University of Groningen
Chemical Modification of Peptide Antibiotics
de Vries, Reinder
DOI:
10.33612/diss.171585325
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Publication date: 2021
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de Vries, R. (2021). Chemical Modification of Peptide Antibiotics. University of Groningen. https://doi.org/10.33612/diss.171585325
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Chapter 6
Characterization of New Antimicrobial NRPs Isolated from
Brevibacillus laterosporus MG64
The discovery and isolation of novel antimicrobial peptides from microorganisms is an important strategy in the search for new treatments in order to overcome antibiotic resistance. In this chapter, the characterization of newly discovered antimicrobial peptides isolated from Brevibacillus laterosporus MG64 via NMR spectroscopy is described. Using 2D NMR spectroscopic techniques the amino acid sequence, as well as important structural features and biosynthetic modifications that were predicted by tandem MS, were confirmed. Elucidation of the exact structures of these peptides, which displayed promising antimicrobial activity profiles, proved to be of crucial importance in the further investigation of their biosynthesis and mechanism of action.
Published as: Z. Li, R. H. de Vries, P. Chakraborty, C. Song, X. Zhao, D.-J. Scheffers, G. Roelfes, O. P. Kuipers, Appl. Environ. Microbiol. 2020, 86, DOI: 10.1128/aem.01981-20.
Z. Li, P. Chakraborty, R. H. de Vries, C. Song, X. Zhao, G. Roelfes, D. J. Scheffers, O. P. Kuipers, Environ. Microbiol. 2020, 22, 5125-5136.
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6.1 Introduction
Antimicrobials are substances that kill or inhibit the growth of microorganisms. They are of great value in different applications. Some of them are being used as agents for plant disease biocontrol,[1] while others are used as antibiotics for preventing and curing bacterial infections in animals, including humans.[2] The discovery of novel antimicrobials is of paramount importance not only for the development of sustainable agriculture but also to overcome antibiotic resistance, which has become one of the biggest threats to human health in recent decades.
Screening of soil microorganisms that harbor novel biosynthetic gene clusters (BGCs) against pathogens is a traditional but efficient way to discover new antimicrobials. In a previous study the rhizosphere bacterium Brevibacillus
laterosporus MG64 was isolated, which displayed potent activity against plant
pathogens and mammalian pathogens.[3] Brevibacillus is a genus of bacteria reclassified from Bacillus based on the 16S rRNA sequence analysis.[4] The species is a rich resource of non-ribosomal peptides (NRPs) that exhibit antimicrobial activity, and many compounds have been isolated and characterized.[5] For instance, gramicidin S, loloatins and tyrocidines were discovered from Brevibacillus brevis,[6–8] while tauramamide, bogorols and laterosporulin were isolated from B. laterosporus.[9–12] Recently, a study was initiated to identify and characterize new antimicrobial NRPs produced by B. laterosporus MG64, which harbors an abundance of novel BGCs.[3]
In this new study, supernatant of B. laterosporus MG64 was subjected to preparative reversed-phase HPLC and the peaks that were collected were screened for antimicrobial activity. The fractions that displayed high activities were isolated and a total of 10 novel antimicrobial peptides belonging to 3 different classes of NRPs were identified by LC-MS/MS (Scheme 6.1). The found amino acid sequences were in
accordance with those predicted from BCGs by antiSMASH (antibiotics & Secondary Metabolite Analysis Shell), a platform capable of identifying biosynthetic gene clusters of secondary metabolite compound classes, including NRPs.[13] Ultimately 4 new members of the bogorol family of NRPs[10,11] were identified, as well as 4 succilins, which are succinylated analogs of these 4 bogorol mutants (Scheme 6.1).[14] Additionally, 2 relacidines, a novel class of cyclic lipopeptides that exhibit high activities against Gram-negative bacteria, were identified (Scheme 6.1).[15] The proposed structures, bioactivity against different kinds of pathogens and the underlying modes of action of the bioactive compounds were further investigated and dissected in order to evaluate their potential in applications.[14,15]
Characterization of New Antimicrobial NRPs Isolated from Brevibacillus laterosporus MG64 121 OH H N O N H O H N O N H HN O H N O N H O H N O NH2 O N H H N O N H O H N NH2 N H O OH H N O O OH O HO O 2-hydroxy-3-methylvaleric acid Dhb1 Succinylation (succilins) Orn3 Vol13 (3) (4) (5) Position
bogorol I and succilin I bogorol J and succilin J bogorol K and succilin K bogorol L and succilin L
(3) Orn Orn Orn Lys (4) Ile Val Ile Ile (5) Val Ile Ile Ile A) N H O H N N H H N N H H N N H H N N H O O O O O O O O OH OH NH NH2 NH2 NH2 NH O NH O N H O HN O N H O O OH 4-methylhexanoic acid Lactone ring
Orn4 Orn5 Orn7
Position relacidine A relacidine B (13) Gly Ala (13) B)
Scheme 6.1: A) Proposed structures of the four bogorols and succilins. B) Proposed structures of the two relacidines.
