Structure Revision of Microginins 674 and 690 from the Cultured

Note Cite This: J. Nat. Prod. XXXX, XXX, XXX−XXX

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Structure Revision of Microginins 674 and 690 from the Cultured Cyanobacterium Microcystis aeruginosa Kevin Calabro,† Greǵ ory Genta-Jouve,*,‡ and Olivier P. Thomas*,†,§ †

Université Côte d’Azur, CNRS, OCA, IRD, Géoazur, 250 Rue Albert Einstein, 06560 Valbonne, France C-TAC UMR-CNRS 8638 COMETE, Université Paris Descartes, 4 Avenue de l’Observatoire, 75270 Paris Cedex 06, France § Marine Biodiscovery, School of Chemistry and Ryan Institute, National University of Ireland Galway (NUI Galway), University Road, H91 TK33 Galway, Ireland Downloaded via UNIV OF CAMBRIDGE on March 20, 2019 at 14:53:03 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: The structures of the commercially available microginins 674 and 690 isolated from a cultured strain of Microcystis aeruginosa and only recently characterized have been revisited. Using NMR and HRMS/MS data, an inversion of two amino acids, N-methylmethionine and tyrosine, in the structure of these metabolites is unambiguously demonstrated. These results highlight the importance of careful examination of spectroscopic data for the proposition of structures of natural products, especially when they are of commercial value.

T

he cyanobacterium Microcystis aeruginosa is mainly known for the production of hepatotoxic microcystins responsible for severe intoxications.1−4 Other bioactive modified peptides such as the protease inhibitors cyanopeptolins and microginins have also been reported from strains of this species.5−9 Microginins are characterized by the presence of three to six amino acid residues starting with the rare 3amino-2-hydroxydecanoic acid (Ahda).10,11 To date, among the 60 microginin analogues, 23 have been fully characterized by NMR spectroscopy.12−15 During a chemical screening performed on a cultured strain of M. aeruginosa (SAG 14.85), the analysis of the UHPLCHRMS data revealed some microginins with characteristic protonated molecules at m/z 675 and 691. These MS data could correspond to the tetrapeptidic microginins 674 and 690 containing the sequence Adha-(N-Me)Met-Tyr-Tyr as shown in the catalogue of companies selling these metabolites such as LKT Laboratories, Biorbyt, or MARBIONC.16−18 Recently, Stewart et al. reported the first spectroscopic data supporting the structures displayed on these Web sites.19 The structure of microginins 674 and 690 was deduced from low-resolution MS/MS fragmentation but also NMR spectroscopy, while the absolute configuration was assessed using Marfey analysis and comparison of NMR data for the Adha amino acid. A deep analysis of the NMR and high-resolution MS/MS data of the metabolites 1 and 2 isolated from our strain proved that the structures of microginins 674 and 690 proposed by the companies and published recently by Stewart et al. are erroneous and correspond to the following sequence: AdhaTyr-(N-Me)Met-Tyr (Figure 1). The structure elucidation of © XXXX American Chemical Society and American Society of Pharmacognosy

Figure 1. Proposed and revised structures of microginins 674 and 690.

microginins has usually been hampered by the presence of additional signals in the NMR spectra of these molecules. The presence of rotamers around the central and tertiary amide was then confirmed applying variable-temperature NMR experiments (VT-NMR). A 12 L culture of M. aeruginosa (SAG-14.85) was centrifuged, and the pellet lyophilized. The dry biomass was then extracted three times with MeOH under sonication. The extract was fractionated by reversed-phase vacuum liquid chromatography, and the hydromethanolic fraction (H2O/ MeOH, v/v, 3:2 and 1:1, combined) was purified by reversedphase HPLC to yield 1 (3.2 mg) and 2 (1.5 mg). Compound 1 was isolated as a yellowish oily solid, and its molecular formula of C34H50N4O8S was inferred from HRESIMS data of a protonated molecule at m/z 675.3432 [M + H]+. The 1H, 13C, and HSQC NMR spectra of 1 did Received: January 6, 2019

