Article pubs.acs.org/jnp
Cite This: J. Nat. Prod. 2018, 81, 1368−1375
Microcystins Containing Doubly Homologated Tyrosine Residues from a Microcystis aeruginosa Bloom: Structures and Cytotoxicity Haiyin He,† ShiBiao Wu,† Paul G. Wahome,† Matthew J. Bertin,† Anna C. Pedone,† Kevin R. Beauchesne,† Peter D. R. Moeller,‡ and Guy T. Carter*,† †
Biosortia Pharmaceuticals, Hollings Marine Laboratory, 331 Ft. Johnson Road, Charleston, South Carolina 29412, United States National Oceanic and Atmospheric Administration, Hollings Marine Laboratory, 331 Ft. Johnson Road, Charleston, South Carolina 29412, United States
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‡
S Supporting Information *
ABSTRACT: Four new microcystin congeners are described including the first three examples of microcystins containing the rare doubly homologated tyrosine residue 2-amino-5-(4hydroxyphenyl)pentanoic acid (Ahppa) (1−4). Large-scale harvesting and biomass processing allowed the isolation of substantial quantities of these compounds, thus enabling complete structure determination by NMR as well as cytotoxicity evaluation against selected cancer cell lines. The new Ahppa-toxins all incorporate Ahppa residues at the 2position, and one of these also has a second Ahppa at position 4. The two most lipophilic Ahppa-containing microcystins showed 10-fold greater cytotoxic potency against human tumor cell lines (A549 and HCT-116) compared to microcystin-LR (5). The presence of an Ahppa residue in microcystin congeners is difficult to ascertain by MS methods alone, due to the lack of characteristic fragment ions derived from the doubly homologated side chain. Owing to their unexpected cytotoxic potency, the potential impact of the compounds on human health should be further evaluated.
A
depending on water chemistry, location, and other environmental influences.5 In fresh water habitats cyanobacteria are often a significant component of the microbiome and are prone to exponential growth when conditions are favorable. In recent years there have been recurrent cyanobacterial blooms in the Great Lakes in the United States, and such events appear to be increasing in frequency.6 The potential impact of these blooms on water quality is a real concern for municipalities throughout America, whether the water is for consumption or for recreational use.7 Although there are some analytical methods for the detection and quantification of microalgal metabolites, the vast majority of the compounds are not monitored, nor are their biological effects well understood.8 Owing to the substantially higher density of microbial growth found under bloom conditions, we have targeted these events for our largescale collection efforts. In this report we describe four new microcystin toxins (1−4) that are responsible for the cytotoxicity associated with a cyanobacterial bloom. Compounds 1, 2, and 3 are the first reported microcystins to contain the rare doubly homologated tyrosine residue 2-amino-5-(4-hydroxyphenyl)pentanoic acid (Ahppa), while 4 is a new congener having a leucine residue at the 1-position. These compounds were isolated from microbial
quatic microbiomes harbor enormous biosynthetic capacity for the production of biologically active secondary metabolites. Historically many bioactive compounds were discovered in chemical studies of sponges or other higher organisms that have co-opted environmental microbes or more recently through culturing of individual isolated microorganisms.1 An alternate approach that has also yielded a substantial number of microbial secondary metabolites is the harvesting and processing of microbial biomass, often under “bloom” conditions.2 The strategy that we have developed at Biosortia is to take the latter approach to an extremely deep level that enables the discovery of biologically active metabolites that are present in the environment down to sub-parts-per-billion. In order to facilitate the “deep-dive” approach, we use proprietary harvesting technologies for the collection of massive quantities of microbial biomass. Processing tens of kilograms of biomass, through solvent partitioning and chromatography, ultimately results in simplified pools of highly concentrated metabolites that are arrayed in bioassay plates for screening. In this way we aim to capture the semiochemicals, antibiotics, and other growth-regulating substances that govern the microbial community.3 Microalgal blooms, i.e., patches of explosive growth within a given aquatic ecosystem, are well known as causative sources of pathogenic events, often with consequences for human health.4 The microbial constitution of a bloom is highly variable © 2018 American Chemical Society and American Society of Pharmacognosy