The newly discovered NRPs have in common that they are of medium length (13 amino acids), contain several cationic ornithine (Orn) residues and D-amino acids, and have an N-terminal lipid tail. Additionally, each class has its own characteristic structural features that are uncommon in peptides and proteins. The bogorols have a dehydrobutyrine (Dhb) resulting from dehydration of a threonine as the N-terminal residue, a 2-hydroxy-3-methylvaleric acid as the fatty acid tail and a reduced valine (valinol, Vol) as its C-terminal residue (Scheme 6.1).[10,11] Introduction of the lipid tail is mediated by activation of the fatty acid by an adenylation domain, while the valinol is the result of a two-step reduction of the C-terminal valine.[14] The succilins share these structural motifs and have an additional succinylation on the ornithine residue at position 3 (Scheme 6.1), which is a novel modification in antimicrobial lipopeptides.[14] The bogorols and succilins display high activities against a variety of Gram-positive and Gram-negative bacteria by forming pores in bacterial cell membranes.
The relacidines consist of a hydrophobic, macrocyclic part that is formed via lactonization of the hydroxyl group of Thr-9 and the C-terminal carboxylic acid, and a linear cationic part that has multiple ornithines and an N-terminal 4-methylhexanoic
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acid fatty acid tail (Scheme 6.1).[15] Relacidines selectively combat Gram-negative bacteria, including human pathogens. In contrast to other cationic lipopeptides, their mechanism of action does not involve membrane disruption, and instead they affect intracellular oxidative phosphorylation, thereby blocking ATP biosynthesis.[15] Significantly, when used in combination with bogorols, which do disrupt membranes, a synergistic antimicrobial effect on different pathogens is observed, suggesting the great potential of these peptides and their producing organism for a broad range of applications.[14]
Initial isolation and identification of the peptides and investigation of their biosynthesis, activity and mechanism of action were carried out in the group of O.P. Kuipers.[14,15] Elucidation of the exact structure of these new antibiotics is crucial for fully understanding their biosynthesis and their mechanism of action, which is an important stepping stone for their clinical application. The aim of the research described in this chapter was to characterize several of the newly discovered bogorols, succilins and relacidines via 1D and 2D NMR spectroscopic techniques in order to confirm their structures as proposed by LC-MS/MS and antiSMASH predictions. The NMR techniques were used to confirm the amino acid sequence, as well as the characterization of important structural features such as modified amino acids. For the bogorols, a Marfey analysis was also performed to investigate the absolute configuration of the different amino acids.
6.2 Results and Discussion
The structures of bogorol K and bogorol L were confirmed with 1H NMR, 1H-1H TOCSY NMR and 1H-1H NOESY NMR spectroscopy. Pure samples of these variants were obtained after preparative reversed-phase HPLC purification and homogeneous samples were prepared in DMSO-d6. 1H-1H TOCSY NMR was used to assign the different spin systems of the amino acids and fatty acid tail, while the amino acid sequence was determined with the assistance of 1H-1H NOESY NMR. The chemical shift assignments are shown in Table S1 and Table S2, respectively. Since bogorols I
and J are very similar mutants (Val4/Ile5 and Ile4/Val5, respectively) they could not be separated and were obtained as a mixture, which made a confident assignment of the signals to the individual peptides via NMR impossible.
