Catechol amide iron chelators produced by a mangrove-derived Bacillus subtilis

Tetrahedron 73 (2017) 5245e5252

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Catechol amide iron chelators produced by a mangrove-derived Bacillus subtilis Jingyan Li, Shaowei Liu, Zhongke Jiang, Chenghang Sun* Institute of Medicinal Biotechnology, Peking Union Medical College and Chinese Academy of Medical Sciences, Beijing 100050, China

a r t i c l e i n f o

a b s t r a c t

Article history: Received 10 March 2017 Received in revised form 30 June 2017 Accepted 4 July 2017 Available online 5 July 2017

A novel triscatecholamide siderophore derivative, linear tribenglthin A (1), together with well known congener, bacillibactin (2) as well as mentioned monomer unit, benglthin were found in the secondary metabolites of a magrove-derived Bacillus subtilis subsp. spizizenii MMB-016. The structure of 1, determined by extensive 1D and 2D NMR studies has an unusual nonproteinogenic amino acid, Z-dehydrobutyrine (Z-Dhb) residue to reveal a putative lantibiotic synthetase function involved in the iterative biosynthesis of non-ribosomal peptide (NRP). Our 1He15N correlation spectroscopy of 2 resulted in a correction of previous NH assignment of bacillibactin, as well as a new evaluation of DPPH free radical scavenging potential suggested antioxidant activity. Benglthin monomer was isolated as a ferrous complex (3), unveiling an interestingly higher affinity for iron Fe(Ⅱ) than trimers, 1 and 2. No dimeric forms were found. © 2017 Elsevier Ltd. All rights reserved.

Keywords: Catecholic siderophore Z-Dhb Ferrous chelate Antioxidant activity

1. Introduction Siderophores are low molecular weight chelators that play an iron transport role in bacteria and fungi.1 People have been studying siderophores for nearly seven decades. From simple molecules of catethol, hydroxamate and acitrate acid to complex cyclic trilactone depsipeptides, all structures have an OO0 donor needed for a bidentate ligation of Fe(Ⅲ).2 Iron is not only essential for almost all crucial life processes such as respiration and DNA synthesis, but its status also strongly implicated in many varieties of diseases related to inflammation such as diabetes, atherosclerosis, neurodegenerative disease, cancer and so on.3 When faced with an iron-deficient conditions, microorganisms always secrete siderophores to sequester iron from around. Catechol-type siderophore is classified by catecholate chelating moiety, most oftenly 2,3-dihydroxybenzoyl(DHB) unit (DHBA). Bacillibactin (BB) and enterobactin (Ent) are two archetypal triscatetholate siderophores first isolated from Gram-positive and

* Corresponding author. E-mail address: [email protected] (C. Sun). http://dx.doi.org/10.1016/j.tet.2017.07.007 0040-4020/© 2017 Elsevier Ltd. All rights reserved.

Gram-negative bacteria respectively.4,5 They both are trisbidentate chelators built on a similar cyclic trilactone scaffold with 2,3-DHB functional groups and differ both in the addition of a glycine (Gly) spacer and methylation of the trilactone ring owning to the threonine (Thr) incorporated in BB.6 They are remarkable for the highest known affinity for iron Fe(Ⅲ) of natural siderophores,7 and BB has been stated as the dominant extracellular ferric ion scavenger of B. subtilis under iron limitation.8 Other Bacilli species can produce, if not only, bacillibactin-type catechol amide siderophores.9 Unlike Ent, BB has not been synthesized successfully yet, due to the symmetrical methylated trilactone ring.7,10 Besides BB, streptobactin/griseobactin11 and paenibactin12 are the only other siderophores with trithreonine lactone reported to date. Although BB was discovered almost twenty years ago6 and the biosynthetic mechanism had been illuminated soon after,4 pity as it until now, to our knowledge, there is no intact structural deduction from the substantial NMR data complemented by other spectroscopic information such as IR absorptions of the compound presented, especially the diagnoses at the two chiral centers of Thr(s) incorporated in the trilactone ring, as well as some flaw was found in the amino NH assignment. The functional study of BB had been almost exclusively focused on its involvement in acquisition of iron from the environment in view of the

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Fig. 1. HPLC-DAD analysis of SPE-fraction of the supernatant of Bacillus subtilis subsp. spizizenii MMB-016 monitored at 314 nm, 1: compound 1; 2: compound 2; 3: benglthin; insert: UV spectra of 1, 2 and 3. Constitutes marked with asterisks are other UV-type compounds.

