Possible Biosynthetic Products and Metabolites of Kainic Acid from the

Article Cite This: J. Nat. Prod. 2019, 82, 1627−1633

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Possible Biosynthetic Products and Metabolites of Kainic Acid from the Red Alga Digenea simplex and Their Biological Activity Yukari Maeno,† Ryuta Terada,‡ Yuichi Kotaki,§ Yuko Cho,† Keiichi Konoki,† and Mari Yotsu-Yamashita*,† †

Graduate School of Agricultural Science, Tohoku University, 468-1 Aramaki-Aza-Aoba, Aoba-ku, Sendai 980-8275, Japan United Graduate School of Agricultural Sciences, Kagoshima University, 1-21-24 Korimoto, Kagoshima 890-0065, Japan § Fukushima College, 1-1 Chigoike Miyashiro, Fukushima 960-0181, Japan Downloaded via RUTGERS UNIV on August 6, 2019 at 22:48:22 (UTC). See https://pubs.acs.org/sharingguidelines for options on how to legitimately share published articles.

‡

S Supporting Information *

ABSTRACT: Four kainic acid (KA, 1)-related compounds, 4hydroxykainic acid (2), allo-4-hydroxykainic acid (3), Ndimethylallyl-L-glutamic acid (4), and N-dimethylallyl-threo-3hydroxyglutamic acid (5), were isolated from the red alga Digenea simplex. The structures of these compounds were elucidated using spectroscopic methods. Compounds 2 and 3 are possible oxidative metabolites of KA and allo-KA (6), respectively. Compound 4 was recently reported as the biosynthetic precursor of KA, but the absolute configuration of 4 has not been previously determined. Herein, we determined the absolute configuration of 4 as 2(S) using advanced Marfey’s method. Compound 5 is similar to Ngeranyl-3(R)-hydroxy-L-glutamic acid (8), which was previously identified in a domoic acid (DA)-containing red alga. Compounds 5 and 8 are predicted to be biosynthetic byproducts of the radicalmediated cyclization reaction to form the pyrrolidine rings of KA and DA, respectively. Furthermore, the toxicities of 1−5 in mice were examined by intracerebroventricular injection. The toxicity of 2 was less than that of KA; however, the mice injected with 2 showed symptoms similar to those induced by KA, while 3−5 did not induce typical symptoms of KA in mice.

K

allo-KA7 (6, Scheme 1A) and 1′-hydroxy KA8 have been reported as major natural analogues of KA. Recently, a two-enzyme biosynthetic pathway for generating KA from L-glutamic acid (L-Glu) and dimethylallyl diphosphate (DMADP) in D. simplex was elucidated by Moore’s group.9 They identified KA biosynthetic (kab) gene clusters, containing an N-prenyltransferase (kabA)- and an αketoglutarate (α-KG)-dependent dioxygenase (kabC), from D. simplex and another red alga, Palmaria palmata. Biosynthesis research on DA, a member of the kainoid family, has also been conducted actively. The first biosynthetic pathway for obtaining DA was proposed by Wright’s group,10 and we also proposed a biosynthetic pathway based on the structures of plausible intermediates isolated from the DA-producing red alga Chondria armata and a precursor labeling experiment using a DA-producing diatom.11 Moore’s group recently also identified DA biosynthetic genes (dabA−D) in the diatom and characterized a geranyl transferase DabA, which catalyzes the N-geranylation of L-Glu to give N-geranyl-L-Glu.12 Moore’s group showed that an α-KG-dependent non-heme iron dioxygenase (DabC) catalyzed the formation of the pyrrolidine

ainic acid (KA, 1), a neuroexcitatory amino acid, was originally isolated as a principal anthelmintic from the red alga Digenea simplex.1 The structure of KA is closely related to those of other excitatory amino acids, such as the amnesic shellfish toxin domoic acid (DA)2 and the mushroom toxin acromelic acid;3 thus, these compounds are categorized as kainoids (Figure 1). All known naturally occurring kainoids

Figure 1. Major kainoids.

have a pyrrolidine ring that is commonly substituted by a COOH at C-2 and by a CH2COOH at C-3, while the structure of the substituent at C-4 varies in kainoid families (2S, 3S, 4S configuration). Kainoids behave as agonists to kainate receptors (GluK1−K5), which are ionotropic glutamate receptors (iGluR).4 Due to such important biological activity and the characteristic structural features, more than 60 routes for the synthesis of KA have been reported.5,6 However, only © 2019 American Chemical Society and American Society of Pharmacognosy

Received: February 11, 2019 Published: May 22, 2019 1627

DOI: 10.1021/acs.jnatprod.9b00128 J. Nat. Prod. 2019, 82, 1627−1633

Journal of Natural Products

Article

Figure 2. Structures of 2−5 with HMBC correlations and observed key NOEs.