Bogorol K was analyzed as a starting point, since it is nearly identical to the previously reported lipopeptide brevibacillin, except that bogorol K has a valine at position 2 instead of a leucine (Figure 6.1A).[16] First, the fatty acid tail of bogorol K was determined to be 2-hydroxy-3-methylvaleric acid. The characteristic signal of the
Characterization of New Antimicrobial NRPs Isolated from Brevibacillus laterosporus MG64
123
α-proton of the fatty acid at 3.79 ppm, the strong correlation with its neighboring –OH signal at 5.61 ppm and the rest of the spin system in the TOCSY NMR were in perfect accordance with those previously reported for brevibacillin.[16] In the NOESY NMR spectrum the α-proton of the acid showed a cross-peak with a signal at 9.15 ppm, which is typical for N-H signals of dehydroamino acids, and the first amino acid in the sequence was determined to be dehydrobutyrine (Dhb) (Figure 6.1B). A NOESY
correlation between the methyl group of Dhb1 (1.78 ppm) and the N-H of the next amino acid in the sequence (7.81 ppm) and a similar correlation of the β-proton of Dhb1 (5.93 ppm) with the N-H of Dhb1 itself (9.15 ppm) confirmed that Dhb1 in bogorol K is in the E-configuration (Figure 6.1B).
With the N-H signal of the next amino acid now known (7.81 ppm), its spin system was examined in the TOCSY NMR spectrum and a distinct signal was observed at 2.11 ppm, which was found to be the β-hydrogen of a valine. This confirmed the Leu2 -> Val2 mutation compared to brevibacillin. The downfield shift of this β-hydrogen compared to typical β-proton signals in valines, which normally appear around 1.90 ppm, is attributed to the electron withdrawing effect of the neighboring Dhb. The next amino acid showed a spin system similar to that of lysine in the TOCSY NMR, however the γ-CH2 appeared at a significantly higher chemical shift (1.55 ppm) compared to that of typical lysines (1.00-1.30 ppm). Moreover, the δ-CH2 signal appeared at 2.76 ppm, a region where normally the ε-CH2 adjacent to the -NH2 of lysine residues is observed. Collectively, these results show that amino acid 3 lacks one methylene compared to lysine and is therefore an ornithine, which is consistent with the LC-MS/MS data and the literature (Figure 6.1A).[16]
Using NOESY NMR the rest of the amino acid sequence was elucidated and the signals of the different amino acids were assigned with the assistance of TOCSY NMR. The sequence of the middle section of the peptide (positions 4-12) was found to be Ile-Ile-Val-Lys-Val-Val-Lys-Tyr-Leu (Figure 6.1A). Finally, the spin system of the
C-terminal amino acid clearly showed a valine carbon skeleton, except for an additional β-carbon signal at 3.34 ppm. This is consistent with the tandem MS data, which showed a double reduction of the C-terminus to a primary alcohol, and therefore amino acid 13 was assigned to be valinol. Collectively, these results and the other assignments are in accordance with the LC-MS/MS data, confirming the proposed structure of bogorol K.
Chapter 6 124 OH H N O N H O H N O N H H2N O H N O N H O H N O NH2 O N H H N O N H O H N NH2 N H O OH H N O O OH Bogorol K 1H-1H TOCSY 1H-1H NOESY OH H N O N H O H N O A) B)
Figure 6.1: A) Structure of bogorol K showing TOCSY (red) and NOESY (blue) correlations used to assign the NMR signals. B) Zoom of NOESY spectrum showing key cross-peaks of the signals of the α-proton of the fatty acid (FA) (3.79 ppm) with the Dhb N-H (9.15 ppm), the Dhb β-proton (5.93 ppm) with the Dhb N-H (9.15 ppm), and the Dhb methyl group (1.78 ppm) with the N-H of Val2 (7.81 ppm).
Bogorol L, which has an additional Orn3 -> Lys3 mutation compared to bogorol K, was analyzed next (Scheme 6.2). It was found that the amino acid in
position 3 in bogorol L indeed has an extra methylene signal compared to the Orn3 found in bogorol K. The γ-CH2 signal appeared at a significantly lower chemical shift (1.28 ppm, Table S2) compared to that of the ornithine in bogorol K (1.55 ppm, Table S1). The signal at 2.70 ppm, which is characteristic for the methylene adjacent to the
Characterization of New Antimicrobial NRPs Isolated from Brevibacillus laterosporus MG64
125
terminal amino group in lysine residues, was assigned to the ε-CH2 instead of the δ-CH2, as was the case with Orn3 in bogorol K. This showed that the amino acid in position 3 was indeed a lysine. The rest of the structural assignments and amino acid sequence (Table S2), including Val2, were in accordance with those found for bogorol
K, confirming the proposed structure of bogorol L.