very high affinity for ferric iron.13,14 During the course of our searching for low-molecular metabolites produced by a marine-derived Bacillus subtilis strain MMB-016, a beige precipitate (2) pumped into our eyes after many times of MeOH washes of one of the carefully collected Sephadex LH-20 fractions (Fr.4). This fraction was chosen mainly by means of HPLC coupled with diode array detector (DAD) and ESI MS detection (LCeDADeESIMS) for desired constituents. A minor constituent (1), immediately preceding 2 on the LC chromatogram, harbours the nearly congruent UV-visible absorptions and the exactly same molecular weight as 2 (Fig. 1, Fig. S1). The structure of 1, as well as 2, were unambiguously elucidated by extensive NMR spectroscopic analyses and a combination of different mass spectrometric measurements, as well as FT-IR microscope transmission detections. Compound 2 is determined to be BB whereas 1 is a new tris-catecholate amide derivative possessing an unusual amino acid residue dehydrobutyrine (ZDhb). The Z-Dhb residue was presumably derived from dehydration of L-Thr. Its occurrence in catecholate siderophore has not been previously reported. Meanwhile, a dark green compound (3) assigned to be a ferrous 2,3-DHBeGlyeThr complex was obtained through the subsequent isolation steps. Although 2,3DHBeGlyeThr monomer, named benglthin this paper, seemed to be produced individually in some origin of Bacillus spp. (with a deprotonated molecular ion at m/z 311.00),15 and many such monomeric catecholic siderophores as vanchrobactin,16 chrysobactin,17 benarthin11 and paenibactin-P112 had been found to accompany both the dimer and trimer production(s), its company only with the trimer(s) (2,3-DHBeGlyeThr)3, as well as the isolated iron-coordination form (3), which unveiled a high affinity for ferrous iron, were not identified before.

Table 1 NMR data for 2 (bacillibactin) (600 MHz for 1H; 150 MHz for 13C; 40 MHz for 15N) in DMSO-d6. Position

dC

1 2 3 4 5 6 7 8a 8b 9 10 11 12 13 N-1 N-2 N-1H N-2H OH (C-2) OH (C-3)

115.9, C 148.5, C 146.2, C 118.9, CH 118.5, CH 118.2, CH 169.3, C 42.6, CH2 169.9, C 56.9, CH 168.5, C 70.8, CH 16.6, CH3

dH (J in Hz)

dN

COSY

6.93, d (7.8) 6.72, t (7.8) 7.33, d (7.8)

5, 6 4, 6 5, 4

4.02, d (14.4) 4.28, dd (5.4, 16.3)

8b, N-1 8a, N-2

4.55, br s

12, N-2

5.30, m 1.18, d (6.0)

10 12 108.0, NH 107.0, NH

9.16, t (5.4) 8.12, br s 11.83, s 9.42, s

HMBC 5 4, 6 4, 5 6 4 6

8a, 8b 13 10, 12 13 12 8b 12

8a, 8b 10

Herein we describe the isolation, structural elucidation, stereochemical assignment of 1 and 2 and present some new evaluations of physiological potentials of BB (2). The isolation and chemical characterization of 3 with possible implication for the significance of monomeric catechol or bidentate catechol amide in understanding endosymbiotic interaction related with iron are also included.

J. Li et al. / Tetrahedron 73 (2017) 5245e5252

2. Results and discussion Strain MMB-016, derived from mangrove silt collected from Zhanjiang, Guangdong province, China, was identified as Bacillus subtilis subsp. spizizenii based on 16S rRNA sequence. While we cultivated it under normal conditions to produce antibiotics, it provided a large amount of 2,3-DHBeGlyeThr-unit siderophores with characteristic UV absorptions corresponding to catechol amide group (lmax 206, 248, 312 nm) under HPLCeDAD chromatography. HPLCeDAD analysis of the ethyl acetate (EtOAc) extract of the culture displayed a predominant peak (2) (data not shown), while after RP-solid phase extraction (RP-SPE), two other catechol amide metabolites (peaks 2, 3) with nearly congruent UV spectra were revealed in the culture (Fig. 1). Constituent 2 showed the same molecular ion at m/z 881 [MH] as 1, and peak 3 presented a molecular ion at m/z 311 [MH] under HPLCeDADeESIMS detections. Fermentation and extraction were carried out, and

