30] 152~154 ℃); ¹H NMR (CDCl₃, 500 MHz) δ: 8.80 (br, 1H), 7.62 (s, 1H), 7.31~7.23 (m, 2H), 4.44 (q, J＝7.2 Hz, 2H), 2.56 (s, 3H), 1.43 (t, J＝7.2 Hz, 3H).

  Ethyl 6-chloro-3-methyl-1H-indole-2-carboxylate (b-2):^[28] white solid, 2.08 g, 88% yield. m.p. 158~159 ℃(lit.^[31] 159~160 ℃); ¹H NMR (CDCl₃, 500 MHz) δ: 8.80 (br, 1H), 7.56 (d, J＝8.4 Hz, 1H), 7.35 (d, J＝1.5 Hz, 1H), 7.10 (dd, J＝8.4, 1.8 Hz, 1H), 4.44 (q, J＝7.2 Hz, 2H), 2.58 (s, 3H), 1.43 (t, J＝7.2 Hz, 3H).

  Ethyl 7-chloro-3-methyl-1H-indole-2-carboxylate (b-3):^[26] white solid, 2.02 g, 85% yield. m.p. 156~157 ℃; ¹H NMR (CDCl₃, 500 MHz) δ: 8.81 (br, 1H), 7.56 (d, J＝8.1 Hz, 1H), 7.32 (d, J＝7.5 Hz, 1H), 7.08 (t, J＝7.8 Hz, 1H), 4.44 (q, J＝7.2 Hz, 2H), 2.60 (s, 3H), 1.43 (t, J＝7.2 Hz, 3H).

  Ethyl 5, 6-difluoro-3-methyl-1H-indole-2-carboxylate (b-4):^[27] white solid, 2.03 g, 85% yield. m.p. 138~139 ℃; ¹H NMR (CDCl₃, 500 MHz) δ: 8.78 (br, 1H), 7.40~7.34 (m, 1H), 7.16~7.11 (m, 1H), 4.42 (q, J＝7.2 Hz, 2H), 2.54 (s, 3H), 1.43 (t, J＝7.2 Hz, 3H).

  Ethyl 6-chloro-5-fluoro-3-methyl-1H-indole-2-carboxylate (b-5):^[26] white solid, 2.09 g, 82% yield. m.p. 163~164 ℃; ¹H NMR (CDCl₃, 500 MHz) δ: 8.67 (br, 1H), 7.41~7.35 (m, 2H), 4.44 (q, J＝7.2 Hz, 2H), 2.54 (s, 3H), 1.43 (t, J＝7.2 Hz, 3H).

  Ethyl 3-methyl-5-(trifluoromethyl)-1H-indole-2-carboxylate (b-6):^[29] white solid, 2.17 g, 80% yield. m.p. 183~184 ℃; ¹H NMR (CDCl₃, 500 MHz) δ: 9.02 (br, 1H), 7.96 (s, 1H), 7.53 (dd, J＝8.4, 1.2 Hz, 1H), 7.44 (d, J＝8.4 Hz, 1H), 4.44 (q, J＝7.2 Hz, 2H), 2.60 (s, 3H), 1.43 (t, J＝7.2 Hz, 3H).

  Ethyl 3-methyl-7-(trifluoromethyl)-1H-indole-2-carbo-xylate (b-7):^[28] white solid, 2.22 g, 82% yield. m.p. 187~188 ℃; ¹H NMR (CDCl₃, 500 MHz) δ: 8.92 (br, 1H), 7.83 (d, J＝8.1 Hz, 1H), 7.57 (d, J＝7.5 Hz, 1H), 7.26~7.18 (m, 1H), 4.44 (q, J＝7.2 Hz, 2H), 2.62 (s, 3H), 1.44 (t, J＝7.2 Hz, 3H).

  4.3   Chemical synthesis of substituted-MIA

  Ethyl substituted 3-methyl-1H-indole-2-carboxylate (5 mmol), NaOH (50 mmol) and EtOH (20 mL) were added together into a round-bottom flask and the mixture was refluxed overnight. After the reaction was finished, the solvent was evaporated in vacuum and the residue was redissolved in water (20 mL). Ethyl acetate (40 mL) was added and the aqueous phase was separated and acidification to pH 3.5 by using 1 mol/L HCl. Ethyl acetate (40 mL) was added and the organic phase was separated, dried over sodium sulphate and evaporated in vacuum to give substituted-MIA in 80%~88% yield.