OH HN O N H O H N O N H O H N O N H O H N O NH2 O N H H N O N H O H N NH2 N H O OH H N O O OH Bogorol L 1H-1H TOCSY 1H-1H NOESY NH2
Scheme 6.2: Structure of bogorol L showing TOCSY (red) and NOESY (blue) correlations used to assign the NMR signals.
A Marfey-type analysis[17] was conducted to determine the absolute configuration of the amino acids in bogorol K and bogorol L (Table S3). Both peptides
showed an L-configuration for all Val, Leu, Ile and valinol residues, and a D-configuration for Tyr in position 11 (Table S3). The Orn in position 3 of bogorol K
was also found to be in the D-configuration, and this is a strong indication that Lys3 in bogorol L is in the D-configuration as well. The other two Lys residues in positions 7 and 10 of both compounds displayed either D- or L-configuration, and these could not be distinguished from each another using this method. However, the results obtained are consistent with Lys7 being in the D-configuration, since epimerization domains were predicted at the 3rd, 7th and 11th modules using antiSMASH.
Another group of peptides with masses 100 Da higher than the bogorols were eluted from reversed-phase HPLC in the same order as bogorols I-L, but with longer elution times. Further analysis by LC-MS/MS revealed that these peptides are likely products of succinylation of the amine residues at Orn3 or Lys3 in bogorols I-L, and the peptides were therefore named succilins I-L, respectively (Scheme 6.1, Figure 6.2A). In the NMR analysis, the succinylation at Orn3 in succilin K was supported by
the appearance of 2 methylene signals at 2.28 and 2.36 ppm (Table S4) which shared
a strong correlation in the 1H-1H TOCSY NMR (Figure 6.2B). Both signals showed NOESY cross-peaks with an amide N-H signal at 7.79 ppm (Figure 6.3), which in turn
showed a correlation with the signal of the δ-CH2 of Orn3 at 2.98 ppm in TOCSY NMR
(Figure 6.2B). The downfield shift of this δ-CH2 signal from 2.76 (in bogorol K) to 2.98
Chapter 6
126
electron-withdrawing amide (Table S4). These results confirm the attachment of a
succinyl group at the end of the side chain of Orn3. The other structural assignments of the NMR signals of succilin K were found to be in agreement with those observed for bogorol K, confirming that the proposed structure of succilin K is correct.
OH H N O N H O H N O N H HN O H N O N H O H N O NH2 O N H H N O N H O H N NH2 N H O OH H N O O OH Succilin K 1H-1H TOCSY 1H-1H NOESY O O OH H N N H HN O O O OH O A) B)
Figure 6.2: A) Structure of succilin K showing TOCSY (red) and NOESY (blue) correlations used to assign the NMR signals. B) Zoom of the TOCSY spectrum showing the key correlations between the two methylenes in the succinyl group (Suc) (2.28 and 2.36 ppm) and between the δ-CH2 signal (2.98 ppm) of Orn3 and the amide N-H of the
Characterization of New Antimicrobial NRPs Isolated from Brevibacillus laterosporus MG64 127 H N N H HN O O O OH O
Figure 6.3: Zoom of the NOESY spectrum showing key correlations between both methylenes in the succinyl group (2.28 and 2.36 ppm) and the amide N-H of the succinyl group (7.79 ppm).
Finally, the structure of the relacidines was investigated. After preparative HPLC purification a pure sample of relacidine B could be obtained, which was analyzed by 1H, 1H-1H-COSY, 1H-1H-TOCSY, 1H-1H-NOESY and 1H-13C-HSQC NMR. Using these techniques, the complete amino acid sequence of relacidine B was elucidated (Figure 6.4A) and all proton and carbon signals were assigned (Table S5). The structures of
the relacidines proposed based the LC-MS/MS data closely resemble those of brevicidine and laterocidine, cationic lipopeptides also produced by B. laterosporus.[18] While the amino acid sequences of the relacidines are more similar to that of laterocidine, the fatty acid chain was proposed to be 4-methylhexanoic acid, which is the same as in brevicidine.[18]
In the NMR analysis of relacidine B, the structure of the lipid tail was investigated first. A signal at 2.11 ppm was identified as the α-proton of the fatty acid, and from there the carbon skeleton of the lipid side chain was assigned using COSY, TOCSY, NOESY and HSQC NMR (Figure 6.4A). The α-proton showed intense cross-peaks with 2
signals at 1.50 and 1.27 ppm in the COSY NMR spectrum. Since the signal of the α-protons integrated for 2 protons, branching at the α-carbon was not possible. Therefore, it was concluded that the signals at 1.50/1.27 ppm belonged to the β-protons, which are diastereotopic due to branching at the γ-carbon. The HSQC data showed that these signals indeed belong to the same carbon, supporting this
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hypothesis. This branching at the γ-carbon, together with the COSY and TOCSY correlations found for the rest of the lipid tail confirmed that the fatty acid is 4-methylhexanoic acid, which is consistent with the LC-MS/MS data.