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purification of metabolites by sequential Sephadex LH-20, flash C18, and RP-C18 HPLC gave 1 (18.0 mg), 2 (2.1 mg) and 3 (1.2 mg) in the end. 2 was isolated as a white powder. HR-ESIMS showed an [MH] at m/z 881.2480, consistent with a molecular formula of C39H42N6O18. The IR spectrum presented absorptions of ester functionality (1752 and 1202 cm1) which could not be observed in the IR spectrum of 1. 1H and 13C NMR spectra of 2 (data in Table 1) were rather clear considering the assigned molecular formula to indicate a highly symmetrical structure with a monomeric structural formula of C13H14N2O6. The structural connections of CeH, CeC, CeNeC or CeOeC could be made up on basis of 1He1H COSY, 1 He13C HSQC, 1He13C HMBC correlations (partial structure of 2 see Fig. 3a). The macrocycle assignment of the (L-)threonine trilactone was supported by the downfield shift of the b-methine proton of the Thr residue (H-12) as well as the degrees of unsaturation and the IR spectral feature. Unambiguous NHs assignments (8.12 ppm for N-2H and 9.16 ppm for N-1H) of 2 were deduced from the 1He15N HSQC measurement, from which the correlated N resonances (108.0 ppm for N-1 and 107.0 ppm for N-2, respectively) corroborating 2 amide groups incorporated in the monomeric structure were settled (Fig. S11a). Additionally, no other N resonances were revealed in the 1 He15N HMBC NMR spectrum except the correlations of N-2 with H-12 (5.30 ppm) (also very weak correlation with vicinal (2J) H10 at 4.55 ppm) and N-1 with the two vicinal (2J) glycine methylene protons (4.02 and 4.28 ppm repectively). An unobserved small coupling value between a- (H-10) and bmethine proton (H-12) of 2 indicated the approximate 90 dihedral angle(s) as well as ROESY NMR spectrum revealed a correlation between these two protons. This 3J coupling manner matches the corresponding known 1H NMR data of BB.6 Thus the relative stereochemistry of the Thr unit(s) assigned as depicted. Many attempts to crystallize 2 were made to determine the absolute stereochemistry with no success. On the basis of biosynthesis recognition4 that L-Thr (2S, 3R) together with the rare L-allo-Thr (2S, 3S) are the only 2 stereoisomers of substrate threonine both can be activated by the synthetase, L-Thr was finally proposed to be incorporated in 2. This spectroscopic elucidation agreed with the result from aminoacid hydrolysis.6 Compound 1 was obtained as a white, light powder. LCeESIMS/ MS analysis of 1 revealed a parent ion mass at m/z 883.4 [MH]þ or at m/z 881.4 [MH] (Fig. S1b) and a similar fragmentation pattern with that of 2 (Fig. 2 and Fig. S2), suggested primarily a trimer analogue of benglthin. The molecular formula of 1 was determined to be C39H41N6O18 on the basis of ESIHRMS data (m/z [MH] 881.2477), the same as 2. The 1H and 13C NMR spectra of 1 (data in Table 2) were rather complicated compared to 2, and the IR absorptions were quite distinguishable. In detail, the 13C NMR and DEPT spectra of 1 showed signals including 9 distinct carbon resonances corresponding to ester/amide carbonyl carbons (dC 162.77 to 170.93), six groups of 18 aromatic carbon resonances representative of three overlapped benzenoid rings (range from dC 115.41 to 149.23) which slight differences in chemical environments were diagnosed by two groups of obvious “splitted”-two signals with an approximated 2:1 abundance ratio: one is at dC118.0 (dC117.82: dC117.90) and the other is at dC149.2 (dC149.18: dC149.23) (Figs. S17c and d). In addition, one sp2 quaternary carbon signal (dC 127.1) and one sp2 methine carbon signal (dC 134.24) which were both absent in the 13C NMR spectrum of 2 were displayed. Meanwhile, signals for 3 methylene carbons bonded to nitrogen (dC 42.0, 42.1 and 42.3, respectively), 4 aliphatic methine carbons (between dC 55.0 and 71.2) as well as 3 methyl carbons (dC13.82, 16.50 and 16.58) were observed (Fig. S17e). The 1H, 1He13C HSQC and 1He15N HSQC NMR spectra of 1 assigned proton resonances including 6 NHs signals (dH

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Fig. 2. LCe ()ESIMS/MS spectrum of 1.

Table 2 NMR data for 1 (600 MHz for 1H; 150 MHz for Position DHBA-1 1 2 3 4 5 6 7 OH (C-2) OH (C-3) Glycine-1 8 9 N-1H (Z)-Dhb 10 11 12 13 N-2H DHBA-2 10 20 30 40 50 60 70 OH (C-20 ) OH (C-30 )

dC/N 115.4, 149.2, 146.2, 119.0, 118.3, 117.9, 169.1,

13

C; 40 MHz for

15

N) in DMSO-d6.

dH (J in Hz) C C C CH CH CH C

6.92, d (7.8) 6.70, dt (7.5, 3.0) 7.31, d (6.6) 12.22, br 9.35, s

42.3, CH2 167.7, C (N-1) 106.5 127.1, C 162.8, C 134.2, CH 13.8, CH3 (N-2) 116.3 115.4, 149.2, 146.2, 119.0, 118.3, 117.8, 169.7,