Table 1. 1H (600 MHz) NMR Data of 1−6 in D2O KA (1) δH (J in Hz)

position 2 3 4 5α 5β 6a 6b 2′a 2′b 3′ a1

2

4.06, 3.04, 2.98, 3.39, 3.59, 2.43, 2.34, 5.00, 4.72, 1.72,

δH (J in Hz)

d (2.9) m m t (11.5) dd (12.0, 7.3) dd (16.7, 6.5) dd (16.7, 8.5) s s s

3

4

δH (J in Hz)

δH (J in Hz)

4.05, d (1.5) 2.94, m

3.93, d (10.9) 2.83, m

3.73, 3.41, 2.24, 2.04, 5.03, 4.86, 1.76,

3.23, 3.65, 2.58, 2.57, 5.21, 5.11, 1.75,

3.55, 2.03, 5.21, 3.62,

d (12.3) d (12.3) dd (15.2, 6.2) dd (15.2, 8.5) s s s

d d d d s s s

(12.3) (12.3) (1.4) (2.6)

allo-KA (6)a,13

5 δH (J in Hz)

t (5.9) m t (7.3) t (8.5)

3.49, 4.26, 5.20, 3.67,

2.38, m

δH (J in Hz)

d (6.8) m t (8.5) t (4.4)

1.75, s

2.72, dd (16.1, 4.1) 2.55, dd (16.1, 7.9) 1.75, s

1.67, s

1.66, s

3.93, d (8.7) 2.68−2.65, m 2.89, dt (10.8, 8.1) 3.36,b t (11.2) 3.57,b dd (11.8, 8.1) 2.70, dd (15.0, 4.4) 2.45, dd (14.8, 7.2) 4.99, s 4.98, s 1.75, s

H NMR (500 MHz). bα and β were not assigned. The signal of the remaining CH3OD (3.30 ppm) was used as the internal reference.

Table 2.

13

C (151 MHz) NMR Data of 1−6 in D2O KA (1)

2

position

δC, type

δC, type

2 3 4 5 6 7 8 1′ 2′ 3′

66.7, CH 41.7, CH 46.7, CH 47.6, CH2 34.2, CH2 176.7, C 174.2, C 140.7, C 114.4, CH2 23.1, CH3

66.4, CH 49.0, CH 83.8, C 53.2, CH2 40.6, CH2 179.7, C 174.4, C 142.3, C 114.3, CH2 17.9, CH3

a

3

4

HMBC

δC, type

HMBC

δC, type

5, 6 2, 5, 6 2, 3, 5, 6, 2′, 3′

64.8, CH 46.7, CH 84.0, C 55.6, CH2 33.1, CH2 177.9, C 174.3, C 142.7, C 115.8, CH2 19.1, CH3

3, 5, 6 2, 5, 6 5, 6, 2′, 3′

60.5, CH 25.7, CH2 112.6, CH 43.9, CH2 32.3, CH2 179.3, C 173.3, C 144.6, C 24.9, CH3 17.2, CH3

a

2 6 2 3′ 3′ 2’

a

2, 3 3, 6 2 2′, 3′ 3′ 2’

a

allo-KA(6)b,13

5 HMBC

δC, type

3, 6 2, 6 5, 2′, 3′ 2 2, 3 3, 6 2, 3 5, 2′, 3′ 3′ 4, 2’

64.5, CH 66.7, CH 112.5 CH 44.4, CH2 40.4, CH2 175.6, C 170.9, C 145.2, C 25.0, CH3 17.5, CH3

a

HMBC 6, 2, 5, 2 2 3, 2 5, 4, 4,

5 6 2′, 3′

6 2′, 3′ 3′ 2’

δC, typea 65.0, CH 42.4, CH 48.3, CH 51.5, CH2 38.9, CH2 178.0, C 173.6, C 140.5, C 115.1, CH2 18.0, CH3

δC were determined based on HSQC and HMBC data. b13C NMR (125 MHz). The signal of the remaining CH3OD (49.8 ppm) was used as the internal reference.

a

■

ring, presumably via a radical reaction mechanism,12 similar to KabC in KA biosynthesis. We are also interested in the biosynthesis of KA and have conducted screening studies for the biosynthetic intermediates of KA. Herein, we report four new KA-related compounds (2− 5) from D. simplex, which support the proposed pathway for biosynthesis of KA9 and the cyclization mechanism to form the pyrrolidine ring of KA by comparison with DA biosynthesis. In addition, we propose a new oxidative metabolism pathway for KA and report the toxicities of KA and 2−5 in mice to which the compounds were administered by intracerebroventricular (i.c.v.) injection.

RESULTS AND DISCUSSION An aqueous extract of dried D. simplex was screened for new KA-related compounds having the molecular formula C10HxNOy using reversed-phase (RP)-high-resolution (HR)LC-MS. Three unknown peaks were detected at [M + H]+ m/z 230.1023 (C10H16NO5+), m/z 216.1230 (C10H18NO4+) (4), and m/z 232.1179 (C10H18NO5+) (5) (Figure S1). Moreover, the same extract was also analyzed using cation exchange (CE)-RP column chromatography. This analysis resulted in detection of two peaks at m/z 230.1023 (C10H16NO5+) (2, 3) (Figure S2). Compounds 2, 3, 4, and 5 (35, 20, 156, and 838 μg, respectively, quantified using LC-MS) were purified from an aqueous extract of dried D. simplex (20, 45, 5, and 5 g, respectively) by sequential column chromatography. Structural characterization of these compounds was performed using 1628



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Figure 3. Determination of absolute configuration of 4. (A) Synthetic scheme of 4 and 4′. (B) HR-LC-MS extracted ion chromatograms ([M + H]+ m/z 510.2195 ± 0.05) of Marfey’s derivatives of natural 4, synthetic 4, and synthetic 4′.