Received: November 20, 2017 Published: May 30, 2018 1368
DOI: 10.1021/acs.jnatprod.7b00986 J. Nat. Prod. 2018, 81, 1368−1375
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(−NHCHCH2CH2CH2−), from amide NH to H2-5. In the COSY spectrum, the NH at δH 7.87 was coupled to the αproton H-2 at δH 3.97, which was in turn coupled to H2-3 at 1.72 and 1.73. H2-3 were coupled to H2-4 at δH 1.68 and 1.49, and the latter two further coupled to H2-5 at δH 2.38 and 2.39. In the HMBC spectrum, a pair of mutually coupled signals at δH 6.96 (2H, 8.3 Hz, H-7/H-11) and 6.64 (2H, 8.3 Hz, H-8/H10) were both respectively correlated to an oxygen-bearing aromatic carbon (C-9) at δC 155.7 and a quaternary carbon (C6) at 132.4. These chemical shift data and multiple-bond 1 H−13C correlations are typically observed for a 1-alkyl-4oxyphenyl system. Additional HMBC correlations from H-2 at δH 3.97 to C-3 at δC 30.7 and C-4 at 28.2, from H-4b at δH 1.49 to C-5 at δC 34.3 and C-6 at 132.4, and finally from H-7/11 at δH 6.96 (2H) to C-5 supported the structural assignment of 2amino-5-(4-hydroxyphenyl)pentanoic acid (Ahppa-2). Ahppa-4. Similar to Ahppa-2, an analysis of the COSY and TOCSY data identified a −NHCHCH2CH2CH2− moiety from amide NH at δH 8.76 to H2-5 at 2.33 and 2.40. In addition, the COSY and HMBC data indicated the presence of a 1-alkyl-4oxyphenyl ring. Finally, the HMBC correlations between the aliphatic section and the aromatic system made the connection of the two moieties and required that this amino acid be 2amino-5-(4-hydroxyphenyl)pentanoic acid (Ahppa-4, Figure 1). The presence of seven amino acids, Adda, Ala, Masp, Glu, Mdha, Ahppa-2, and Ahppa-4, accounted for 24 degrees of unsaturation, and the final one, required by the molecular formula, was attributed to the macrocycle. The sequence of AlaAhppa-2-Masp was established by the HMBC correlations from NH (Ahppa-2) at δH 7.87 to C-1 (Ala) at δC 173.2 and from NH (Masp) at δH 8.02 to C-1 (Ahppa-2) at δC 172.3. On the other hand, the sequence of Masp-Ahppa-4-Adda was indicated by strong NOESY correlations from NH (Ahppa-4) at δH 8.76 to H-2 (Masp) at 4.50 and H3-5 (Masp) at 0.97 and from NH (Adda) at δH 6.91 to H-2 (Ahppa-4) at 4.23. Additional crosspeaks observed in the NOESY and HMBC spectra (Table 1) supported the connections of these and the remaining amino acid units and established the structure of a cyclic heptapeptide. The advanced Marfey’s method was utilized to determine absolute configurations, and the experimental data (Table 3) indicated that 1 contained Adda, D-Ala, D-Masp, D-Glu, and two units of L-Ahppa. As a result, compound 1 was determined to be cyclo(-D-Ala1-L-Ahppa2-D-Masp3-L-Ahppa4-Adda5-D-Glu6Mdha7). The high-resolution MS data of 2 indicated a molecular formula of C54H76N10O13. Inspection of the NMR data (Table S2) showed 2 to be a microcystin congener containing Ala, Masp, Arg, Adda, Glu, Mdha, and a single Ahppa moiety. Analysis of the 2D correlations shown in Figure 1 placed the Ahppa unit at position 2, and the complete sequence was confirmed through additional 2D correlations (Table S2). The results of the advanced Marfey’s method gave the absolute configurations of the component amino acids, and therefore the structure of 2 is revealed as cyclo(-D-Ala1-L-Ahppa2-D-Masp3-LArg4-Adda5-D-Glu6-Mdha7). Compound 2 is analogous to 1, the only difference being that L-Ahppa4 in 1 is replaced by L-Arg4 in 2. The high-resolution MS data of 3 indicated a molecular formula of C58H75N7O14. Analysis of the NMR data (Table S3) showed 3 to be a microcystin congener containing homotyrosine (Hty) and Ahppa in addition to Ala, Masp, Adda, Glu, and Mdha. The sequence as confirmed by 2D NMR correlations, and configurations established by the advanced
biomass collected from storage lagoons in a wastewater management facility in Muskegon, Michigan, in which Microcystis aeruginosa was the dominant organism. M. aeruginosa is frequently associated with harmful algal blooms and is a prolific producer of microcystins as well as other classes of toxic compounds.9