C C C CH CH CH C

4.06, m 9.07, br s

6.70, m 1.68, d (7.2) 9.35, s

6.92, d (7.8) 6.70, dt (7.5, 3.0) 7.31, d (6.6) 12.22, br 9.35, s

8.33 to 9.35) in which the sharp singlet at dH 9.35 overlapped one group of 3 OH protons to indicate an a, b-unsaturated amino acid. The other group of 3 overlapped OH protons resonated at dH 12.22. Nine aromatic proton signals presented in two groups of doublets (dH 6.93 and 7.31) and one group of triplet (dH 6.70) were indicative of three overlapped 1,2,3-trisubstituted benzenoid ring systems. An additional olefinic proton resonated in the aromatic triplet at dH 6.70, which was deficient in 2, besides 3 methyl doublets (dH 1.69,

position Glycine-2 80 90 N-10 H Threonine-1 100 110 120 130 N-20 H DHBA-3 100 200 300 400 500 600 700 OH (C-200 ) OH (C-300 ) Glycine-3 800 900 N-100 H Threonine-2 1000 1100 1200 1300 N-200 H

dC/N

dH (J in Hz)

42.0, CH2 169.8, C (N-10 ) 106.5

4.06, m

55.0, CH 168.2, C 71.0, CH 16.5, CH3 (N-20 ) 106.8 115.4, 149.2, 146.2, 119.0, 118.3, 117.8, 169.9,

C C C CH CH CH C

9.07, br t 4.80, d (9.3) 5.38, m 1.17, d (6.0) 8.46, d (9.3)

6.92, d (7.8) 6.70, dt (7.5, 3.0) 7.31, d (6.6) 12.22, br 9.35, s

42.1, CH2 169.4, C (N-100 ) 106.2 55.0, CH 170.9, C 71.2, CH 16.6, CH3 (N-200 ) 108.3

4.06, m 9.02, br t 4.57, d (9.3) 5.34, m 1.06, d (6.6) 8.33, br s

1.17 and 1.06), 3 overlapped methylene units (dH 4.06) and 4 aliphatic methine protons (dH 4.57, 4.80, 5.34 and 5.38) were also displayed. The 1He15N HSQC analysis revealed 6 amide nitrogen actors (dN 106.5 to 116.3), and the remaining 3 oxygen-bonded carbonyl carbons (dC 162.8, 168.2 and 170.9) could be assigned later. Extensive 1He1H COSY, 1He13C HSQC, HMBC and ROESY NMR studies in DMSO-d6 allowed the assignments of the simple substructures Ⅰ/Ⅰ0 /Ⅰ00 eⅢ/Ⅲ0 /Ⅲ00 (shown in Fig. 3) for 1. Partial structures I

J. Li et al. / Tetrahedron 73 (2017) 5245e5252

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Fig. 3. Partial structures a) of 2 and b) of 1 (ⅠeⅢ) with COSY and key HMBC correlations.

(C-1 through C-7),Ⅰ0 (C-10 through C-70 ) and Ⅰ00 (C-100 through C-700 ) were determined to be three 2,3-DHBs overlapped. The 1,2,3trisubstituted aromatic spin systems were clearly confirmed by the COSY correlations of H-5 (50 , 500 ) at dH 6.70 with H-4 (40 , 400 ) at dH 6.92 and H-6 (60 , 600 ) at dH 7.31, respectively. The observation of HMBC correlations of H-6 (60 , 600 ) with carbonyl carbons (C-7, C7'and C-700 ) verified the carbonyl substituents at C-1 (10, 100 ). Partial structures Ⅱ (N-1, C-8, C-9),Ⅱ0 (N-10, C-80 , C-90 ) and Ⅱ00 (N-100, C-800 , C900 ) were readily assigned as glycyl units, from methylene protons H-8 (80 , 800 ) COSY correlations with N-1 (N-10, N-100 ) H and the HMBC correlations to their neighboring vicinal (2J) carbonyl C-9 (C90 , C-900 ). Partial structure Ⅲ (N-2, C-10 through C-13), a peculiar unsaturated amino acid (Z-)Dhb, which distinguished compound 1 from 2, was established by COSY correlation between the b-olefinic proton at dH 6.70 (H-12) and the g-methyl protons at dH1.69 (H-13) in addition to the HMBC cross peaks of H-12 with carbonyl carbon C-11 (dC 162.8) and H-13 with the a-carbon C-10 (dC 127.1), as well as H-13 with the vicinal (2J) b-carbon C-12 (dC 134.2) of (Z-)Dhb (Fig. 3b, Fig. S20). The Z-configuration of Dhb was determined by the ROESY interactions of the a-NH proton at dH 9.35 (N-2H) with gmethyl protons (H-13) due to their proximity (Fig. 4, Fig. S23a). The remaining two partial structures Ⅲ0 (N-20 , C-100 through C-130 ) and Ⅲ00 (N-200 , C-1000 through C-1300 ) were deduced as Thr residues (Thr1 and Thr-2, respectively) from sequential COSY correlations from the secondary methyl group H-130 (H-1300 ) to N-20 (N-200 ) H, and