A

DOI: 10.1021/acs.jnatprod.9b00015 J. Nat. Prod. XXXX, XXX, XXX−XXX

Journal of Natural Products

Note

further supported by MS/MS fragmentation with a key fragment at m/z 494.2688 (Figure 2). First, we were confident that the structure Ahda-(NMe)Met-Tyr-Tyr displayed on the Web sites of the three companies supplying microginin 674 did correspond to the structure of 1.16−18 However, when trying to confirm the identity of 1 by comparison of its HRMS/MS spectrum (Figure 2) with the predicted spectrum of the proposed sequence, a key fragment at m/z 349.2122 (b2) in the MS/MS spectrum of 1 was absent in the predicted spectrum of the proposed microginin 674 (Figure 3A). A closer inspection of the HMBC spectrum of 1 then revealed key H-N(Tyr1)/C-1 and CH3-N(Met)/C-11 correlations that were consistent with a Tyr and not a methionine residue next to the N-terminus Ahda. A better agreement between the MS/MS spectra of 1 and the predicted spectrum of the revised sequence Ahda-Tyr-(N-Me)Met-Tyr came as a first confirmation of this assumption (Figure 3B). In this case the key fragment at m/z 349.2122 (b2) was present and represented the Ahda-Tyr fragment. We then wondered whether 1 could just be an isomer of the proposed structure of microginin 674 and not the same compound even if they were isolated from the same cyanobacterium species. The absence of clear references and spectroscopic data from the suppliers prevented us from addressing this question. Gratifyingly, Stewart et al. recently provided some useful spectroscopic data on microginin 674, presented with the same proposed structure as by the suppliers.19 With all fragments of the low-resolution MS/MS spectrum of microginin 674 being in perfect agreement with those of the high-resolution MS/MS spectrum of 1, we could therefore conclude that 1 is the revised structure of microginin 674 proposed by the suppliers and Stewart et al. (Figure 4). While the authors used a fragment at m/z 343 to propose the Tyr-Tyr terminal dipeptide, we were not able to observe this fragment in both sets of MS/MS data. The NMR data of microginin 674 performed in DMSO-d6 were also in very good agreement with those of 1, therefore confirming the same relative configurations for both compounds. Finally, the specific rotations of 1 and microginin 674 have the same sign and order of magnitude; therefore we can conclude on the same absolute configuration for these molecules.19 At room temperature, most 1H NMR signals of 1 were duplicated, indicating the possible presence of rotamers. We also wanted to clarify this observation, as to our knowledge there are no clear mentions of this phenomenon. Acquisition of the 1H NMR spectrum of 1 in MeOH-d4 revealed a 2:1 ratio of the two rotamers 1a/1b, which changed to 5:4 in DMSO-d6, suggesting the occurrence of rotamers. Complete assignment of spectroscopic data of the two isomers was made after analysis of 1D, NOESY, HSQC, and HMBC NMR spectra in DMSO-d6 (Table 1). Compound 1 was then subjected to VTNMR experiments to observe the coalescence. The temperature of the NMR analysis was gradually increased from 293 K to a final temperature of 405 K. A 1H NMR spectrum was recorded for each selected temperature (Figure 5). Analysis of VT-NMR data evidenced the presence of two rotamers with a coalescence reached at 376.8 K (Tc). Below Tc the slow exchange leads to a duplication of the signals, while above Tc the fast exchange induces the observation of the four aromatic doublets of the two tyrosines.

confirm the presence of a peptide with characteristic signals of α-amino acid methines at δH 4.30 (dd, 3J = 8.5, 5.1 Hz, 1H, H27), 4.98 (dd, 3J = 9.4, 5.7 Hz, 1H, H-21), and 4.72 (dd, 3J = 8.2, 5.3 Hz, 1H, H-12) 1J coupled to δC 54.2 (C-27), 55.7 (C21), and 50.8 (C-12) (Table 1). Inspection of MS/MS, Table 1. 1H (500 MHz) and 13C (125 MHz) NMR Spectroscopic Data for Both Rotamers of 1 in DMSO-d6 1a (major) residue Ahda