NMR techniques; 1H NMR, COSY, TOCSY, HSQC, HMBC, and NOESY1D spectra were acquired for 2−5. The molecular formulas of 2 and 3, C10H15NO5, have one additional oxygen than that for KA (C10H15NO4). The 1H NMR signals and 1H connectivities indicated by the COSY and TOCSY spectra (Figures S9, S10, S23, S24) suggest that 2 and 3 are analogues of KA that have a pyrrolidine ring substituted by COOH, CH2COOH, and an isopropenyl group at C-2, C-3, and C-4, respectively. In the COSY spectra of 2 and 3, allylic coupling correlations of H3-3′/H-2′ were observed, as also observed in the spectrum of KA. However, the H-4 signal for KA was lacking for 2 and 3, and the signals corresponding to 5-CH2-N appeared as two doublet signals due to geminal coupling. In the case of KA, one double doublet and one triplet signal appeared for 5-CH2-N, where this group was also coupled with H-4 (Table 1). Therefore, in 2 and 3, C4 is suggested to be a nonprotonated carbon. Moreover, C-4 in 2 and 3 is assigned as a carbon bonded to an oxygen atom, with signals at δC 83.8 ppm and δC 84.0 ppm, respectively, based on the HMBC correlations (H2-5/C-4, H2-6/C-4, H-2′/ C-4, and H-3′/C-4; Figure 2). In addition, key HMBC correlations (H-2/C-4, H-3/C-4) were observed in the spectrum for 2, whereas these correlations could not be observed for 3 due to the limited quantity of sample. Other HMBC correlations (Table 2) also support the assignment that the planar structures of 2 and 3 are different from that of KA in that C-4 is hydroxylated. Compounds 2 and 3 are suggested to be stereoisomers. The H-2/H-3-trans and H-3/4-isopropenyltrans configurations in 2 are suggested by the observed NOEs from H2-6 to H3-3′, from H-5α to H-6b, from H-2 to H3-3′, and between H-6b and H-2 using NOESY1D analysis (Figure 2). These NOEs were also observed for KA (Figures S61, S62, S74, S75). In a previously reported method for determining the relative configuration of kainoids using 1H NMR chemical shifts,14 an H-2 signal below 4.2 ppm correlates with the H-2/ H-3-trans configuration, whereas an H-2 signal above 4.2 ppm corresponds to the H-2/H-3-cis configuration. The present data suggest the H-2/H-3-trans configuration in 2, because the chemical shift of H-2 in 2 is δH 4.05 ppm. In addition, the value of 3JH‑2/H‑3 (1.5 Hz) for 2, which is close to that of KA (2.9 Hz) but far from that of allo-KA (Scheme 1A; 6) (8.7 Hz)13 (Table 1), also suggests the H-3/4-isopropenyl-trans configuration. The absolute configuration of 2 is predicted as 2S, 3S, 4R, because C-2 (2S) possibly originated from the α-carbon of L-Glu, similar to the case of KA. The relative configuration of 3

was predicted as follows: the H-2/H-3-trans and H-3/4isopropenyl-cis configurations in 3 are supported by the observed NOEs between H2-6 and H-2, between H-3 and H33′, between H3-3′ and H-5β, from H-2 to H-5α, from H-3 to H-5β, and from H2-6 to H-5α using NOESY1D analysis (Figure 2). The chemical shift of H-2 (3.90 ppm) also supports the H-2/H-3-trans configuration according to the reported method as described above.14 Furthermore, the H-3/4isopropenyl-cis configuration is suggested by the value of 3 JH‑2/H‑3 (10.6 Hz), which is closer to that of allo-KA (8.7 Hz) than that of KA (2.9 Hz) (Table 1). Therefore, 3 was assigned as allo-4-hydroxy KA. For further confirmation, the stable conformers of KA, 2, 3, and allo-KA were simulated using the MM2 force field in the Chem3D modeling software (Figure S83). In such conformers, the dihedral angles of H2−C2−C3− H3 are 107° and 149° for KA and allo-KA, respectively, and the calculated values of 3JH‑2/H‑3 based on these dihedral angles using the Karplus equation15 are estimated as 2.8 and 9.7 Hz for KA and allo-KA, respectively, which are close to the values measured using 1H NMR (2.9 Hz for KA and 8.7 Hz for alloKA, Table 1). In a like manner, the dihedral angles and the calculated 3JH‑2/H‑3 values for 2 and 3 are 102° and 2.3 Hz, and 156° and 10.7 Hz, respectively. These values are also close to the values measured using 1H NMR: 1.5 Hz for 2 and 10.6 Hz for 3. These results support the assigned relative configurations of 2 and 3 and also indicate that the relative configuration of the H-3/4-isopropenyl groups causes a large difference in the values of 3JH‑2/H‑3; the value for the trans configuration is smaller than that of the cis configuration. The absolute configuration of C-2 in 3 is predicted as 2S for the same reason as discussed for 2, although this assignment has not been independently confirmed. Compound 4 has the molecular formula C10H17NO4. The COSY, TOCSY, HSQC, and HMBC correlations (Figure 2) suggest that 4 is N-dimethylallylglutamic acid (DMA-Glu), which was recently reported by Moore’s group as a substrate that could be cyclized by KabC to form KA.9 They mentioned the presence of 4 in the aqueous extract of D. simplex based on the exact mass and retention time by HR-LC-MS analysis. However, the absolute configuration of 4 has not been previously determined. It is necessary to determine it, because both L-Glu and D-Glu can be substrates of KabA, which can produce both enantiomers of 4 (Figure S6 in ref 9). Herein, the absolute configuration of 4 was determined as 2S by derivatization with Marfey’s reagent, Nα-(5-fluoro-2,4-dinitro1629