confirmed by HMBC correlations from H-130 (H-1300 ) to both C-100 (C-1000 ) at dC 55.0 and vicinal (2J) C-120 at dC 71.0 (C-1200 at dC 71.2). The HMBC correlation from threonine a-methine proton H-100 /H1000 to neighboring (2J) C-110 /C-1100 was responsible for the connectivity of a-carbon C-100 /C-1000 to carbonyl C-110 /C-1100. A TOCSY (Fig. S21) experiment corroborated the assignments obtained from the COSY spectrum. The TOCSY correlations unambiguously resolved the amine- and a-proton resonances of each of the 5 common amino acid residues (3 Glys and 2 Thrs) in the structure of 1. HMBC and ROESY experiments of 1 could establish the connectivity and sequence of the amino acids. Connectivity of the DHBA unit (partial structure Ⅰ/Ⅰ0 /Ⅰ00 ) to the Gly unit (partial structure Ⅱ/Ⅱ0 /Ⅱ00 ) on the N-terminus to form substructure Ⅰ/Ⅰ0 /Ⅰ00 -Ⅱ/Ⅱ0 /Ⅱ00 was confirmed by a HMBC correlation from the Gly NH proton (N-10 H/ N-100 H) to the adjacent (2J) DHB carbonyl carbon (C-70 /700 ) (Fig. S20) in addition to the ROESY cross peaks between Gly NH proton and H6/60 /600 , H-8/80 /800 . ROESY correlations between H-8, 80 , 800 and the NH proton(s) in Z-Dhb, Thr-1, -2 (partial structure Ⅲ, Ⅲ0 , Ⅲ00 ) established the amide linkage(s) from Gly unit to Z-Dhb (Thr-1, -2) unit providing substructure(s)Ⅱ (Ⅱ0 , Ⅱ00 ) -Ⅲ (Ⅲ0 , Ⅲ00 ). Two threonine ester linkages corroborated by downfield shifts of b-methine protons of Thr-1 (H-120 at dH 5.38) and Thr-2 (H-1200 at dH 5.34) relative to b-methine proton (at ~ 4.3 ppm) with free threonine hydroxy terminal11 completed the establishment of 1. The deshielded

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Fig. 4. Key ROESY correlations of 1.

character of H-100 (dH 4.80) and the vicinal a-NH proton (dH 8.46) of Thr-1 compared to H-1000 (dH 4.45) and the vicinal a-NH proton (dH 8.33) of Thr-2 were consistent with the ester carbonyl group in Thr1 versus a free terminal carboxylic acid functionality in Thr-2 of 1. This lack of triester-macrocycle backbone resulted in a quite different IR spectrum of 1 from 2 with the deficit of typical IR absorptions of ester functionalities (1752 and 1202 cm1). In this study, tribenglthin A (1), was the only polymeric derivative accompanying BB (2) production. No dimer form of benglthin was detected or isolated, which was unlike the generations of other cyclic triscatecholate siderophores such as streptobactin,11 trichrysobactin17 and paenibactin.12 It's true that the linear trimerictriscatecholate siderophore with a free hydroxyl can be stably detected without the presence of the cyclic trimer in such cases as trivanchrobactin16 and turnerbactin.18 They both are fabricated by serine esters, and the latter has congener(s) containing unusual dehydroalanine (Dha) residue(s). BB was proved to be much more resistant to acid hydrolysis than the triserine counterpart, Ent.10 If there were some hydrolysis and/or elimination of the threonine macrocycle happened, chemical elimination reaction could not conduct to form the unsaturated (Z-)Dhb under the conditions of bacterial growth and subsequent chemical isolation.8,19,20 As one of the signature structural motifs of the well known lantibiotics, Dhb has also been reported as a composition of non-ribosomal peptides (NRPs) most oftenly isolated from cyanobacteria21e25 and marine animals,26e29 with occasions of Streptomycete,30 Bacillus,31 Pseudomonas32 and Halomonas.33 But the mechanism(s) of their formation of Dhb(s) is not known. Previous studies on the biosynthesis of lantibiotics/lantipeptides acknowledged lanthionine biosynthetic enzymes B (LanB) responsible for the dehydration of threonine or serine in such class of lantipeptides as from Bacillus subtilis.34e37 More broadly, homologous LanB-like enzymes may take part in the biosynthesis of many other nonlanthipeptide natural products.38,39 So, to the knowledge of the function of LanB dehydratase and the mechanism of iterative NRP synthesis of BB, we presume that 1 is probably natively released from the terminal Te domain in the NRP-synthetase (NRPS) catalyzing by assumed NRPS-associated small LanB function, which needs further investigation in this bacterium, to form the Z-Dhb residue during the last step of the biosynthesis, while 2, BB, is released by the other