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RESULTS AND DISCUSSION With a few exceptions, structures of the microcystins can be represented by cyclo(-D-Ala1(Leu)-X2-D-Asp3(Masp)-Y4-Adda56 7 D-Glu -Dha (Mdha)), where X and Y are variable L-amino acids at the 2- and 4-positions, respectively; D-Masp is D-erythro-ßiso-aspartic acid; Adda is (2S,3S,8S,9S)-3-amino-9-methoxy2,6,8-trimethyl-10-phenyldeca-4,6-dienoic acid; Dha and Mdha are respectively dehydroalanine and N-methyldehydroalanine. The residues of the heptapeptide are conventionally numbered beginning with D-Ala (1) and ending with Dha (7).10 The high-resolution MS data of 1 indicated a molecular formula of C59H77N7O14, requiring 25 degrees of unsaturation. An initial analysis of the 13C and 1H NMR spectra aided by phase-sensitive HSQC correlations identified nine carbonyl signals from δC 163 to 177, seven heteroatom-attached methine carbons between δC 49 and 87, and six amide proton signals between δH 6.9 and 8.8. In addition, it revealed three substituted phenyl rings, along with three olefinic groups. Detailed analyses of the 1D and 2D NMR data including COSY, HMBC, HSQC, TOCSY, and NOESY (Table 1 and Supporting Information Table S1) clearly indicated the presence of an Adda unit, characteristic to microcystins. The NMR data also implied four other amino acids, Ala, Masp, Glu, and Mdha. The remaining signals in the NMR spectra pointed to the presence of two units of a rare amino acid, 2-amino-5-(4hydroxyphenyl)pentanoic acid (Ahppa-2 and Ahppa-4). Ahppa-2. As shown in Figure 1, the COSY and TOCSY spectra identified a 1H−1H spin system 1369
DOI: 10.1021/acs.jnatprod.7b00986 J. Nat. Prod. 2018, 81, 1368−1375
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Table 1. NMR Data for Compound 1 in DMSO-d6 (1H 700 MHz, 13C 175 MHz) AA unit Ala
Ahppa-2
Masp
Ahppa-4
Adda
Glu
position
δ Ca
1 2 3 NH 1 2 3a 3b 4a 4b 5a 5b 6 7/11 8/10 9 9-OH NH 1 2 3 4 5 NH 1-OH 1 2 3a 3b 4a 4b 5a 5b 6 7/11 8/10 9 9-OH NH 1 2 3 4 5 6 7 8 9 10a 10b 11 12/16 13/15 14 17 18 19 20 NH 1 2
173.2, C 49.0, CH 17.1, CH3 172.3, C 55.0, CH 30.7, CH2 28.2, CH2 34.3, CH2 132.4, 129.6, 115.4, 155.7,
C CH CH C
169.7, C 53.7, CH 39.1, CH 176.7, C 15.3, CH3
172.4, C 51.5, CH 30.4, CH2 27.8, CH2 33.8, CH2 132.2, 129.3, 115.4, 155.6,
C CH CH C
174.4, C 44.2, CH 53.6, CH 126.2, CH 136.2, CH 132.4, C 136.1, CH 35.9, CH 86.2, CH 37.5, CH2 139.7, C 129.7, CH 128.6, CH 126.3, CH 16.7, CH3 13.0, CH3 16.4, CH3 57.8, CH3 173.7, C 51.5, CH
δH (J in Hz)a
1
H−13C HMBC
NOESY
4.31, m 1.14, d (6.8) 7.72, d (6.2)
1, 3 1, 2
NH(Ahppa-2) NH H3-3
3.97, 1.73, 1.72, 1.68, 1.49, 2.39, 2.38,
3, 4 2
NH(Masp) NH NH
m m m m m m m
2, 2, 3, 3,
3, 3, 4, 4,
5, 5, 6, 6,
6 6 7/11 7/11
NH
6.96, d (8.3) 6.64, d (8.3)
5, 6, 8, 9 6, 9
H-4a 9-OH
9.1, brs 7.87, d (8.8)
9 1(Ala)
H2-3, H-4a
4.50, m 2.99, m
3, 4 4
NH(Ahppa-4) NH(Ahppa-4)
0.97, d (7.4) 8.02, d (8.4) 12.9, br
2, 3, 4 1(Ahppa-2)
NH, NH(Ahppa-4) H3-5
2, 5
H-5a, NH(Adda) NH
4.23, 1.90, 1.40, 1.46, 1.40, 2.40, 2.33,
m m m m m m m
2, 2, 3, 3,
3, 3, 4, 4,
5, 6 5, 6 6 6
NH
6.86, brd (8.0) 6.63, d (8.0)
5, 9 6, 9
H-4b 9-OH
9.1, brs 8.76, d (8.4)
9
H-8/10 H2-3b, H-2(Masp), H-3(Masp), H3-5(Masp), NH(Adda)
2.53, 4.51, 5.36, 6.12,
m m dd (16, 7.8) d (16)
3, 1, 3, 3,
5.47, 2.57, 3.26, 2.74, 2.69,
d (9.0) m m dd (5.0, 14.0) dd (7.0, 14.0)
5, 6, 8, 18, 19 6, 7, 9, 19
17 2, 4, 5, 1(Ahppa-4) 6 4, 6, 7, 18
NH(Glu) NH, H3-17, H3-18 NH H-9 H-7
8, 9, 11, 12/16 7, 8, 10, 11, 19, 20
7.20, m 7.28, m 7.19, m 0.97, d (7.5) 1.56, s 0.98, d (7.4) 3.2, s 6.91, m
10, 11, 13/15, 14 11, 12/16, 14 12/16, 13/15 1, 2, 3 5, 6, 7 7, 8, 9 9
4.38, m
1, 3, 4
H-4, H-5, H-7, NH H-5
H-2, H-2(Ahppa-4)
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H-4b DOI: 10.1021/acs.jnatprod.7b00986 J. Nat. Prod. 2018, 81, 1368−1375
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Table 1. continued AA unit
Mdha
a
1
position
δ Ca
δH (J in Hz)a
3a 3b 4a 4b 5 NH 1-OH 1 2 3a 3b NCH3
27.5, CH2 32.0, CH2
2.0, m 1.67, m 2.54, m 2.42, m
1
H−13C HMBC
1, 2 1, 2
NOESY NH NH H-2
5
175.0, C 7.19, m 12.9, br 163.4, C 145.6, C 113.9, CH2 36.3, CH3
H3-17(Adda), H-3b(Glu), H-3a(Glu), H-2(Adda)
5.78, brs 5.40, brs 3.23, s
1, 2 1 2, 5(Glu)
H3-4 H3-4, NH(Ala), H-5(Adda) H-3b, NH(Ala)
13
Direct H− C correlations and the multiplicities of the 13C signals were assigned by a phase-sensitive HSQC spectrum.
Figure 1. NMR assignments and correlations for the Ahppa units in 1, 2, and 3.