N H O H N N H H N N H H N N H H N N H O O O O O O O O OH OH NH NH2 NH2 NH2 NH O NH O N H O HN O N H O O OH 1H-1H TOCSY 1H-1H NOESY Relacidine B N H O NH O N H O HN O N H O O OH O A) B)
Figure 6.4: A) Structure of relacidine B showing TOCSY (red) and NOESY (blue) correlations used to assign the NMR signals. B) Zoom of the NOESY spectrum (bottom) showing the key correlations between the methyl protons of Ala13 (1.41 ppm) and the β-protons of Thr9 (5.00 ppm), and between the α-proton of Ala13 (4.39 ppm) and the γ-methyl protons of Thr9 (0.98 ppm).
Characterization of New Antimicrobial NRPs Isolated from Brevibacillus laterosporus MG64
129
The α-proton of the N-terminal lipid also showed a NOESY cross-peak with the N-H of the first amino acid in the sequence (7.95 ppm), which was shown to be a serine by TOCSY NMR. From there, the sequence of the linear middle part of the peptide was found to be Ser-Tyr-Trp-Orn-Orn-Gly-Orn-Trp by NOESY NMR. The found sequence was in accordance with that of laterocidine and our LC-MS/MS data. The C-terminal part of the peptide was predicted to be a lactone macrocycle, since b- and
y-ions could not be observed beyond Trp8 in LC-MS/MS, unless the lactone was first
hydrolyzed using strong aqueous base.[15] After hydrolysis, the sequence of the C-terminal fragment could be determined using tandem MS and was found to be Thr-Ile-Gly-Ser-Ala.[15] TOCSY and NOESY NMR were used to confirm this sequence and the presence of the lactone ring in the natural product.
From Trp8, the next amino acid was indeed found to be a Thr using NOESY and TOCSY NMR. A NOESY correlation was observed between the α-proton of the Thr (4.64 ppm) and the N-H of the next amino acid (7.73), which turned out the be an Ile. In a similar fashion, the rest of the amino acid sequence of the macrocycle was found to be consistent with the LC-MS/MS data. Significantly, NOESY correlations were also observed between the methyl protons of Ala13 (1.41 ppm) and the β-protons of Thr9 (5.00 ppm), and the α-proton of Ala13 (4.39 ppm) and the γ-methyl protons of Thr9 (0.98 ppm) (Figure 6.4B). These key cross-peaks, together with the LC-MS/MS data,
verified the presence of a macrolactone formed between the alcohol residue of Thr9 and the C-terminal carboxylic acid of Ala13.
Collectively, these results show the characterization of newly discovered antimicrobial peptides belonging to the bogorol, succilin and relacidine classes of NRPs via NMR spectroscopy. The amino acid sequences proposed based on tandem MS data and antiSMASH predictions were confirmed in all cases. Moreover, important structural features, such as a novel succinylation modification in succilins and a macrolactone moiety in relacidines, were identified.
6.3 Conclusion
NMR spectroscopy has proven to be a powerful tool for the complete characterization of newly discovered bogorols, succilins and relacidines isolated from B. laterosporus MG64. Using 2D NMR techniques, their amino acid sequences were established and important structural features and biosynthetic modifications were characterized successfully. Detailed knowledge of the structure of these compounds proved to be valuable for understanding their biosynthesis and mechanism of action, which is
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crucial for the clinical application of these promising antibiotic candidates in the future.
6.4 Acknowledgements
We would like to acknowledge Zhibo Li and Oscar P. Kuipers for the pleasant collaboration on this challenging project. The other co-authors are also thanked for their contribution in making the study to a success, and their input in the writing of the two resulting papers. Johan Kemmink and Pieter van der Meulen are acknowledged for their technical support with the NMR experiments and help with the analysis of the 2D NMR data.