cyclization way leading to the cyclic lactone. Thus, it can be safely assumed that the L-Thr skeletons in 1 have the same stereochemistry as those in 2. The relative stereochemistry of the Thr residues of 1 could be elucidated by analysis of different 1He1H couplings and interpretation of the ROSEY NMR correlations. The ROESY interaction(s) between the a-NH proton(s) and g-methyl proton(s) revealed that a-N atom is syn to g-methyl carbon. No ROESY correlations between a-NH protons and adjacent a-methine protons suggested an anti orentation(s) of amine proton(s) and a-proton(s) H-100 /H-1000 , which was coincident with a large 3J (N-20 H/N-200 H, H100 /H-1000 ) value (Table 2, DMSO-d6). The unobserved coupling between a- and b-methine proton(s) as well as ROESY correlation(s) between them established the gauche orentation(s) of these two protons. Thus the threo relationship of C-100 /C-1000 and C-120 /C1200 was determined. The absolute stereochemistry of threonine is still under investigation. Compound 3 was isolated as an intense green, amorphous powder. IR microsanalysis of 3 revealed absorption bands at 3366.7, 1749.3, 1621.8, 1261.4 and 1064.6 cm1 owning to hydroxyl, amide carbonyl and carboxylic acid functional groups. The ESIMS spectrum showed pseudomolecular ion peaks at m/z 338.35 [MHCOþH]þ, 360.41 [MHCOþNa]þ and 336.54 [MHCOH] indicative of a 1:1 complex of Fe2þ with the monomer of 2,3dihydroxybenzoyleGlyeThr (benglthin), corresponding to a molecular formula of C13H14FeN2O7 for 3 (Fig. S27b). The strong ion peak at m/z 249.05 [Mþ2HFe2þH2OCO2H] in the negative ESIMS spectrum could be attributed to the deprotonated ion of the ligand after neutral loss of H2O and CO2. The iron existence in 3 was confirmed by a couple of 2u less 54Fe isotopic molecular ion peaks of [M(54Fe)HCOþH]þ at m/z 336.69 and [M(54Fe)HCOH] at m/ z 334.17 corresponding to the 56Fe isotopic molecular ion peaks at m/z 338.35 and 336.54, repectively, shown by ESIMS measurement. The abundance ratio of the isotopic peaks shown was approximately 1/10 which was coincident with the natural 54Fe/56Fe isotopic composition. For benglthin, it displayed an ESIMS molecular ion at m/z 311.00 [MH] (m/z 623.15 [2MeH]e) and a congruent 2,3-DHB amide UV absorptions to compound 1 and 2 per se (Fig. 1, Fig. S27a). Coordination of benglthin with Fe(Ⅱ) resulted in a red shift into the visible region around 400 nm due to the charge transfer (CT) transition observed in the UV-vis spectrum (Fig. S26). The source of the iron may be contamination from the glassware used for the culture and isolation procedures not surprisingly. Anyway, the ferrous chelate of benglthin obtained in this experiment, but not that of compound 1 and 2 during the same course, unveils a higher binding constant for ferrous ion of the 2,3DHBeGlyeThr monomer than that of the cyclic and linear trimers. That may be one of the explanations for the prevalence of monomeric 2,3-catecholamide siderophores in host tissue during endosymbiosis and sequentially proved to be responsible for bacterial virulence,18,40 for that when under a low oxygen circumstances when Fe(Ⅱ) form predominates over Fe(Ⅲ) state of iron, such as reducing environment of the cell cytoplasm or anaerobic conditions in the gut, microbe(s) reasonably tend to excrete more efficient ferrous iron chelator for competition for nutritional iron (data in this study are not shown).15 Such a high-affinity siderophore as BB is, it is not required for pathogenicity, though.41,42 Nor does Ent.43 Due to sample limitations, only 1H NMR spectrum of 3, although with very weak resonances, could be measured (data not shown). BB (2) is believed to be the strongest iron chelator with a high specificity for Fe(Ⅲ). In our study, 2 showed (to our knowledge, for the first time) a much stronger antioxidant activity with an IC50 value of 1.8 mM than the positive control ascorbic acid with IC50 value of 27.7 mM in a concentration dependent way in a DPPH free radical scavenging assay. This result suggests physiological roles