The high-resolution MS data of 4 indicated a molecular formula of C55H77N7O13. Analysis of the NMR data (Table S4) indicated the compound to be a microcystin congener with Leu in place of Ala in position 1, as well as a second Leu and Masp, Tyr, Adda, Glu, and Mdha. The advanced Marfey’s method indicated both D- and L-Leu to be present in 4. Based on the fact that the vast majority of microcystins, including all examples from this biomass, have a D configuration at position 1 and L at position 2, compound 4 was assigned as cyclo(-DLeu1-L-Leu2-D-Masp3-L-Tyr4-Adda5-D-Glu6-Mdha7). A number of other microcystins with alternative amino acids in the 1position have been reported, such as [D-Leu1]LR 6, which was one of the first examples11 (also found in this biomass), although these remain relatively rare. Cytotoxicity of the New Microcystin Congeners against HCT-116 and A549 Cells. Some microcystins have
Table 2. Concentration (μM) of Compound Causing 50% Reduction in the Viability (EC50) of HCT-116 or A549 Cells EC50 (μM) compound
HCT-116
A549
1 2 3 4 5
2.9 >35 3.3 11 ∼35
0.45 ∼18 0.36 1.3 >35
Marfey’s analysis led to the structure of 3 as cyclo(-D-Ala1-LHty2-D-Masp3-L-Ahppa4-Adda5-D-Glu6-Mdha7). Compound 3 is essentially a lower homologue of 1 in which the amino acid residue at position 2 is Hty in 3 versus Ahppa in 1. 1371
DOI: 10.1021/acs.jnatprod.7b00986 J. Nat. Prod. 2018, 81, 1368−1375
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11 μM, respectively (Table 2 and Figure S10A). Similarly, these compounds showed dose-dependent cytotoxicity on A549 cells with estimated EC50 values of 0.45, 0.36, and 1.3 μM, respectively (Table 2 and Figure S10B). In contrast, microcystins 2 and 5 were essentially inactive on both cells lines (Table 2, Figure S10C). Discussion. Microcystins are cyclic heptapeptides recognized as being harmful to mammals owing to severe liver toxicity.10 Even at subacute concentrations, long-term exposure to microcystins appears to enhance the incidence of liver cancer.15 More than 100 microcystin congeners have been reported to date, and since they are recognized as environmental toxicants, there are mandated tests to determine the concentration of microcystins in critical water supplies. The most commonly employed test method is an antibody-based assay that recognizes the Adda side chain and is therefore generic for the core structure of microcystins.16 On the other hand, the potency of the toxic effects exerted by microcystins is highly dependent on the structures of the amino acid residues arrayed around the macrocycle.17 Although powerful LC-MS/ MS methods have been developed for the identification and quantification of many microcystins,18 some congeners fail to give structurally unique fragment ions, leading to ambiguous results where the amino acid residues are not readily distinguishable by mass spectrometric methods alone.19 In this study, we have evaluated the cytotoxicity of four newly isolated microcystins (1−4) on HCT-116 and A549 cells. Microcystins 1, 3, and 4 showed strong dose-dependent cytotoxicity on both cell lines. In contrast, microcystins 2 and 5 were essentially inactive. The differences in the potency of the compounds must be attributed to their different constituent amino acid residues. Compounds 1 and 3 contain the Ahppa residue at position 4, but differ at position 2 (Ahppa in 1 and Hty in 3). On the other hand, both 2 and 5 contain Arg at position 4 but differ at position 2 (Ahppa in 2 and Leu in 5). The presence of Ahppa at position 4 and either Hty or Ahppa at position 2 correlated with stronger cytotoxicity, while the presence of Arg at position 4 correlated with reduced potency. Compound 4 is notably different from the other four microcystins owing to the presence of Leu at position 1. Like LR (5), 4 has Leu at position 2 but has Tyr at position 4, which renders it more lipophillic. This combination of amino acid residues correlates with enhanced cytotoxic potency compared to that given by 2 and 5. Microcystins have shown stronger cytotoxicity on hepatocytes or other mammalian cells with elevated levels of the organic anion transporting polypeptides (OATPs).20−22 In one report the more hydrophobic microcystins LF and LW were shown to be considerably more toxic to cells than the polar congeners LR (5) and RR owing to enhanced active transport of the nonpolar compounds.23 It appears that new compounds 1, 2, and 3 also demonstrate this trend, showing enhanced cytotoxicity compared to more polar congeners, presumably due to enhanced OATP-mediated transport. Homologated amino acid side chains are found with some frequency in cyanobacterial secondary metabolites and can be regarded as biosynthetic signatures.24 The homologated tyrosine units presumably arise through two sequential net one-carbon additions of acetate units to a 2-keto tyrosine derivative, formed via transamination, followed by oxidative decarboxylation and transamination back to the 2-amino group.25 There has been no experimental evidence for this sequential homologation mechanism; however a single