6.5 Experimental
Extraction and purification of antimicrobial compounds from B. laterosporus MG64
A fresh colony of B. laterosporus MG64 was inoculated in 3 mL Lennox broth (LB) and incubated at 28 ˚C overnight with shaking. The overnight culture was then diluted with fresh LB to an OD600 of 1.0 as inoculum. 1 mL of inoculum was inoculated into 100 mL of fresh LB broth and
incubated at 28 ˚C with shaking of 220 rpm for 24 h. After centrifugation at 10,000 × g for 10 min, the supernatant was collected and applied to a column filled with 10 g C18 silica gel (Sigma-Aldrich, 97727-U). After washing with 20 mL 20 % acetonitrile (aq.) with 0.1 % trifluoroacetic acid (TFA), the crude extract was eluted with 20 mL 95 % acetonitrile (aq.) with 0.1 % TFA. The crude extract was lyophilized and re-dissolved in Milli-Q water. After filtering over a 0.45 µm cellulose acetate membrane, an aliquot of crude extract was applied to high-performance liquid chromatography (HPLC) equipped with an analytical Phenomenex C18 column (3.6 µm particle size, 250 × 4.6 mm) for purification. A linear gradient of water with 0.1 % TFA (solvent A) and acetonitrile with 0.1 % TFA (solvent B) was used to separate the compounds. In each run, solvent B was linearly increased from 15 % to 60 % over 45 min, using a flow rate of 0.5 mL/min. The effluent was monitored with a UV-detector at a wavelength of 214 nm. Every single peak was collected and lyophilized before testing activity against
Xanthomonas campestris pv. campestris (Xcc), after which the active compounds were analyzed
by tandem MS.
NMR sample preparation
Prior to NMR analysis, the active peptides were purified by RP-HPLC as described above and the pure fractions were pooled and lyophilized. The peptides (0.5 -1 mg) were dissolved in 0.5 mL DMSO-d6 and 1H NMR, 1H-1H-COSY NMR, 1H-1H-TOCSY NMR, 1H-1H-NOESY NMR and 1H-13C-HSQC NMR spectra[15] were recorded on a Brüker Ascend 600 MHz spectrometer.
Characterization of New Antimicrobial NRPs Isolated from Brevibacillus laterosporus MG64
131 Chemical shifts in NMR spectra were internally referenced to solvent signals (DMSO-d6 at δH =
2.50 ppm, δC = 39.51 ppm). Marfey analysis[17] 6M HCl (aq) H2N R1 O OH H 2N R2 O OH H N F O2N NO2 NH2 O H N HN O2N NO2 NH2 O OH O R1 H N HN O2N NO2 NH2 O OH O R2
Separable by UPLC-MS (UV @ 340 nm)
Marfey's Reagent 160 °C MW N H R1 O H N N H O R2
Peptide L-amino acid D-amino acid
The peptides (0.5-1 mg) were dissolved in 300 μL 6M HCl (aq.) and heated under microwave irradiation at 160 °C (50 W) for 10 minutes. The mixture was concentrated in vacuo and redissolved in 100 μL 1M NaHCO3 (aq.). 12 μL 1 % (w/v) Marfey’s reagent
(1-fluoro-2,4-dinitrophenyl-5-L-alanine amide (FDAA)) in acetone was added and the mixture was shaken for 1 hour at 40 °C. The mixture was acidified using 60 μL 2M HCl (aq.) and supplemented with 800 μL MeOH. The clear yellow solution was analyzed directly by UPLC-MS (Aqcuity UPLC HSS T3 1.8 μm 2.1x150 mm column, solvent A: 0.1 % FA in ddH2O, solvent B: 0.1
% FA in ACN, gradient: 80 % A for 1 minute, then to 40 % A over 15 minutes, total runtime: 20 min., flow: 0.3 mL/min., UV 340 nm). D,L configurations were determined by comparing retention times of the mixtures with retention times of Marfey-derivatized amino acids of known configuration. Marfey-derivatized L-Phe (not present in any of the peptides) was used as an internal standard.