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aside from those in iron acquisition siderophores may have. Disappointed poor antiatherosclerotic property of this Fe(Ⅲ)-selective chelator, 2, was realized from a negative result on upregulation of ATP-binding cassette transporter A1 (ABCA1) expression besides no cytotoxicity against HeLa cell line (data not shown). 3. Conclusions A novel triscatechol amide siderophore, linear tribenglthin A (1) as well as the known cyclic BB (2) and the monomer unit, benglthin were discovered from the secondary metabolites of Bacillus subtilis subsp. spizizenii MMB-016. Tribenglthin A has an unusual Z-Dhb amino acid residue which has not been previously reported in catechol-type siderophores. The integration of Z-Dhb in 1 reveals an assumed NRPS-associated LanB-like dehydrase present in the biosynthetic pathway of trilactone depsipeptide. Whether other type of NRPs with this peculiar Z-Dhb amino acid residue could be produced by this bacterium is wondering. The ferrous form of benglthin (3) isolated under our experimental conditions comes up with a much higher affinity for soluble Fe2þ than tribenglthins to hint at the fit of monomeric catechol for playing crucial role in endosymbiotic interaction related with iron. 4. Experimental 4.1. General procedures A solid phase extraction (SPE) using reversed-phase (RP) column (Waters Oasis HLB, 60 mg) was applied on a SUPELCO Solid Phase Extraction Production device before transferring to HPLC analysis. Analytical as well as preparative HPLC were performed on an Agilent 1200 series instrument with a DAD detector, RP C18 columns (Agilent ZORBAX Eclipse XDB, 5 mm, 150 or 250  4.6 mm i.d. for LC-UV detection and 250  10.0 mm i.d. for compound preparation, respectively). IR spectra were recorded on a Thermo Nicolet 5700 Centaurms FT-IR microscope instrument. 1H, 13C and 2D NMR (COSY, HSQC, HMBC, TOCSY, ROESY) measurements were carried out in DMSO-d6 solution on a Bruker AVⅢHD 600 spectrometer, and chemical shifts were referenced to solvent residual signal. 1H-15N HSQC and HMBC spectra were acquired on a Bruker AVANCEⅢ 400 spectrometer. High-resolution ESI-FT-ICR mass spectra were recorded on a 9.4 T Bruker Solarix FTICR mass spectrometer. Low-resolution LC-ESIMS experiments were performed on a Shimadzu LCMS-2020 system with RP C18 column (Agilent ZORBAX Eclipse XDB, 150 or 250  4.6 mm i.d., 5 mm) at a flow rate of 1.0 mL/min. LC-ESIMS/MS data were measured using a Thermo LTQ Orbitrap X coupled to an Agilent 1200 HPLC system. A Yamazen ultra pack ODS-SM-50A column (300  11.0 mm i.d., 50 mm) equipped on a Yamazen smart flash EpCLC AI-580s system and Sephadex LH-20 (Pharmacia, USA) were used for column chromatography. 4.2. Bacterial strain The marine-derived bacterium, strain MMB-016, was isolated from a sea mudsample collected from the mangrove in Zhanjiang, Guangdong province, China and isolated by Difco marine agar 2216 (BD, USA) media using standard dilution-plate method. Genomic DNA was extracted using TIANamp Bacteria DNA Kit (TIANGEN, Beijing, China) and was amplified by PCR using the universal 16S rRNA primers 27F and 1492R with Pfu DNA polymerase (TIANGEN, Beijing, China). By comparing the resulting 16S rRNA sequence with sequences available in the EzTaxon database, strain MMB-016 revealed to have 99% identity to Bacillus subtilis subsp. spizizenii.