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Scheme 1. (A) Previously Proposed Biosynthetic Pathway to 4 and KA9 and Proposed Pathway to 2, 3, and 5 in This Study; (B) Previously Proposed Biosynthetic Pathway to DA Analogue (9)11,12 and DA12a

a

The compounds that have been isolated from natural sources are enclosed in squares with broken lines.

phenyl)-L-leucinamide (L-FDLA),16 and comparison with the derivatives of synthesized 4 and its enantiomer (4′) using HRLC-MS (Figure 3). The synthetic standards were prepared by reductive amination of 3-methyl-2-butenal and L- or D-Glu with NaBH3CN (Figure 3). This result suggests that 4 originates from L-Glu. Compound 5 has the molecular formula C10H17NO5. The COSY, TOCSY, HSQC, and HMBC correlations (Figure 2) suggest that 5 has a linear structure similar to that of 4; however, hydroxylation of 5 at C-3 is indicated by the oxymethine HSQC correlation (δH/δC 4.26/66.7) and by the HMBC correlations (C-3/H-2, C-3/H2-6; Figure 2). Therefore, 5 is determined to be N-dimethyl allyl-3-hydroxyglutamic acid (DMA-3-hydroxy-Glu). The relative configuration of C-2 and C-3 in 5 was suggested as threo by the similarity of the 3 JH‑2/H‑3 value to that of 8 (5: 6.8 Hz, 8: 6.7 Hz). Compound 8 (Scheme 1B) is a previously reported possible precursor of DA, for which the configuration was determined as 2S, 3R based on chemical synthesis in our previous study.11 The 1H NMR chemical shifts and coupling patterns of H-4 and H2-6 for 5 [H-4, 5.20 t (8.5 Hz); H2-6, 2.72 dd (16.1, 4.1 Hz), 2.55 dd (16.1, 7.9 Hz) in D2O] were similar to those of 8 [H-4, 5.29 t (7.6 Hz); H2-6, 2.82 dd (16.3, 3.7 Hz), 2.60 dd (16.1, 7.9 Hz) in CD3OD] (Figure S77). Furthermore, the coupling patterns and chemical shifts of the methylene group in the 3hydroxy-Glu segment of 5 and 8 are close to those of threo-3hydroxy-Glu [nonequivalent; Ha, 2.88 dd (16.5, 4.6 Hz), Hb, 2.73 dd (16.5, 8.7 Hz) in D2O], whereas these coupling patterns are not similar to those of erythro-3-hydroxy-Glu

[equivalent; 2H, 2.82 d (6.7 Hz) in D2O].17 Based on these data, 5 was assigned as DMA-threo-3-hydroxy-Glu. The absolute configuration of 5 was predicted as 2S, 3R, because 2S should biosynthetically originate from L-Glu, although direct experimental evidence is needed to determine it. The NMR data for 2−5 are summarized in Tables 1 and 2 along with those of 1 and 6 for comparison. The first step in the biosynthesis of KA was recently reported by Moore’s group as condensation of L-Glu and DMADP to form 4, catalyzed by KabA (Scheme 1 A).9 They also confirmed that the recombinant KabC, a non-heme iron(II)- and α-KG-dependent dioxygenase from D. simplex, catalyzes the direct cyclization of 4 to form KA (route a).9 However, the absolute configuration of 4 has not been determined. In this study, 4 was isolated from D. simplex and was determined to be DMA L-Glu, supporting the proposed biosynthetic pathway to KA. In addition, the pathway for oxidative metabolism of KA and allo-KA (6)7 to form 2 and 3 was predicted herein based on the structures of these KA analogues (Scheme 1A). Moore’s group hypothesized that a radical would be produced by converting the FeIVO species to an FeIII−OH species in the enzyme and that the radical would be localized at C-3 of 4 in the first step of the cyclization (I in Scheme 1A; see also Figure S9 in ref 9). In this pathway, 5 could be interpreted as a byproduct for which the radical at C-3 in 4 (I in Scheme 1A, route b) traps a hydroxyl radical, plausibly originating from the FeIII−OH species in the enzyme (Scheme 1A, route b, Figure S84). Hence, the identification of 5 in D. simplex in this study would support the KabC-catalyzed 1630



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substitution of the hydrogen at C-4 with a hydroxy group decreases the toxicity, but this decrease is not significant. The linear compounds 4 and 5, used as references, did not induce any symptoms when administered at 4.4 nmol.