Table 3. Results of Amino Acid Analyses for Compounds 1− 4 retention time in minutes (reference sample retention time) compound 1
amino acid identified D-Glu
a
D-Masp
a
L-Ahppa a D-Ala
a
Addae 2
L-Arg
g
D-Glu
a
D-Maspa L-Ahppa a D-Ala
a
Addae 3
D-Glu
a
D-Masp
a
a L-HTyr a L-Ahppa a D-Ala
Addae 4
L-Tyr
a
D-Glu
a
D-Masp a L-Leu
D-Leu
a
Addae
a
L-FDLA
D-FDLA
adduct
adduct
8.1 (D-Glu, 8.1) 8.8 (D-Maspb, 8.8) 10.1d 10.9 (D-Ala, 10.9) 11.0 (Addaf, 11.1) 14.4 (L-Arg, 14.5) 8.1 (D-Glu, 8.1) 8.8 (D-Masp, 8.8) 10.1d 10.8 (D-Ala, 10.9) 11.1 (Adda, 11.1) 8.1 (D-Glu, 8.1) 8.9 (D-Masp, 8.8) 9.7d 10.1d 10.9 (D-Ala, 10.9) 11.0 (Adda, 11.1) 9.0 (L-Tyr, 9.0) 8.1 (D-Glu, 8.1) 8.8 (D-Masp, 8.8) 11.4 (L-leu, 11.4) 15.1 (D-Leu, 15.2) 11.1 (Adda, 11.1)
7.6 (D-Glu, 7.6) 7.8 (D-Masp, 7.8) 11.4 9.2 (D-Ala, 9.2) 14.7 (Adda, 14.7) 14.0 (L-Arg, 14.0) 7.6 (D-Glu, 7.6) 7.8 (D-Masp, 7.8) 11.4 9.2 (D-Ala, 9.2) 14.7 (Adda, 14.7) 7.6 (D-Glu, 7.6) 7.8 (D-Masp, 7.8) 10.3 11.4 9.3 (L-Ala, 9.3) 14.7 (Adda, 14.7) 9.7 (L-Tyr, 9.7) 7.6 (D-Glu, 7.6) 7.8 (D-Masp, 7.8) 15.2 (L-Leu, 15.2) 11.4 (D-Leu, 11.3) 14.7 (Adda, 14.7)
MS data m/z (MH)+ 442.1 442.1 504.1 384.1 594.3 469.1 442.1 442.1 504.1 384.1 594.3 442.1 442.1 490.1 504.1 384.1 594.3 476.1 442.1 442.1 426.1 426.1 594.3
a Gradient A. bReference D-Masp was derived from hydrolysis of 6. cIn 1, the respective MS intensities for adducts L-FDLA-L-Ahppa and DFDLA-L-Ahppa were about 2-fold greater than those for 2 and 3. dLConfiguration assigned based upon the earlier elution of the L-FDLA derivatives. eGradient B. fReference Adda was derived from hydrolysis of 6. gGradient C.
been reported to be cytotoxic to mammalian cells.12−14 These reports encouraged us to evaluate the new microcystins for cytotoxicity on HCT-116 and A549 cells. We also included microcystin-LR 5, the most common microcystin, in our assay to serve as a benchmark for cytotoxicity. As described in the Supporting Information, the cells were seeded in 96-well microplates, incubated overnight at 37 °C, and then treated with varying concentrations (∼35 to 0.1 μM) of the microcystins. After a further incubation at 37 °C for ∼72 h, the viability of the cells was assessed using the MTT method. Microcystins 1, 3, and 4 showed dose-dependent cytotoxicity on HCT-116 cells with estimated EC50 values of 2.9, 3.3, and 1372
DOI: 10.1021/acs.jnatprod.7b00986 J. Nat. Prod. 2018, 81, 1368−1375
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eluted with MeCN/H2O (0.1% TFA) at 3 mL/min as follows: 30% MeCN for 5 min; gradient elution from 30% to 52% MeCN from 5 to 45 min; gradient from 52% to 88% MeCN from 45 to 50 min. An HPLC chromatogram with UV detection at 220 nm (Figure S9) shows that these new microcystins are major contituents in the biomass. New microcystins eluted as indicated on the chromatogram: 2 (27 min), 3 (44 min), 1 (45.5 min), 4 (49.2 min). Quantification of the compounds on a weight basis in the biomass was imprecise owing to variability across the harvest. The estimated content of the major microcystin 2 based on isolated yield is in the range of 30−80 mg per kg of freeze-dried biomass. Isolation of 1, 3 and 4. Fraction C18−B (800 mg) was dissolved in dimethylformamide/MeOH (1:1) 8 mL and subjected to reversedphase chromatography. Four replicate samples of 1.8 mL were injected onto a C18 column (Phenomenex Luna 250 × 50 mm, 12 μm, 100 Å) eluted with a gradient of MeCN in H2O (each with 0.1% FA) from 65 to 70% over 30 min at 40 mL/min. The effluent containing compounds 1 and 3 was collected between 17 and 25 min, yielding a residue of 20.2 mg upon evaporation of the solvents. Compound 4 was contained in effluent eluting between 29 and 30 min; its residual weight was 4.6 mg. Separation of 1 and 3. The mixture (20.2 mg) was dissolved in dimethylformamide/MeOH (1:1, 0.4 mL), and four replicate injections of 0.1 mL were used for the final chromatographic step. This mixture was resolved by reversed-phase chromatography on a YMC-ODS A 250 × 10 mm, 5 μm column eluted with