  5-Chloro-3-methyl-1H-indole-2-carboxylic acid (a-1): 0.88 g, 85% yield. ¹H NMR (500 MHz, DMSO-d₆) δ: 13.05 (s, 1H), 11.59 (s, 1H), 7.70 (dd, J＝2.0 Hz, 1H), 7.39 (dd, J＝8.7, 0.6 Hz, 1H), 7.24 (dd, J＝8.7, 2.1 Hz, 1H), 2.50 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ: 163.74 (s, C-8), 134.86 (s, C-7a), 129.28 (s, C-3a), 126.03 (s, C-5), 125.13 (s, C-2), 124.24 (s, C-3), 119.93 (s, C-6), 117.81 (s, C-4), 114.40 (s, C-7), 10.10 (s, C-9); HRMS(EI) calcd C₁₀H₇ClNO₂ (M－H)^－ 208.0171, found 208.0171.

  6-Chloro-3-methyl-1H-indole-2-carboxylic acid (a-2): 0.89 g, 86% yield. ¹H NMR (500 MHz, DMSO-d₆) δ: 13.05 (s, 1H), 11.53 (s, 1H), 7.65 (d, J＝8.6 Hz, 1H), 7.41 (d, J＝1.9 Hz, 1H), 7.06 (dd, J＝8.6, 1.9 Hz, 1H), 2.52 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ: 163.42 (s, C-8), 136.39 (s, C-7a), 129.36 (s, C-6), 126.75 (s, C-3a), 125.20 (s, C-2), 122.06 (s, C-3), 119.91 (s, C-5), 118.12 (s, C-4), 111.75 (s, C-7), 9.8(s, C-9); HRMS(EI) calcd C₁₀H₇ClNO₂ (M－H)^－ 208.0171, found 208.0169.

  7-Chloro-3-methyl-1H-indole-2-carboxylic acid (a-3): 0.86 g, 83% yield. ¹H NMR (500 MHz, DMSO-d₆) δ: 13.10 (s, 1H), 11.45(s, 1H), 7.63 (dd, J＝8.0, 0.8 Hz, 1H), 7.33 (dd, J＝7.5, 0.9 Hz, 1H), 7.07 (t, J＝7.8 Hz, 1H), 2.52 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ: 163.61 (s, C-8), 133.62 (s, C-7a), 130.25 (s, C-3a), 126.26 (s, C-2), 124.73 (s, C-3), 120.66 (s, C-5), 119.80 (s, C-7), 119.57 (s, C-6), 117.02 (s, C-4), 10.42 (s, C-9); HRMS(EI) calcd C₁₀H₇ClNO₂ (M－H)^－ 208.0171, found 208.0173.

  5, 6-Difluoro-3-methyl-1H-indole-2-carboxylic acid (a-4): 0.84 g, 80% yield. ¹H NMR (500 MHz, DMSO-d₆) δ: 12.99 (s, 1H), 11.55 (s, 1H), 7.66 (dd, J＝11.1, 8.1 Hz, 1H), 7.27 (dd, J＝11.0, 7.0 Hz, 1H), 2.48 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ: 162.98 (s, C-8), 148.84 (d, J＝240, C-5), 145.55 (d, J＝240, C-6), 131.27 (d, J＝10.8 Hz, C-7a), 125.81 (d, J＝3.8 Hz, C-3a), 123.24 (d, J＝7.8 Hz, C-2), 118.23 (m, C-3), 106.92 (d, J＝18.5 Hz, C-4), 99.63 (d, J＝21.4 Hz, C-7), 9.75 (s, C-9); ¹⁹F NMR (470 MHz, DMSO-d₆) δ: －140.25 (dt, J＝20.3, 9.5 Hz), －147.38 (ddd, J＝21.6, 11.2, 7.0 Hz); HRMS(EI) calcd C₁₀H₆F₂NO₂ (M－H)^－ 210.0372, found 210.0372.

  6-Chloro-5-fluoro-3-methyl-1H-indole-2-carboxylic acid (a-5): 0.93 g, 83% yield. ¹H NMR (500 MHz, DMSO-d₆) δ: 13.09 (s, 1H), 11.59 (s, 1H), 7.65 (d, J＝10.1 Hz, 1H), 7.48 (d, J＝6.4 Hz, 1H), 2.48 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ: 163.06 (s, C-8), 152.66 (d, J＝240, C-5), 150.78 (s, C-7a), 132.31 (s, C-3a), 126.4 (s, C-2), 117.97 (d, J＝5.2, C-3), 117.16 (d, J＝21.8, C-4), 113.23 (s, C-7), 106.35 (d, J＝22.7, C-6), 9.70 (s, C-9); ¹⁹F NMR (470 MHz, DMSO-d₆) δ: －127.19 (dd, J＝10.2, 6.2 Hz); HRMS(EI) calcd C₁₀H₆ClFNO₂ (M－H)^－ 226.0077, found 226.0075.