radical-mediated cyclization mechanism (Scheme 1A, route a) in the biosynthesis of KA. In the biosynthetic pathway to DA, Moore’s group confirmed that a non-heme iron(II)- and αKG-dependent dioxygenase (DabC) in the diatom catalyzes the direct cyclization of a 4-like compound, N-geranyl-L-Glu (7)11 (Scheme 1B) to form 9 as one of the products, although the major substrate of this enzyme was assigned as 7′-carboxy7, which was converted to isodomoic acid A (IA), with subsequent isomerization to DA.12 Compound 8, a similar compound to 5, identified in our previous study12 was also predicted to be a byproduct in the radical cyclization of DA analogue 9. This was supported by a labeling experiment with synthetic [2,5-D2] 8 using the DA-producing diatom Nitzschia navis-varingica (see S83, Figure S87). In this experiment DA was not labeled, although this negative result could have occurred from lack of uptake into the cells rather than lack of incorporation into the pathway. A linear compound structurally similar to 4 and 7 was also isolated from an acromelic acid (Figure 1)-containing mushroom.18 Thus, this cyclization mechanism for formation of the pyrrolidine ring is common in kainoids. The amounts of 1−6 in frozen D. simplex were quantified using RP-LC-MS. The ratio of 2 to 3 was analyzed using CERP-LC-MS. As summarized in Table 3, 2−6 were the minor

■

CONCLUSION Both epimers of hydroxy KA at C-4 (2, 3) and the linear compounds 4 and 5 were isolated from the KA-containing red alga D. simplex. Compounds 2 and 3 can be regarded as the oxidized metabolites of KA and allo-KA, respectively. The absolute configuration of 4 was determined as 2(S), supporting the proposed biosynthetic pathway toward KA. The structure of 5 was similar to that of the DA-related compound 8. Identification of 3-hydroxylated compounds 5 and 8 in KAand DA-containing red alga, respectively, supports the radicalmediated cyclization reaction for formation of the pyrrolidine rings of KA and DA. Hydroxylation at C-4 of KA decreased the toxicity in mice, but this decrease was not significant in 2, in which the isopropenyl group at C-4 is α-oriented.

■

Table 3. Content of 1−6 in D. simplex content (nmol/g wet weight)a

compound

4500 0.53 0.062 3.9 32 0.66

1 2 3 4 5 6

± ± ± ± ± ±

170 0.014 0.0016 0.30 0.44 0.020

Results are expressed as the means ± SD (n = 3).

a

components compared with 1, constituting only 0.012−0.73% (mol/mol) of 1. Notably, the ratio of 2 to 1 (0.012%) was significantly lower than that of 3 to 6 (9.1%), suggesting that 6 is more easily oxidized at the C-4 position than 1, probably due to the stereochemical preference of the enzyme involved in this oxidative reaction. The toxicities of 1−5 in mice were investigated by i.c.v. injection19 (Table 4). These compounds were quantified by Table 4. Toxicities of 1−5 in Mice (i.c.v.)a compound

dose (nmol)

death time

symptoms

KA (1)

1.1

15−30 min

2 3 4 5

2.2 2.2 4.4 4.4

no no no no

frequent, scratching, spasms, sudden jumping same as for 1 no adverse symptoms no adverse symptoms no adverse symptoms

deaths deaths deaths deaths

EXPERIMENTAL SECTION

General Experimental Procedures. Most of the NMR spectra were acquired with an Agilent 600 MHz NMR spectrometer in 0.4 mL of D2O (deuteration degree: 99.95%) at 20 °C. HSQC, HMBC (3JCH = 8 Hz), and NOESY1D spectra of 2 and 3 were acquired using 0.5 mL (for a 5 mm Super grade, Kanto Chemical Co. Ltd.) and 0.2 mL (for a micro bottom tube, cat. #SP504, Shigemi) of D2O, respectively, on a Bruker AVANCE III 600 instrument with a 5 mm CryoProbe. The data were referenced to residual solvent signals with resonances at δH/C = 3.30/49.8 ppm (remaining CH3OD). LC-MS analysis was performed with a micrOTOF-Q II mass spectrometer (HR, ESI, Q-TOF; Bruker Daltonics) and an API2000 (ESI, triple quadrupole; AB SCIEX) apparatus. HRMS data were acquired with a micrOTOF-Q II mass spectrometer. The reagents for isolation, organic synthesis, and Marfey’s derivatization were purchased from Wako Pure Chemical Industries, Ltd., Sigma-Aldrich Co., and Tokyo Chemical Industry Co. Ltd. LC-MS-grade CH3CN, formic acid (Wako Pure Chemical Industries, Ltd.), and MeOH (Kanto Chemical) were used for HR-LC-MS. Distilled and purified H2O (Milli-Q) by Simplicity UV (Merck Millipore Corporation) was used for all the experiments. Plant Materials and Diatoms. The red alga Digenea simplex (wet) was collected by snorkeling at Hanasezaki, Ibusuki, Kagoshima, Japan, on May 30, 2018, at a depth of approximately 1 m during low tide and identified by R.T. (one of the authors). The collected alga was immediately frozen at −30 °C and kept at this temperature before use. The dried D. simplex was purchased from Uchida Wakanyaku Ltd. and kept at room temperature. The diatom Nitzschia navisvaringica was isolated from Onna Villege, Kunigami-Gun, Okinawa Prefecture, Japan, on December 28, 2017, by Y.K. (one of the authors) and identified by Dr. Nina Lundholm, Natural History Museum of Denmark, University of Copenhagen. Screening of KA-Related Compounds Using HR-LC-MS. The dried red alga, D. simplex (5 g), was homogenized with H2O (200 mL) and centrifuged for 5 min at 12000g at 4 °C. The supernatant was concentrated under vacuum and loaded onto an activated charcoal column (54 mL). The column was washed with H2O (500 mL), and then the compounds were eluted from the column with H2O−EtOH−AcOH (50:50:3, v/v/v, 200 mL). The eluate was evaporated under vacuum and dissolved in H2O−HCOOH (100:0.1, v/v, 4 mL). The solvent was loaded onto a Cosmosil 140C18-OPN column (6 mL, Nacalai Tesque) pre-equilibrated with H2O− HCOOH (100:0.1, v/v). After washing the column with the same solvent (12 mL), the two solvents, H2O−HCOOH (100:0.1, v/v, 30 mL) and H2O−MeOH−HCOOH (95:5:0.1, v/v/v, 30 mL), were supplied to the resin in a stepwise manner, and an aliquot of each eluate was subjected to HR-LC-MS using a micrOTOF-QII mass