a gradient of MeCN in H2O (each with 0.025% trifluoroacetic acid, TFA) from 45% to 50% over 25 min at 3.5 mL/min. Compound 1 was obtained as a peak centered at 20.4 min (6.6 mg), and compound 3 at 19.0 min (2.8 mg). Purification of 4. The mixture containing 4 (4.6 mg) was dissolved in MeOH (1.0 mL) and purified on a YMC-ODS A 30 × 250 mm, 10 μm column eluted with a gradient of MeCN in H2O (each with 0.025% TFA) from 40% to 100% over 26 min at 20 mL/min. Compound 4 was obtained as a peak centered at 18.0 min (3.7 mg). Isolation of 2. A 5 g portion of fraction C18-B was subjected to LH-20 chromatography in MeOH (200 g; column 10 × 110 cm; flow 10 mL/min). After a 500 mL forerun, 100 fractions of 16 mL were collected. A series of fractions (10−24) was combined owing to their microcystin content as determined by LC/MS analysis. The residual weight of the material after solvent removal was 1.1 g. A portion of this LH-20 fraction (0.15 g) was dissolved in 2 mL of MeOH and subjected to reversed-phase chromatography on a C18 column (Phenomenex Luna 50 × 250 mm, 12 μm, 100 Å) eluted with a gradient of MeCN in H2O (each with 0.1% FA) from 35% to 42% over 30 min at 40 mL/min. The peak containing 2 was collected between 20.5 and 21.9 min. This process was repeated with four additional 0.15 g portions of the LH-20-derived fraction. The combined peak fractions were concentrated under reduced pressure to remove the bulk of the MeCN and then extracted with n-butanol. Evaporation of the butanol yielded 2 (80 mg). Microcystin-LR 5 was obtained in a broad peak that eluted prior to 2, between 19.0 and 20.5 min. Processing of that effluent yielded a mixture of components with a residual weight of 61 mg. Isolation and characterization of microcystin-LR is described in the Supporting Information. [D-Leu1] microcystin-LR ([D-Leu1]LR) 6 was obtained as a single component eluting at 24.8 min (74 mg); its identity was confirmed by comparison with published NMR spectroscopic data.11 Compound 1: colorless solid; [α]20D −55 (EtOH, c 0.20); UV (MeOH) λmax (log ε) 203 (4.4), 238 (4.4) nm; NMR data, Tables 1 and S1; HRMS data: m/z 1108.5594 [M + H]+ (calcd for C59H78N7O14, 1108.5607). Compound 2: colorless solid; [α]20D −40 (EtOH, c 0.10); UV (MeOH) λmax (log ε) 203 (4.4), 238 (4.4) nm; NMR data, Table S2; HRMS data m/z 1073.5669 [M + H]+ (calcd for C54H77N10O13, 1073.5672). Compound 3: colorless solid; [α]20D −10 (EtOH, c 0.10); UV (MeOH) λmax (log ε) 202 (4.3), 238 (4.3) nm; NMR data, Table S3; HRMS data m/z 1094.5443 [M + H]+ (calcd for C58H76N7O14, 1094.5450).
homologation sequence for phenylalanine residues is supported by labeling data in the case of the cyanobacterial metabolite pahayokolide.26 Although homotyrosine is well known in microcystin congeners,27 this is the first report of microcystins containing the rare doubly homologated tyrosine residue (Ahppa). In this report we highlight the enhanced cytotoxic potency of two microcystins containing the Ahppa residue compared to those containing more common amino acids. The enhanced cytotoxic potency of these hydrophobic microcystins against cancer cell lines suggests there is potential for unanticipated adverse effects toward humans, wildlife, and domesticated animals when these compounds are abundant in microbial blooms. We believe that compound-specific assays should be developed that enable quantification of these chemical species in critical environmental samples.
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EXPERIMENTAL SECTION
General Experimental Procedures. Optical rotations were measured using a Rudolph Research Analytical Autopol I automatic polarimeter. UV spectra were measured using a Beckman Coulter DU800 spectrophotometer. LC/MS was performed using a Waters 1525 system equipped with the 2767 sample manager and a ZQ mass spectrometer. NMR spectroscopy was performed using a Bruker Avance 2 700 MHz spectrometer equipped with a 5 mm triple nucleus gradient probe, and data processing was performed using TopSpin 2.1 software. Compounds were dissolved in dimethyl sulfoxide-d6 with deuterium serving as the lock nucleus. High-resolution mass spectrometry was carried out with a Waters UPLC/QTOFMS operated using an Acquity UPLC system, controlled by MassLynx v4.1 software. Cytotoxicity against cancer cell lines was determined by a standard MTT assay (see Supporting Information).28 L-Leucine amide and D-leucine amide used to synthesize the FDLA reagents were purchased from Santa Cruz Biotechnologies. The standard amino acids and other chemicals