Chapter 6 132
Supplementary data
OH H N O N H O H N O N H H2N O H N O N H O H N O NH2 O N H H N O N H O H N NH2 N H O OH H N O O OH Bogorol K 1H-1H TOCSY 1H-1H NOESY Residue NH 1Hα 1Hβ Others FA 3.79 OHβ1, 5.61; CHβ2, 1.71 CH 2γ1, 1.38/1.12; CH3γ2, 0.88; CH3δ1, 0.80 Dhb-1 9.15 5.93 CH3γ1, 1.78 Val-2 7.81 4.24 2.11 CH3γ, 0.87 Orn-3 8.08 4.42 1.68 CH2γ, 1.55; CH2δ, 2.76; NH2ε, 7.63 Ile-4 7.85 4.28 1.70 CH2γ1, 1.35/1.02* Ile-5 8.00 4.17 1.69 CH2γ1,1.43/1.04 Val-6 7.81 4.18 1.93 Lys-7 7.93 4.38 1.63 CH2γ, 1.26; CH2δ, 1.48; CH2ε, 2.70; NH2ζ,7.64 Val-8 7.86 4.22 1.91 Val-9 7.88 4.11 1.94 Lys-10 7.78 4.24 1.43/1.32* CH2γ, 1.05; CH2δ, 1.42; CH2ε, 2.65; NH2ζ, 7.65 Tyr-11 8.09 4.47 2.84/2.60 CHδ, 6.99; CHε, 6.62 Leu-12 8.08 4.24 1.38 CH3δ1, 0.82; CH3δ2, 0.76* Valinol-13 7.40 3.53 CH2β1, 3.34; CHβ2, 1.80 CH3γ, 0.81Table S1. Partial chemical shift (ppm) assignments of bogorol K (DMSO-d6). *Tentative assignment due to overlap, but in accordance with the literature.[16]
Characterization of New Antimicrobial NRPs Isolated from Brevibacillus laterosporus MG64 133 OH H N O N H O H N O N H O H N O N H O H N O NH2 O N H H N O N H O H N NH2 N H O OH H N O O OH Bogorol L 1H-1H TOCSY 1H-1H NOESY NH2
Table S2. Partial chemical shift (ppm) assignments of bogorol L (DMSO-d6). *Tentative assignment due to overlap, but in accordance with the literature.[16]
Residue NH 1Hα 1Hβ Others FA 3.79 OHβ1, 5.61*; CHβ2, 1.72 CH2γ1, 1.38/1.13; CH3γ2, 0.88; CH3δ1, 0.80 Dhb-1 9.11 5.97 CH3γ1, 1.79 Val-2 7.79 4.24 2.11 CH3γ, 0.87 Lys-3 8.08 4.34 1.63/1.55 CH2γ, 1.28; CH2δ, 1.48; CH2ε, 2.70; Ile-4 7.79 4.24 1.70 Ile-5 7.99 4.17 1.71 CH2γ1,1.41/1.05 Val-6 7.78 4.17 1.94 Lys-7 7.94 4.37 1.63 CH2γ, 1.27; CH2δ, 1.48; CH2ε, 2.70; Val-8 7.86 4.21 1.93 Val-9 7.88 4.11 1.94 Lys-10 7.78 4.24 1.42/1.31* CH2γ, 1.05; CH2δ, 1.42; CH2ε, 2.65; Tyr-11 8.09 4.47 2.84/2.61 CHδ, 6.99; CHε, 6.62 Leu-12 8.09 4.24 1.39 CHγ, 1.35*; CH 3δ1, 0.81; CH3δ2, 0.76 Valinol-13 7.40 3.54 CH2β1, 3.34; CHβ2, 1.81 CH3γ, 0.81
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Table S3. LC-MS results of Marfey analysis of bogorol K and L. Retention times from UV
chromatogram (340 nm).