Tyr1

(N-Me) Met

Tyr2

position

δC, type

1 2 3 4 5 6 7 8 9 10 NH2 11 12

171.1, C 70.3, CH 53.4, CH 28.9, CH2 23.7, CH2 28.8, CH2 28.6, CH2 31.2, CH2 22.2, CH2 14.1, CH3 172.2, C 50.8, CH

13a 13b 14 15/19 16/18 17 NH OH 20

35.9, CH2

21

55.7, CH

22a 22b 23 24 25 26 27

27.9, CH2

28a 28b 29 30/34 31/33 32 NH OH

127.4, C 130.0, CH 114.9, CH 156.0, C

169.8, C

29.8, CH2 14.7, CH3 30.7, CH3 172.2, C 54.2, CH 35.7, CH2 127.2 C 130.1, CH 115.2, CH 156.0, C

1b (minor)

δH, mult. (J in Hz) 3.94, 2.99, 1.38, 1.18, 1.20, 1.22, 1.23, 1.26, 0.86,

d (5.4) m m m m m m m m

δC, type 171.6, C 70.0, CH 53.2, CH 28.9, CH2 23.7, CH2 28.8, CH2 28.6, CH2 31.2, CH2 22.2, CH2 14.1, CH3

171.5, C 4.72, dd (8.2, 49.5, CH 5.3) 2.85, m 36.7, CH2 2.78, m 127.2, C 7.01, m 130.2, CH 6.63, m 115.1, CH 155.9, C 8.18, d (7.6) 9.20, br s 168.1, C 4.98, dd (9.4, 5.7) 2.04, m 1.69, m 2.25, t (7.6) 2.00, s 2.65, s

58.4, CH 27.7, CH2

29.7, CH2 14.7, CH3 29.0, CH3 173.4, C 4.30, dd (8.5, 55.5, CH 5.1) 2.98, m 36.7, CH2 2.81, m 128.7, C 6.98, m 130.4, CH 6.63, m 114.8, CH 155.6, C 7.80, br s 9.20, br s

δH, mult. (J in Hz) 3.89, 2.91, 1.39, 1.18, 1.20, 1.22, 1.23, 1.26, 0.86,

d (5.0) m m m m m m m m

5.01, m 2.90, m 2.78, m 7.02, m 6.64, m 8.33, d (8.2) 9.22 br s

4.61, dd (8.4, 5.6) 2.10, m 1.49, m 2.16, t (7.6) 2.00, s 2.38, s 4.06, m 2.88, m 2.77, m 6.86, m 6.57, m 7.50, br s 9.19, br s

TOCSY, HSQC, and HMBC spectra revealed the presence of three amino acid residues, Tyr1, Tyr2, and (N-Me)Met. The alkyl chain was identified as the β amino acid residue 3-amino2-hydroxydecanoic acid with a key MS/MS fragment at m/z 128.1430, confirming that 1 belongs to the microginin family.19 HMBC data proved that Tyr2 was the terminal amino acid through the key H-27/C-26 2J correlation with a terminal carboxylic acid at C-26. This interpretation was B



Note

Figure 2. MS/MS fragmentation pattern of 1 (Frag = 120.0 V and CE = 30.3 V).

Figure 4. Comparison between the MS/MS spectra of 1 and microginin 674.19

Figure 3. (A) Comparison between the experimental HRMS/MS spectrum of 1 and the predicted spectrum of the proposed structure of microginin 674. (B) Comparison between the experimental HRMS/MS spectrum of 1 and the predicted spectrum of the revised structure of microginin 674.

Figure 5. VT-NMR experiments showing the slow exchange (396 K) of 1 in the aromatic region (6.50−7.15 ppm) between the two rotamers.