a

n = 3.

LC-MS using the standard curve of KA. Although 2 (2.2 nmol) was not lethal, the injected mice exhibited the typical symptoms induced by KA,19 whereas the mice injected with the same dose of 3 (2.2 nmol) did not show any such symptoms. This suggests that the α-orientation of the isopropenyl group at C-4 in KA is crucial to the biological activity, consistent with a previous study.20 In addition, 1631



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TCI Dual ODS CX15 column (4.6 × 250 mm, 5 μm, Tokyo Chemical Industry Co. Ltd.) and H 2 O−CH 3 CN−HCOOH (90:10:0.1, v/v/v) at a flow rate of 0.3 mL/min. Toxicities of 1−5 in Mice. The mouse assays were approved by the Animal Ethical Committee of Tohoku University (protocol approval number 2016AgL-003). Male ddY mice (11−13 g, 3 weeks old) were purchased from Japan SLC. Each compound was tested using three mice. Compounds 1−6 were isolated from D. simplex in this study. Compounds 2−6 were quantified using LC-MS as described above using weighed 1 (3.7 mg) as the standard. Intracerebroventricular injection was performed according to the method reported by Sakai and co-workers.19 An aqueous solution (10 μL) of 1 (1.1 nmol), 2 (2.2 nmol), 3 (2.2 nmol), 4 (4.4 nmol), and 5 (4.4 nmol) was injected slowly over 5 s at 2 mm lateral to the midline of the skull, 3 mm rostal to a line down through the anterior base of the ears, at a depth of 3 mm. Water was used as a control vehicle. Synthesis of 4 and Its Enantiomer 4′. MeOH (1.5 mL) and 3methyl-2-butenal (30 μL, 0.31 mmol) were added to a solution of Lor D-glutamic acid (for 4 or 4′) (15 mg, 0.10 mmol) in 1.5 mL of H2O. After stirring for 0.5 h at room temperature, NaBH3CN (9 mg, 0.14 mmol) was added to the mixture, which was continuously stirred for 3 h at room temperature. The reaction mixture was concentrated, dissolved in H2O, and then loaded onto a Cosmosil 140C18-OPN column (1 mL) pre-equilibrated with H2O−HCOOH (100:0.1, v/v). The compounds were eluted from the column with H2O−MeOH− HCOOH (90:10:0.1, v/v/v, 3 mL). The eluate was concentrated and purified using an InertSustain C18 column (7.6 × 250 mm, 5 μm) with H2O−MeOH−HCOOH (95:5:0.1, v/v/v). Synthetic 4 (8.9 mg, 0.041 mmol, yield 41%): white powder; [α]20D +15 (c 0.43, H2O); 1H NMR (600 M Hz, D2O) δ 5.21 (1H, t, J = 7.3 Hz, H-4), 3.64−3.59 (3H, m, H-2, H2-5), 2.47 (2H, m, H-6), 2.11 (1H, m, H-3a), 2.03 (1H, m, H-3b) 1.75 (3H, s, H-2’), 1.67 (3H, s, H-3′); 13C NMR (D2O, 151 MHz) δ 176.7 (COOH, C-7), 172.9 (COOH, C-8), 144.6 (C, C-1′), 112.5 (CH, C-4), 59.7 (CH, C-2), 44.1 (CH2, C-5), 29.8 (CH2, C-6), 24.9 (CH2, C-3), 24.8 (CH3, C-2′), 17.2 (CH3, C-3′); HRESITOFMS m/z 216.1229 [M + H]+ (calcd for C10H18NO4+, 216.1230). Synthetic 4′ (9.1 mg, 0.042 mmol, yield 42%): white powder; [α]20D −14 (c 0.41, H2O); 1H NMR (600 M Hz, D2O) δ 5.21 (1H, t, J = 7.3 Hz, H-4), 3.64−3.59 (3H, m, H-2, H2-5), 2.47 (2H, m, H-6), 2.11 (1H, m, H-3a), 2.01 (1H, m, H-3b), 1.75 (3H, s, H-2′), 1.67 (3H, s, H-3′); 13C NMR (D2O, 151 MHz) δ 176.7 (COOH, C-7), 172.9 (COOH, C-8), 144.6 (C, C-1′), 112.5 (CH, C4), 59.7 (CH, C-2), 44.1 (CH2, C-5), 29.8 (CH2, C-6), 24.9 (CH2, C3), 24.8 (CH3, C-2′), 17.2 (CH3, C-3′); HRESIMS m/z 216.1233 [M + H]+ (calcd for C10H18NO4+, 216.1230). Synthetic 4 and natural 4 were analyzed by RP-LC-MS. RP-LC-MS was performed on a micrOTOF-Q II mass spectrometer using a Mightysil RP-18GP column (2.0 × 150 mm, 5 μm) and a mobile phase composed of H2O−MeOH−HCOOH (95:5:0.1, v/v/v) at a flow rate of 0.15 mL/ min. LC-MS chromatograms are shown in Figure S80. Marfey’s Analysis of 4. Approximately 60 μg of natural 4 (400 μg of synthetic 4 or 4′) was dissolved in 0.1 mL of H2O. The solutions were added to 40 μL of sodium bicarbonate (1 M) and 100 μL of LFDLA (1%) in acetone.16 The solution was stirred at 40 °C for 90 min, after which the reaction was quenched with 40 μL of 1 M HCl. The reaction mixture was partitioned between EtOAc and H2O, and the organic layer was concentrated. To purify the Marfey’s derivative, the crude mixture was subjected to RP-HPLC (Mightysil RP 18 GP column, 4.6 × 250 mm, 5 μm) at a flow rate of 0.4 mL/min using the following method: 0% B (10 min), 0−100% B (45 min), 100% B (30 min), where A = H2O−CH3CN−HCOOH (5:95:0.1, v/v/v), and B = H2O−CH3CN−HCOOH (95:5:0.1, v/v/v). Elution of the components was monitored with a diode array detector, Chromaster 5430 (Hitachi High-Technologies). The purified fraction containing the derivative was analyzed by HR-LC-MS, detected at m/z 510.2195 ± 0.02, using a Mightysil RP 18 GP column (2.0 × 150 mm, 5 μm) and H2O−MeOH−HCOOH (25:75:0.1, v/v/v) at a flow rate of 0.2 mL/ min.