  3-Methyl-5-(trifluoromethyl)-1H-indole-2-carboxylic acid (a-6): 1.02 g, 85% yield. ¹H NMR (500 MHz, DMSO-d₆) δ: 13.17 (s, 1H), 11.84 (s, 1H), 8.04 (dd, J＝1.9, 1.0 Hz, 1H), 7.63~7.45 (m, 2H), 2.57 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ: 163.21 (s, C-8), 137.34 (s, C-7a), 127.05 (s, C-3a), 126.19 (s, C-2), 125.42(q, J＝272.2 Hz, C-10), 120.62 (q, J＝3.3 Hz, C-5), 120.06 (d, J＝31.1 Hz, C-3), 118.88 (s, C-4), 118.32 (d, J＝4.4, C-6), 113.21 (s, C-7), 9.58 (s, C-9); ¹⁹F NMR (470 MHz, DMSO-d₆) δ: －58.74 (s); HRMS(EI) calcd C₁₁H₇F₃NO₂ (M－H)^－ 242.0434, found 242.0432.

  3-Methyl-7-(trifluoromethyl)-1H-indole-2-carboxylic acid (a-7): 0.99 g, 82% yield. ¹H NMR (500 MHz, DMSO-d₆) δ: 13.22 (s, 1H), 11.18 (s, 1H), 7.97 (dd, J＝8.0, 1.7 Hz, 1 H), 7.62 (dt, J＝7.4, 1.0 Hz, 1H), 7.23 (ddd, J＝8.2, 7.3, 0.8 Hz, 1H), 2.56 (s, 3H); ¹³C NMR (125 MHz, DMSO-d₆) δ: 163.03 (s, C-8), 130.80 (s, C-7a), 129.88 (s, C-3a), 126.44 (s, C-2), 125.20 (s, C-3), 124.20 (q, J＝273.5 Hz, C-10), 122.39 (s, C-4, 5), 118.76 (d, J＝3.6 Hz, C-5), 113.10 (d, J＝32.7 Hz, C-6, 7), 9.63 (s, C-9); ¹⁹F NMR (470 MHz, DMSO-d₆) δ: －59.63 (s); HRMS (EI) calcd C₁₁H₇F₃NO₂ (M－H)^－ 242.0434, found 242.0434.

  4.4   Fermentation and chemical feeding

  4.4.1   Fermentation

  For sporulation, the Streptomyces actuosus strains were cultured on MS agar plates (mannitol, ω＝2.0%; soybean cake meal, ω＝2.0%; agar, ω＝2.0%) at 30 ℃ for 3 d. The S. actuosus spores were inoculated into a 500-mL flask containing 100 mL of primary fermentation medium (sucrose, ω＝2.0%; corn steep liquor, ω＝3.0%; peptone, ω＝0.5%; CaCO₃, ω＝0.5%; pH 7.2~7.6) and incubated at 30 ℃ and 220 r/min for 30 h.

  4.4.2   Chemical feeding

  0.2 mL substituted-MIA (1 mmol/L dissolved in DMSO) was fed to 100 mL fermentation broth of ΔnosL, which would be incubated at 30 ℃ and 220 r/min for 48 h.

  4.5   Compound analysis

  The fermentation broth was centrifuged, and the supernatant was discarded. The mycelia cake was soaked with acetone and sonicated for 30 min. The acetone sample was then centrifuged for high performance liquidchromatography (HPLC) analysis on an Agilent Zorbax column. The column was eluted with solvents A (H₂O+1 mmol/L HCOOH) and B (CH₃CN+1 mmol/L HCOOH) at a flow rate of 1 mL•min^－1 as follows: t＝0 min, φ(B)＝15%; t＝3 min, φ(B)＝15%; t＝6 min, φ(B)＝45%; t＝12 min, φ(B)＝45%; t＝19 min, φ(B)＝55%; t＝22 min, φ(B)＝85%; t＝28 min, φ(B)＝85%; t＝30 min, φ(B)＝15%. UV absorbance was monitored at 330 nm.

  Supporting Information   ¹H NMR and ¹³C NMR spectra of compounds a-1, a-2, a-3, a-4, a-5, a-6, a-7, and UV absorbance and HRMS analysis of substituted-NOS and substituted-NOSint. The Supporting Information is available free of charge via the Internet at http://sioc-journal.cn.

  
  Dedicated to the 40th anniversary of Chinese Journal of Organic Chemistry.
  
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• 

  Figure 1  (A) Biosynthetic gene cluster of NOS and (B) amino acid sequence of NosM

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  Scheme 1  proposed NOS biosynthetic pathway

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  Scheme 2  (A) Chemical synthesis of the substituted-MIA and (B) enzymes involved in NOS biosynthesis pathway to generate NOS analogues or NOSint in NosL mutant, by chemically feeding substituted-MIA

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  Figure 2  HPLC analysis of the fermentation products from the wild-type NOS-producing strain streptomyces actuosus ATCC25421 (Ⅱ), the nosL-deficient mutant strain ΔnosL (Ⅰ) and ΔnosL complemented by chemically feeding substituted-MIA (Ⅲ~X).

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  Scheme 3  Proposed mechanism of NosN

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•