Compound 2 was isolated as a yellowish oily solid, and its molecular formula was established by HRESIMS as C34H50N4O9S. Based on the related NMR data and an additional oxygen, compound 2 is therefore an oxygenated

analogue of 1. Due to the overall complexity of the NMR spectra, the structure elucidation of 2 was performed mainly by MS/MS fragmentation (Figure 6). The presence of the Ahda C



Note

Figure 6. MS/MS fragmentation pattern of 2 (Frag = 120.0 V and CE = 30.8 V).

residue was confirmed by the key fragment at m/z 128.1432, confirming the microginin nature of 2. While a strong peak at m/z 180.0689 was in accordance with an oxidized Nmethylmethionine, the fragment ion at m/z 627.3378 [M + H − CH3SO]+ confirmed the presence of this sulfoxide. Accordingly, the 1H NMR spectrum displayed two singlet methyls at δH 2.35 and 2.60, corresponding to the methyls of both epimers of the sulfoxide. The fragment ion at m/z 510.2635 (b3) evidenced a loss of a terminal tyrosine Tyr2, while the strongest fragment ion y2 at m/z 343.1328 was characteristic of a tyrosine linked to a (N-Me)Met(O) residue. We believe here that there was a confusion about the presence of this fragment in microginin 674 by Stewart et al. Finally, the fragment at m/z 349.2104 (b2) corresponding to a tyrosine linked to the Ahda residues confirmed the final amino acid sequence Ahda-Tyr1-(N-Me)Met(O)-Tyr2 for 2. Metabolites isolated from cyanobacteria have proven to be of interest as toxins but also as potential drugs in the cosmetics and pharmaceutical industries. The possibility to obtain large cultures of some cyanobacteria is very attractive to produce and sell metabolites of interest. Combined with the advances in molecular biology that allow the identification of biosynthetic gene clusters for some metabolites, the limitations for their production have been largely overcome, and therefore these metabolites can be sold in the marketplace. Microginins isolated from the very common cyanobacterium M. aeruginosa belong to these families of interesting metabolites that exhibit protease inhibitor activities. Even though they were placed on the market during the past decade by some companies with proposed structures for microginins 674 and 690, no spectroscopic data were reported in the cited references, and therefore no confirmation of these structures could be made. The first literature data on microginin 674 were found in a MSc thesis of the University of North Carolina Wilmington.20 Only recently Stewart et al. from the same institution reported

the isolation and structure elucidation of microginins 674 and 690, even though they were already proposed in the market.19 The structures they proposed in this article were different from those of the same compounds in the MSc thesis. Importantly, the authors provided some spectroscopic data that allowed comparison with data of other isolated natural products. When we isolated these metabolites from a strain of M. aeruginosa grown in our laboratory, our MS/MS data did match perfectly those published by Stewart et al. but not those of the predicted fragments of the proposed structure using CFM-ID (competitive fragmentation modeling for metabolite identification). A careful inspection of NMR and MS data unveiled some discrepancies in the assignment of the MS fragments, and we are now able to propose a revised structure of these metabolites with an inversion of two amino acid residues also based on interpretation of key HMBC correlations. We also believe that similar corrections should be made for all compounds published by Stewart et al., namely, microginins 511, 527, and 704, and for latter, inspection of the published data in the Supporting Information suggests a methyl ester instead of a methylated phenol for the first tyrosine when compared to microginin 690. Even if we are not able to provide experimental data to support these modifications, we believe that a metabolome consistency in this family is expected. This misinterpretation reveals the importance of HRMS data interpretation of fragments for structure elucidation. Finally, the methionine-containing microginins reported by Stewart et al. were isolated from M. aeruginosa UTEX LB-2385, while we report the same metabolites from another strain, M. aeruginosa UTEX LB-2386 (related to SAG 14-85). Even if both cyanobacteria share the same origin (Little Rideau Lake, Ontario, Canada), it is worth mentioning that the strain UTEX LB-2385 is a microcystin producer, while ours is reported as a nontoxic strain. D



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Notes

EXPERIMENTAL SECTION

The authors declare no competing financial interest.