spectrometer. LC was performed using a Mightysil RP-18GP column (2.0 × 150 mm, 5 μm, Kanto Chemical) with H2O−MeOH− HCOOH (95:5:0.1, v/v/v) as the mobile phase at a flow rate of 0.15 mL/min at 28 °C using two LC-30AD pumps (Shimadzu), a CTO20AC column oven (Shimadzu), a SIL-30AC autosampler (Shimadzu), and a CBM-20A communications bus module (Shimadzu). The data acquisition parameters for the mass spectrometer were as follows: ion polarity, positive; capillary, 4500 V; nebulizer, 1.6 bar; dry heater, 180 °C; dry gas, 7.0 L/min (N2). The extracted ion chromatograms of the predicted molecular formula for the KA-related compounds were analyzed with Smart Formula software (Bruker Daltonics). Extraction and Purification of 1−5. Dried D. simplex was used for extraction. Extraction with H2O (200 mL) and partial purification using a Cosmosil 140C18-OPN column were performed using the same procedures employed for screening. The eluate with H2O− HCOOH (100:0.1, v/v, 30 mL) was concentrated and applied to an InertSustain C18 column (7.6 × 250 mm, 5 μm, GL Sciences) with H2O−MeOH−HCOOH (97:3:0.1, v/v/v). Compounds 2 and 3 were eluted first as a mixture, and then, 1, 4, and 5 were subsequently eluted in almost pure form. The mixture of 2 and 3 was separated into the respective components by CE-RP chromatography using a TCI Dual ODS CX15 column (4.6 × 250 mm, 5 μm) with H2O− CH3CN−HCOOH (90:10:0.1, v/v/v), and 2 and 3 were obtained in almost pure form. The eluted compounds 1−5 were analyzed by routine LC-MS. Eventually, almost pure 1, 2, 3, 4, and 5 (3710, 35, 2.4, 156, and 838 μg, respectively) were obtained from dried D. simplex (5, 20, 40, 5, and 5 g dry weight, respectively). Further extraction and purification were performed to obtain a larger amount of 3 as follows. Extraction and partial purification using a Cosmosil 140C18-OPN column were performed using the procedures described above. The eluate was concentrated and applied to an InertSustain AQ C18 column (7.6 × 250 mm, 5 μm, GL Sciences) with H2O− HCOOH (100:0.1, v/v). The mixture of 2 and 3 was separated into the respective components by CE-RP chromatography using the same conditions described above. Almost pure 3 (20 μg) was obtained from dried D. simplex (45 g dry weight). Compound 1 was assigned based on HRESIMS m/z 214.1069 [M + H]+ (calcd for C10H16NO4+ 214.1074), along with 1H NMR, HSQC, and HMBC, where the data were largely consistent with the previously reported 1H and 13C NMR data.5e 4-Hydroxykainic acid (2): white powder; 1H and 13C data (Tables 1 and 2); HRESIMS m/z 230.1012 [M + H]+ (calcd for C10H16NO5+, 230.1023). allo-4-Hydroxykainic acid (3): white powder; 1H and 13C data (Tables 1 and 2); HRESIMS m/z 230.1014 [M + H]+ (calcd for C10H16NO5+, 230.1023). N-Dimethylallyl-L-glutamic acid (4): white powder; 1H and 13C data (Tables 1 and 2); HRESIMS m/z 216.1240 [M + H]+ (calcd for C10H18NO4+, 216.1230). N-Dimethylallyl-threo-3-hydroxyglutamic acid (5): white powder; 1H and 13C data (Tables 1 and 2); HRESIMS m/z 232.1173 [M + H]+ (calcd for C10H18NO5+, 232.1179). Quantitative Analysis of 1−6 in D. simplex by LC-MS. The frozen D. simplex (20 g) was used for quantification. Extraction with H2O (200 mL) and partial purification using a Cosmosil 140C18-OPN column were performed using the same procedures used for screening as described above. Two eluates with H2O−HCOOH (100:0.1, v/v, 30 mL; Fr.A, mainly containing 1 and 6) and H2O−MeOH− HCOOH (95:5:0.1, v/v/v, 30 mL; Fr.B, containing part of 1 and 6 and 2−5) from the Cosmosil 140C18-OPN column were individually evaporated and applied to an InertSustain C18 column with H2O− MeOH−HCOOH (97:3:0.1, v/v/v). The respective desalted compounds (1−6) obtained by this procedure were properly diluted and mixed, then subjected to RP-LC-MS three times. Compound 1 isolated from D. simplex was weighed (3.7 mg) and used as the standard for 1−6 for LC-MS quantitation. RP-LC-MS was performed on an API2000 mass spectrometer using a Mightysil RP-18GP column (2.0 × 150 mm, 5 μm) and a mobile phase comprising H2O− MeOH−HCOOH (95:5:0.1, v/v/v) at a flow rate of 0.2 mL/min. The abundance ratio of 2 to 3 was analyzed by CE-RP-LC-MS using a 1632



