602 Chem. Pharm. Bull. 64, 602–608 (2016) Vol. 64, No. 6 Regular Article

Identification of Cyclopropylacetyl-(R)-carnitine, a Unique Chemical Marker of the Fatally Toxic subnigricans

Masanori Matsuura, Suguru Kato, Yoko Saikawa, Masaya Nakata, and Kimiko Hashimoto* Department of Applied Chemistry, Faculty of Science and Technology, Keio University; 3–14–1 Hiyoshi, Kohoku-ku, Yokohama 223–8522, Japan. Received December 20, 2015; accepted March 17, 2016

A toxic mushroom, Russula subnigricans, causes fatal poisoning by mistaken ingestion. In spite of the potent bioactivity, the responsible toxin had not been identified for about 50 years since its first documenta- tion. Recently, we isolated an unstable toxin and determined the structure. The slow elucidation was partly due to the instability of the toxin and also due to misidentification of R. subnigricans for similar . To discriminate genuine Russula subnigricans from similar unidentified Russula species, we searched for a unique chemical marker contained in the mushroom. Cyclopropylacetyl-(R)-carnitine specific to R. subnigri- cans was identified as a novel compound whose 1H-NMR signals appearing in the upfield region were easily recognizable among the complicated signals of the crude extract. Key words cyclopropylacetyl-(R)-carnitine; cycloprop-2-ene carboxylic acid; russuphelin G; mushroom poisoning; Russula subnigricans;

Mushroom poisonings attributable to the Russulaceae cause of accidental poisonings. The three representative Rus- mushroom Russula subnigricans were first documented in sula species identified to date in Japan are R. subnigricans, R. 1954 in Japan.1) In the past 50 years, the following seven nigricans, and R. densifolia; the latter two are considered to poisonings have occurred: [deaths/cases (year, place where be edible after cooking. The most useful characteristic feature poisoning occurred)] unknown/unknown (1954, Kyoto); 2/4 to discriminate R. subnigricans from the other two species is (1958, Osaka); 1/3 (1958, Osaka); 0/2 (1970, Toyama); 2/2 the color change that occurs after scratching fruiting body. All (2005, Aichi); 1/1 (2006, Miyazaki); 1/3 (2007, Osaka). In three species have whitish flesh that are tinged reddish brown addition, nine individuals in Taiwan were identified with on scratching. After that, the colors of R. nigricans and R. symptoms of R. subnigricans poisoning in 1998.2) Typical densifolia turn black, whereas the color of R. subnigricans is symptoms following ingestion of R. subnigricans are vomiting persistent. However, classifying these species based on only and diarrhea, which first appear approximately 30 min after this color change is difficult and unreliable. Although discrim- ingestion, followed by stiff shoulders, back ache, and bloody ination of R. subnigricans from R. densifolia is rather easy, as urine that is reddish-brown in color due to high levels of the latter has crowded gills compared to the former, the clas- myoglobin, not hemoglobin. Myoglobin is an oxygen-binding sification of Russula species becomes more complicated due to protein pigment of striated muscles, such as skeletal and car- the existence of several similar unclassified species in addition diac muscles, and is discharged following the breakdown of to the three aforementioned Russula species. Unclassified Rus- myocells, a condition termed rhabdomyolysis. In severe cases sula spp. have distant gills and fruiting bodies that undergo of R. subnigricans poisoning, further symptoms develop, in- color changes similar to those of R. subnigricans. cluding speech impediment, chronic convulsion, contraction Here, to discriminate genuine R. subnigricans from similar of pupils, loss of consciousness, and weakening of the heart, unclassified Russula species, we searched for a compound that resulting in death.3) could serve as a unique chemical marker. In spite of the strong toxicity of R. subnigricans, the re- sponsible toxin remained unknown until recently, when we Results and Discussion isolated and identified a small, unstable compound, cycloprop- Prior to our identification of cycloprop-2-ene carboxylic 2-ene carboxylic acid (1; Fig. 1), as the fatal toxin of this acid as the compound responsible for R. subnigricans toxic- mushroom.4) The toxicity of this compound was demonstrated ity,4) several candidate molecules had been reported. Using on administration to mice, which displayed severe rhabdo- fruiting bodies collected in Miyagi Prefecture, Japan, 3-hy- myolysis. It appears that the difficulty in identifying this droxybaikiain (2),5) russuphelins A–F (3A–F),6,7) and a related compound as the responsible toxin was due in part to its in- compound, russuphelol,8) were isolated (Fig. 1). Among these stability; concentration of solutions of this toxin by drying, a compounds, russuphelins A–D showed cytotoxic acitivity. To common technique in chemical separation and isolation steps, confirm the results of these experiments, we also collected R. promotes its polymerization. Polymerized cycloprop-2-ene subnigricans candidate in Miyagi Prefecture in a broadleaf carboxylic acid lacks toxicity. forest including the Japanese oak, Quercus serrata (one of In addition to inactivation, the long period required for the generally proposed host trees of R. subnigricans; Japanese the determination of cycloprop-2-ene carboxylic acid as the name: konara), and subjected fruiting bodies to methanol responsible toxin is attributable to difficulties discriminating extraction. According to a previous report,5) we isolated 3-hy- R. subnigricans from similar species, which is the primary droxybaikiain (2) from the aqueous layer of the methanol ex-