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General Experimental Procedures. Optical rotations were recorded with a PerkinElmer 343 polarimeter equipped with a 10 cm microcell and a sodium lamp (589 nm). UV and CD measurements were obtained on a Jasco J-815 spectropolarimeter (Jasco). NMR experiments were performed on a 500 MHz spectrometer (Bruker, Avance). Chemical shifts are referenced to the carbon δC 49.0 and residual proton δH 3.31 signals of MeOH-d4 and δC 39.26/δH 2.50 for the signals of DMSO-d6. High-resolution mass spectra were obtained from an Agilent 6540 mass spectrometer. HPLC purifications were carried out on a Jasco LC-2000 series equipped with a UV detector. Biological Material. Microcystis aeruginosa was obtained from the Culture Collection of Algae at Goettingen University (CCAGU). The strain SAG-14.85 was collected in September 1954 by B. Simpson and P. R. Gorham and deposited at the CCAGU in April 1984. A similarity with the strains UTEX 2386 and PCC 7941 has been reported. Cells were cultured at the Laboratoire d’Océanographie de Villefranche-sur-Mer (France) in a BG-11 medium at 25 °C under a light intensity of 120 μE·m−2·s−1 and a 14:10 h light/dark cycle. Extraction and Isolation. A 12 L culture (20 L carboy, Nalgene) was harvested after 19 days of growth and centrifuged at 4500 rpm. The pellet was freeze-dried (2.2 g) and then extracted three times with MeOH (3 × 100 mL) under sonication, yielding 347 mg of extract. The extract was then fractionated using reversed-phase C18 vacuum liquid chromatography, giving nine fractions. The hydromethanolic (H2O/MeOH, v/v, 3:2 and 1:1) fractions were combined and purified by HPLC on a NUCLEODUR C18 HTec semipreparative column, 5 μm, 10 × 250 mm (Macherey Nagel). The mobile phases consisted of A (H2O + 0.1% TFA (trifluoroacetic acid)) and B (CH3CN + 0.1% TFA). The optimal separation and therefore the purification were assessed in isocratic mode at 32% B to yield 1 at 14.3 min (3.2 mg, 0.14% w/w) and 2 at 9.8 min (1.5 mg, 0.07% w/w). Microginin 674 (1): yellowish, oily solid; [α]20D − 22 (c 0.2, MeOH); UV (MeOH) λmax (log ε) 225 (3.09), 277 (2.73) nm; 1H NMR and 13C NMR data, Table 1; HRESIMS (+) m/z 675.3432 [M + H]+ (calcd for C34H51N4O8S, 675.3422, Δ +1.5 ppm). Microginin 690 (2): yellowish, oily solid; [α]20D −16 (c 0.1, MeOH); UV (MeOH) λmax (log ε) 225 nm (3.02), 276 (2.72) nm; HRESIMS (+) m/z 691.3377 [M + H]+ (calcd for C34H51N4O9S, 691.3371, Δ +0.9 ppm). MS/MS Spectra Prediction. The CFM-ID software was used for the prediction of the MS/MS spectra using the following parameters: adduct = [M + H]+, prob_thresh = 0.003, param_file = param_output0.log, config_f ile = param_conf ig.txt.21 The spectra were plotted using Matlab 2014a (Mathworks).

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ACKNOWLEDGMENTS The scholarship of K.C. has been supported by the French ANRT and the company COSMO Int. Ingredients. This publication has emanated from research supported in part by a research grant from the Marine Institute and the project NMBLI (Grant-Aid Agreement PBA/MB/16/01). We would like to thank R. Laville (CII) for fruitful discussion and support and M. Gaysinski (Plateforme Chimie de Nice) for help in recording the NMR spectra.

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ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00015. HRMS, MS/MS, 1H, 13C, COSY, TOCSY, NOESY, ROESY, HSQC, and HMBC NMR data for compound 1 as well as HRMS, MS/MS, and 1H data for compound 2 (PDF)

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REFERENCES

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AUTHOR INFORMATION

Corresponding Authors

*E-mail: [email protected]. *E-mail: [email protected]. ORCID

Kevin Calabro: 0000-0001-8962-4810 Grégory Genta-Jouve: 0000-0002-9239-4371 Olivier P. Thomas: 0000-0002-5708-1409 E


Structure Revision of Microginins 674 and 690 from the Cultured

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