were bought from SigmaAldrich. Collection of Biological Material. During September 21−24, 2014, biomass from a holding lagoon at the Muskegon County Wastewater Management facility on Maple Island Road, Muskegon, Michigan, USA, was collected and concentrated on a filter belt press to approximately 15% solids. The resulting paste was then packaged in 2 kg batches in plastic bags and immediately frozen. A water sample was evaluated microscopically, which showed that the bloom was dominated by coccoid cyanobacteria, predominantly Microcystis aeruginosa.29 Extraction and Purification. Frozen biomass (500 kg) was lyophilized to yield a friable solid (43 kg) that was used for solvent extraction. A 10 kg portion of the freeze-dried biomass was extracted with MeOH with gentle agitation (1 × 30L, 2 × 20L). The resulting solution was evaporated under reduced pressure to yield an oily residue (1310 g). The bulk of this material was adsorbed onto HP-20 resin (8 kg) and then sequentially eluted with H2O, MeOH−H2O mixtures, MeOH, and finally acetone. The material that eluted with 70−80% MeOH−H2O was combined and concentrated to yield a fraction that contained metabolites of medium polarity (253 g). This medium-polarity HP-20-processed material was adsorbed onto Celite (510 g) and subjected to reversed-phase chromatography on bulk C18 packing (POLYGOPREP 60-50 C18 [Machery & Nagel], 60 Å, 40− 63 μm). The bulk C18 process was conducted in nine equal batches on a column of 50 × 250 mm eluted with a gradient of MeCN/H2O, 10% to 80% (each with 0.1% formic acid (FA)) at a flow rate of 50 mL/min. The components that eluted with MeCN in the 40 to 60% range were collected sequentially as three separate fractions and concentrated under reduced pressure: C18-A (67 g), C18−B (50 g) and C18−C (31 g). HPLC Analysis of C18−B. A sample of fraction C18−B (1 mg/mL MeOH) was separated on HPLC: Phenomenex Synergi C18 column (250 × 10 mm, 4 μm particle size, 80 Å pore size). The column was 1373
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Compound 4: colorless solid; [α]20D −10 (EtOH, c 0.10); UV (MeOH) λmax (log ε) 203 (4.4), 238 (4.4) nm; NMR data, Table S4; HRMS data m/z 1044.5643 [M + H]+ (calcd for C55H78N7O13, 1044.5658). Determination of the Absolute Configuration of Constituent Amino Acids in 1−4. The advanced Marfey’s analysis was employed to establish absolute configuration.30 Each of the peptides 1−4 (1 mg) was mixed with 6 N HCl (0.5 mL) by sonication. The suspension was heated at 85 °C for 16 h. At the end of the hydrolysis, the aqueous solution was evaporated at 50 °C under reduced pressure to dryness, and the residue was redissolved in distilled H2O (200 μL). A portion of this solution (20 μL) was mixed with 40 μL of 0.5% acetone solution of either L- or D-FDLA, prepared according to the literature,31 and 8 μL of 1 M sodium bicarbonate. The resulting solution was incubated at 40 °C for 1 h, cooled, and neutralized with 1 M HCl. After the CO2 was removed by vortexing, the solution was subjected to LC/MS analysis. Amino acid standards were derivatized with the FDLA reagents in a similar manner. In order to generate reference materials for D-Masp and Adda, we subjected [D-Leu1]LR (6) to the same hydrolysis/derivatization procedure. The results of the analyses for both L- and D-FDLA derivatives are listed in Table 3. The LC/MS analyses were carried out using an Agilent 1100 LC/G1956B MSD system in the positive mode, with a YMC ODS-A 250 × 4.6 mm, 5 μm, 120 Å column eluted with either gradient A [(60% MeCN in H2O) + 0.025% TFA for 2 min, then (60−100% MeCN in H2O) + 0.025% TFA in 15 min, 0.5 mL/min] or B [(80% MeCN in H2O) + 0.025% TFA for 2 min, then (80−100% MeCN in H2O) + 0.025% TFA in 15 min, 0.5 mL/min], or C [(20% MeCN in H2O) + 0.025% TFA for 2 min, then (20−100% MeCN in H2O) + 0.025% TFA in 15 min, 0.5 mL/min].
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(3) Venuleo, M.; Raven, J. A.; Giordano, M. New Phytol. 2017, 215, 516−530. (4) Carmichael, W. W.; Azevedo, S. M.; An, J. S.; Molica, R. J.; Jochimsen, E. M.; Lau, S.; Rinehart, K. L.; Shaw, G. R.; Eaglesham, G. K. Environ. Health Perspect 2009, 109, 663−668. (5) Wells, M. L.; Trainer, V. L.; Smayda, T. J.; Karlson, B. S. O.; Trick, C. G.; Kudela, R. M.; Ishikawa, T. J.; Bernhard, S.; Wulff, A.; Anderson, D. M.; Cochlan, W. P. Harmful Algae 2015, 49, 68−93. (6) Michalak, A. M.; Anderson, E. J.; Beletsky, D.; Boland, S.; Bosch, N. S.; Bridgeman, T. B.; Chaffin, J. D.; Cho, K.; Confesor, R.; Daloglu, I.; DePinto, J. V.; Evans, M. A.; Fahnenstiel, G. L.; He, L.; Ho, J. C.; Jenkins, L.; Johengen, T. H.; Kuo, K. C.; LaPorte, E.; Liu, X.; McWilliams, M. R.; Moore, M. R.; Posselt, D. J.; Richards, R. P.; Scavia, D.; Steiner, A. L.; Verhamme, E.; Wright, D. M.; Zagorski, M. A. Proc. Natl. Acad. Sci. U. S. A. 2013, 100, 6448−6452. (7) Backer, L. C.; McNeel, S. V.; Barber, T.; Kirkpatrick, B.; Williams, C.; Irvin, M.; Zhou, Y.; Johnson, T. B.; Nierenberg, K.; Aubel, M.; LePrell, R.; Chapman, A.; Foss, A.; Corum, S.; Hill, V. R.; Kiesak, S. M.; Cheng, Y.