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The strain was routinely grown on Mueller Hinton (MH) agar (Oxoid, USA) under standard growth conditions (24e72 h, at 30  C), in an aerobic condition and the storage was kept at 80  C in 20% glycerol (vol/vol) stock solution. 4.3. Fermentation and extraction Strain MMB-016 grown on a slant culture was inoculated into 500 mL Erlenmeyer flasks each containing 100 mL of liquid starch medium (5 g soluble starch, 2.5 g peptone and 0.25 g NaCl) and grown for 72 h at 30  C in an aerobic atmosphere on a rotary shaker at 175 rpm. Difference between the cultures for our primary SPEHPLC-DAD screening and eventual isolation of the targeted constituents was that the former was harvested in 50 mL polypropylene centrifuge tube (Corning, USA). In total, approximately 19 L fermented broth were obtained for further isolation. The culture was applied onto Diaion HP-20 (Mitsubishi Chemical Co.) column after cells were removed by centrifugation (9000 rpm, 15 min at 4  C) followed by subsequent thorough washing with deionized water, and a gradient elution with acetone and H2O from 30:70 to 50:50 and to 80:20 (vol/vol). As for primary screening, a 3 mL aliquot of the culture supernatant was filtered through a SPE column equilibrated with water and MeOH, respectively, by a step gradient elution with aqueous MeOH (30%, 60% and 90%MeOH). Each fraction was applied to RP HPLC analysis with a MeCN-0.05% trifluoroacetic acid (TFA) gradient elution (10:90 to 50:50 in 8 min, 50:50 to 20:80 in 12 min and 100:0 hold for the next 2 min) for capture of constituents with typical 2,3-dihydroxybenzoic acid amide UV absorption pattern. The UV traces were observed at 314 nm and UV spectra (DAD) were recorded between 190 and 800 nm on-line at a flow rate of 1 mL/min. 4.4. Isolation The 50% acetone eluate was lyophilized after the acetone was evaporated. The dried material was dissolved in MeOH for next Sephadex LH-20 purification using MeOH as the eluent, and 5 fractions (Fr.1eFr.5) were collected. Fraction containing mainly the targeted constituents with the characteristic UV absorptions, that is Fr.4, was further purified by flash column chromatography on ODS, eluting with a step gradient of MeOH and 0.1% formic acid (40:60 to 60:40 in 10 min, 60:40 to 80:20 in 40 min and 100:0 hold for 20 min) at a flow rate of 2 mL/min monitoring at 254 nm. Three sub-fractions (Fr.4-1eFr.4-3) were collected. Sub-fractions Fr.4-1 and Fr.4-2 were further purified by RP HPLC (Agilent ZORBAX Eclipse XDB C18, 250  10.0 mm i.d., 2.0 mL/min) with MeCN-0.01% TFA (33:67) as the mobile phase, separately, to afford pure compounds. In the end, tribenglthin A (1, 2.1 mg), BB (2, 18 mg) and benglthin-Fe (Ⅱ) (3, 1.2 mg) were obtained. Tribenglthin A (1) white powder; IRnmax 3394.4, 3187.8, 3010.4, 2920.8, 2849.9, 1647.1, 1468.9, 1420.4, 1324.4, 1300.6, 1245.6, 1214.8, 1119.1 cm1; 1H, 13C and 15N NMR data, see Table 2; ESIMS m/z 883.2 [MþH]þ, m/z 881.2 [MH]; HRESIMS m/z 881.2477 [MH] (calcd for C39H41N2O18, 881.2483). Bacillibactin (2) light beige powder; IRnmax 3276.9, 3077.0, 1751.7, 1674.7, 1590.0, 1545.8, 1459.4, 1384.9, 1342.3, 1266.2, 1202.3, 1139.0, 1066.9, 1021.0, 992.6, 964.5, 923.6 cm1; 1H, 13C and 15N NMR data, see Table 1; ESIMS m/z 883.7 [MþH]þ, m/z 881.5 [MH]; HRESIMS m/z 881.2480 [MH] (calcd for C39H41N2O18, 881.2483). Benglthin-Fe(Ⅱ) (3) dark green powder; IRnmax 3366.7, 2255.1, 1749.3, 1621.8, 1547.8, 1452.4, 1386.0, 1261.4, 1233.7, 1160.6, 1064.6, 1031.0, 854.4, 798.0, 749.0, 670.5, 604.1 cm1; ESIMS m/z 338.4 [MHCOþH]þ, m/z 336.5 [MHCOH].

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4.5. Antioxidant assay The antioxidant activity of BB (2) was tested by 1,1-diphenyl-2picrylhydrazyl (DPPH) free radical scavenging assay.44 Different dilutions (6.25, 12.5, 25, 50 mM final in DMSO) of 2 (100 mL) were added each by 100 mL ethanolic solution of DPPH (Sigma-Aldrich) solution (final concentration 200 mM) and mixed. After reaction in the dark for 30 min, the absorbance was measured at 517 nm using EnVision multilabel plate reader (PerkinElmer). The percent scavenging activity was calculated as: [(A517 of control -A517 of sample)/A517 of control]  100. Ascorbic acid was used as positive control (IC50 ¼ 27.7 mM). 4.6. Antiatherosclerotic assay Compound 2 (BB) was examined for its activity as antiatherosclerotic agent using the upregulators of ABCA1 high-throughput screening assay as described previously.45,46 Acknowledgements The authors are indebted to Prof. Shuyi Si, Drs.Yanni Xu and Xiao Wang for support for atherosclerosis assay and to Dong Dou for aids of fermentation and extraction. We thank Mrs. Juanjuan Han and Prof. Bin Xin for HRMS assistance and Dr. Yanan Wang for NMR performance. This work was supported by the National Natural Science Foundation of China (Nos: 81373308 and 81402834) and the Beijing Natural Science Foundation (No. 7154223). Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.tet.2017.07.007. References 1. 2. 3. 4. 5. 6.

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