Standard Retention Time (min.) bogorol K bogorol L
L-Val 10.30 10.25 10.21 D-Val 12.05 - - L-Leu 12.06 12.02 11.98 D-Leu 13.76 - - L-Ile 11.76 11.71 11.68 D-Ile 13.60 - - L-Phe (standard) 11.98 11.93 11.89 L-Tyr 6.59 / 9.14 - - D-Tyr 6.58 / 9.77 6.44 / 9.69 6.41 / 9.65 L-Orn 4.55 / 4.91 - - D-Orn 4.04 / 4.89 3.80 / 4.69 - L-Lys 5.14 / 5.61 4.84 / 5.39 4.85 / 5.38 D-Lys 5.12 / 4.78 4.78 / 4.50 4.79 / 4.51 L-(S)Vol 9.94 9.87 9.84 D-(R)Vol 12.05 - -
Characterization of New Antimicrobial NRPs Isolated from Brevibacillus laterosporus MG64 135 OH H N O N H O H N O N H HN O H N O N H O H N O NH2 O N H H N O N H O H N NH2 N H O OH H N O O OH Succilin K 1H-1H TOCSY 1H-1H NOESY O O OH
Table S4. Partial chemical shift (ppm) assignments of succilin K (DMSO-d6). *Tentative assignment due to overlap, but in accordance with the literature.[16]
Residue NH 1Hα 1Hβ Others FA 3.77 OHβ1, 5.61; CHβ2, 1.72 CH2γ1, 1.37/1.12; CH3γ2, 0.88; CH3δ1, 0.79 Dhb-1 9.08 5.99 CH3γ1, 1.79 Val-2 7.76 4.25 2.10 CH3γ, 0.86 Orn-3 (Suc) 8.12 4.31 1.63/1.52 CH2γ, 1.41/1.33; CH2δ, 2.98; NHε, 7.79; CH2suc1, 2.28; CH2suc2, 2.36 Ile-4 7.78 4.21 1.71 Ile-5 8.01 4.16 1.72 CH2γ1,1.43/1.05 Val-6 7.84 4.16 1.94 Lys-7 8.01 4.35 1.63 CH2γ, 1.27; CH2δ, 1.48; CH2ε, 2.70; Val-8 7.89 4.20 1.93 Val-9 7.89 4.10 1.94 Lys-10 7.82 4.24 1.41/1.32* CH2γ, 1.04; CH2δ, 1.41; CH2ε, 2.65; Tyr-11 8.11 4.47 2.84/2.61 CHδ, 6.99; CHε, 6.62 Leu-12 8.10 4.24 1.39 CHγ, 1.35*; CH 3δ1, 0.82; CH3δ2, 0.76 Valinol-13 7.40 3.53 CH2β1, 3.34; CHβ2, 1.80 CH3γ1, 0.82; CH3γ2, 0.80
Chapter 6 136 N H O H N N H H N N H H N N H H N N H O O O O O O O O OH OH NH NH2 NH2 NH2 NH O NH O N H O HN O N H O O OH F-1 F-2 F-3 F-4 F-5 F-6 1 2 3 4 5 5 6 6 7 8 9 11 12 13 14 10 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 35 34 33 32 3637 38 39 40 41 42 43 44 45 46 47 48 relacidine B
Amino acid Position δ 13C NMR δ 1H NMR Amino acid Position δ 13C NMR δ 1H NMR
Ser1 1-NH 7.95 Trp8 28-NH 8.28 1 54.6 4.29 28 52.9 4.68 2 61.4 3.50 29 27.1 3.11 / 2.96 Tyr2 3-NH 8.04 30 123.8 7.11 3 54.5 4.32 31 10.84 4 35.9 2.81 / 2.64 32 111.0 7.31 5 129.8 6.88 33 120.6 7.04 6 114.6 6.56 34 118.0 6.96 Trp3 7-NH 8.14 35 118.2 7.57 7 53.3 4.51 Thr9 36-NH 7.98 8 27.1 3.14 / 2.93 36 53.2 4.64 9 123.6 7.14 37 69.4 5.00 10 10.81 38 14.1 0.98 11 111.0 7.31 Ile10 39-NH 7.73 12 120.6 7.04 39 56.5 4.15 13 118.0 6.96 40 35.4 1.64 14 118.2 7.57 41 24.7 1.53 / 1.10 Orn4 15-NH 8.04 42 10.5 0.86 15 51.5 4.32 43 14.8 0.85 16 29.0 1.72 / 1.57 Gly11 44-NH 9.20 17 24.4 1.51 44 43.5 3.90 / 3.35 18 38.6 2.73 Ser12 45-NH 8.47 Orn5 19-NH 8.23 45 56.3 4.10 19 51.6 4.34 46 60.4 3.80 / 3.68 20 29.0 1.72 / 1.58 Ala13 47-NH 7.74 21 24.4 1.50 47 48.0 4.39 22 38.6 2.69 48 16.9 1.41 Gly6 23-NH 8.32 FA F-1 32.6 2.11 23 41.7 3.77 / 3.72 F-2 31.5 1.50 / 1.27 Orn7 24-NH 8.05 F-3 33.3 1.26 24 51.5 4.32 F-4 28.4 1.28 / 1.09 25 29.2 1.42 / 1.29 F-5 10.9 0.81 26 24.4 1.27 F-6 18.6 0.81 27 overlap 2.51
Table S5. 1H and 13C chemical shift (ppm) assignments of relacidine B (DMSO-d 6).
Characterization of New Antimicrobial NRPs Isolated from Brevibacillus laterosporus MG64
137
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Chapter 6