-S. Toxicon 2010, 55, 909−921. (8) Chorus, I.; Bartram, J. Toxic Cyanobacteria in Water: A Guide to Their Public Health Consequences, Monitoring, and Management; E & FN Spon: London, 1999. (9) Falconer, I. R. Toxicon 2007, 50, 585−588. (10) Pearson, L.; Mihali, T.; Moffitt, M.; Kellmann, R.; Neilan, B. Mar. Drugs 2010, 8, 1650−1680. (11) Park, H.; Namikoshi, M.; Brittain, S. M.; Carmichael, W. W.; Murphy, T. Toxicon 2001, 39, 855−862. (12) Piyathilaka, M. A.; Pathmalal, M. M.; Tennekoon, K. H.; De Silva, B. G.; Samarakoon, S. R.; Chanthirika, S. Microbiology 2015, 161, 819−828. (13) Shimizu, K.; Sano, T.; Kubota, R.; Kobayashi, N.; Tahara, M.; Obama, T.; Sugimoto, N.; Nishimura, T.; Ikarashi, Y. Toxins 2014, 6, 168−179. (14) Dias, E.; Andrade, M.; Alverca, E.; Pereira, P.; Batoreu, M. C.; Jordan, P.; Silva, M. J. Toxicon 2009, 53, 487−495. (15) Svirčev, Z.; Baltić, V.; Gantar, M.; Juković, M.; Stojanović, D.; Baltić, M. J. of Environ. Sci. Health, PartC: Environ. Carcinog. Ecotoxicol. Rev. 2010, 28, 39−59. (16) www.epa.ohio.gov/ddagw/HAB.aspx. (17) Rinehart, K. L.; Namikoshi, M.; Choi, B. W. J. Appl. Phycol. 1994, 6, 159−176. (18) Benke, P. I.; Vinay Kumar, M. C. S.; Pan, D.; Swarup, S. Analyst 2015, 140, 1198−1206. (19) Teta, R.; Della Sala, G.; Glukhov, E.; Gerwick, L.; Gerwick, W. H.; Mangoni, A.; Costantino, V. Environ. Sci. Technol. 2015, 49, 14301−14310. (20) Monks, N. R.; Liu, S.; Xu, Y.; Yu, H.; Bendelow, A. S.; Moscow, J. A. Mol. Cancer Ther. 2007, 6, 587−598. (21) Fischer, A.; Hoeger, S. J.; Stemmer, K.; Feurstein, D. J.; Knobeloch, D.; Nussler, A.; Dietrich, D. R. Toxicol. Appl. Pharmacol. 2010, 245, 9−20. (22) Niedermeyer, T. H.; Daily, A.; Swiatecka-Hagenbruch, M.; Moscow, J. PLoS One 2014, 9, e91476. (23) Puddick, J.; Prinsep, M. R.; Wood, S. A.; Cary, S. C.; Hamilton, D. P.; Wilkins, A. L. Phytochem. Lett. 2013, 6, 575−581. (24) Plaza, A.; Bewley, C. A. J. Org. Chem. 2006, 71, 6898−6907. (25) Walsh, C. T.; O’Brien, R. V.; Khosla, C. Angew. Chem., Int. Ed. 2013, 52, 7098−7124. (26) Liu, L.; Bearden, D. W.; Rein, K. S. J. Nat. Prod. 2011, 74, 1535−1538. (27) Harada, K.; Ogawa, K.; Kimura, Y.; Murata, H.; Suzuki, M.; Thorn, P. M.; Evans, W. R.; Carmichael, W. W. Chem. Res. Toxicol. 1991, 4, 535−540. (28) Wahome, P. G.; Beauchesne, K. R.; Pedone, A. C.; Cavanagh, J.; Melander, C.; Zimba, P.; Moeller, P. D. R. Mar. Drugs 2015, 13, 65− 75. (29) Species identification by Dr. Paul V. Zimba, Texas A & M University−Corpus Christi.
ASSOCIATED CONTENT
S Supporting Information *
The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.7b00986. NMR and HRMS general procedures; 1H and 13C NMR spectra for compounds 1−4; tabulated NMR data for compounds 1−4; HPLC chromatograms for compounds 1−4; cytotoxity (MTT) assay procedure; HCT-116 and A549 cell viability data; isolation procedure for microcystin-LR (5); LC/MS chromatogram and MS/MS spectrum of microcystin-LR (5) (PDF)
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AUTHOR INFORMATION
Corresponding Author
*E-mail:
[email protected]. ORCID
Matthew J. Bertin: 0000-0002-2200-0277 Guy T. Carter: 0000-0002-9386-067X Notes
The authors declare no competing financial interest.
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ACKNOWLEDGMENTS We are very grateful to Dr. S. Boussert and the College of Charleston Department of Chemistry and Biochemistry for use of their polarimeter. We also thank Dr. D. Bearden of NIST at Hollings Marine Laboratory for technical assistance and helpful discussions on NMR.
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REFERENCES
(1) Gulder, T. A. M.; Moore, B. S. Curr. Opin. Microbiol. 2009, 12, 252−260. (2) Sasso, S.; Pohnert, G.; Lohr, M.; Mittag, M.; Hertweck, C. FEMS Microbiol. Rev. 2012, 36, 761−785. 1374
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(30) Fujii, K.; Ikai, Y.; Oka, H.; Suzuki, M.; Harada, K. Anal. Chem. 1997, 69, 5146−5151. (31) Bhushan, R.; Vashistha, V. K. J. Chromatogr. A 2015, 1379, 43− 50.
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