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Oborník, M.; Smith, G. J.; Hutchins, D. A.; Allen, A. E.; Moore, B. S. Science 2018, 361, 1356−1358. (13) Arena, G.; Chen, C. C.; Leonori, D.; Aggarwal, V. K. Org. Lett. 2013, 15, 4250−4253. (14) Hashimoto, K.; Konno, K.; Shirahama, H. J. Org. Chem. 1996, 61, 4685−4692. (15) Hoch, J. C.; Dobson, C. M.; Karplus, M. Biochemistry 1985, 24, 3831−3841. (16) Harada, K.; Fujii, K.; Hayashi, K.; Suzuki, M.; Ikai, Y.; Oka, H. Tetrahedron Lett. 1996, 37, 3001−3004. (17) Broberg, A.; Menkis, A.; Vasiliauskas, R. J. Nat. Prod. 2006, 69, 97−102. (18) Yamano, K.; Shirahama, H. Chem. Lett. 1993, 22, 21−24. (19) Sakai, R.; Swanson, G. T.; Shimamoto, K.; Green, T.; Contractor, A.; Ghetti, A.; Tamura-Horikawa, Y.; Oiwa, C.; Kamiya, H. J. Pharmacol. Exp. Ther. 2001, 296, 650−658. (20) (a) Biscoe, T. J.; Evans, R. H.; Headley, P. M.; Martin, M.; Watkins, J. C. Nature 1975, 255, 166−167. (b) Biscoe, T. J.; Evans, R. H.; Headley, P. M.; Martin, M. R.; Watkins, J. C. Br. J. Pharmacol. 1976, 58, 373−382.

ASSOCIATED CONTENT

S Supporting Information *

The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jnatprod.9b00128.

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1D and 2D NMR spectra of 1−5, HRMS spectra of 2− 5, HPLC chromatograms, simulated stable conformers of 1−3 and 6, and labeling experiments for 8 (PDF)

AUTHOR INFORMATION

Corresponding Author

*Tel/Fax: +81 22 7574425. E-mail: mari.yamashita.c1@ tohoku.ac.jp. ORCID

Keiichi Konoki: 0000-0001-5788-5426 Mari Yotsu-Yamashita: 0000-0002-5009-0409 Notes

The authors declare no competing financial interest.

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ACKNOWLEDGMENTS This work was funded by the Japan Society for the Promotion of Science (JSPS) through its KAKENHI Grant-in-Aid for Scientific Research no. JP17H03809 and by an Innovative Area, Frontier Research on Chemical Communications grant (no. JP17H06406) and on Redesigning Biosynthetic Machineries (no. JP19H04636) to M.Y.Y. The study was also supported by a Grant-in-Aid from Tohoku University, Division for Interdisciplinary Advanced Research and Education, to Y.M. Y.M. is a research fellow of the JSPS (DC1) (no. JP19J20430). We are grateful to Prof. R. Sakai, Hokkaido University, for assistance with intracerebroventricular injections to mice.

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REFERENCES

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