* To whom correspondence should be addressed. e-mail: [email protected] © 2016 The Pharmaceutical Society of Japan Vol. 64, No. 6 (2016) Chem. Pharm. Bull. 603

Fig. 1. Chemical Structures Appeared in Text

Fig. 2. Photos of Three Russula spp. Used in This Study (a) R. subnigricans collected in Kyoto, (b) Russula sp. collected in Miyagi, (c) Russula sp. collected in Saitama.

tract, which partitioned between water (H2O) and ethyl acetate carboxylic acid (1), from the Kyoto specimen, but could not (EtOAc), while russuphelins A and D (3A, D)6,7) were isolated isolate the toxin in the other two R. subnigricans candi- from the organic layer. Thus, we confirmed that the Russula dates. Taken together, these results indicated that the Kyoto sp. collected in Miyagi Prefecture was the mushroom materi- specimen was genuine R. subnigricans, whereas the other two als previously analyzed and reported in refs. 5–8. Notably, in specimens collected in Miyagi and Saitama were not.4) Thus, addition to these compounds, we isolated a new russuphelin the existence of two unidentifiable mushroom species that congener which we named russuphelin G (3G). On compari- shared nearly identical features with R. subnigricans prompted son of the spectroscopic data (see Experimental) with those of us to search for a unique chemical marker among these three other russuphelins, the structure of 3G was determined to be a species. The photos of R. subnigricans and unidentified two demethyl congener of russuphelin E or F (3E, F). In addition, Russula spp. collected in Miyagi or Saitama are cited in the position of the methoxy group was determined by nuclear Fig. 2. Overhauser effect (NOE) analysis (see Experimental). Genuine R. subnigricans contains cycloprop-2-ene car- We also collected two additional R. subnigricans candidate boxylic acid (1), which could potentially serve as a chemical specimens in Kyoto and Saitama Prefectures for analysis, be- marker to distinguish the fatally toxic mushroom from other cause the first poisoning of R. subnigricans occurred in Kyoto, similar species by chemical analysis. However, as mentioned, and Saitama Prefecture is located between Miyagi and Kyoto toxic compound 1 is easily polymerized under concentrated Prefectures with respect to latitude. In Kyoto Prefecture, conditions, limiting the usefulness of this toxin as a unique mushrooms were collected in a chinquapin forest including marker of genuine R. subnigricans. We next compared the Castanopsis cuspidate (Japanese name: tsuburajii or kojii), 1H-NMR spectra of the crude water extracts of R. subni- whereas in Saitama Prefecture, mushrooms similar to those of gricans and the two unidentifiable species. In the 1H-NMR Miyagi Prefecture were collected in a broadleaf forest includ- spectrum of the R. subnigricans (Kyoto) extract, characteristic ing Quercus serrata, similar forest to that of Miyagi Prefec- signals of a cyclopropane unit were observed in the upfield ture. Our analysis revealed that neither of these two candidate region (0.15, 0.52 ppm). As this compound was not found in specimens contained either russuphelins or 3-hydroxybaikiain the 1H-NMR spectra of the extracts from the two examined (2). Among the three candidate R. subnigricans specimens species, we attempted to isolate the compound indicated by obtained from Miyagi, Kyoto, and Saitama Prefectures, only the 1H-NMR measurement. the Kyoto candidate exhibited fatal toxicity to mice on oral The fruiting bodies of genuine R. subnigricans were cut administration of the water extract. Following further separa- into small pieces (approximately 5 mm) and extracted with tion, we isolated the responsible lethal toxin, cycloprop-2-ene 0.3% acetic acid (AcOH). After filtration and dialysis, the 604 Chem. Pharm. Bull. Vol. 64, No. 6 (2016)

Table 1. NMR Data of Cyclopropylacetyl-(R)-carnitine (4)

1H-NMR (300 MHz, D O) 13C-NMR (75 MHz, D O) Position 2 2 HMBC correlation HOD=4.79 DSSa)=−2.04

1 — 177.1 C2-Ha, Hb 2 a: 2.49 (1H, dd, J=8.0, 16.0 Hz) 40.9 C3-H, C4-Ha b: 2.63 (1H, dd, J=5.6, 16.0 Hz) 3 5.63 (1H, m) 67.5 C2-Ha, Hb, C4-Hb 4 a: 3.60 (1H, d, J=14.0 Hz) 68.9 C2-Ha, Hb, C3-H, NMe b: 3.86 (1H, dd, J=9.0, 14.0 Hz)

NMe3 3.18 (9H, s) 54.5 C4-Ha, Hb 1′ — 175.6 C2′-Ha, Hb 2′ a: 2.27 (1H, dd, J=7.0, 16.0 Hz) 39.7 C4′-Ha, C5′-Ha b: 2.36 (1H, dd, J=7.4, 16.0 Hz) 3′ 0.98 (1H, m) 6.7 C4′-Ha, C5′-Ha, C2′-Ha, Hb 4′ a: 0.15 (1H, m) 4.2 C2′-Ha, Hb b: 0.52 (1H, m) 5′ a: 0.15 (1H, m) 4.4 C2′-Ha, Hb b: 0.52 (1H, m) a) DSS=sodium 2,2-dimethyl-2-silapentane-5-sulfonate. extracts were concentrated in vacuo, and the resulting residue was confirmed to be cyclopropylacetyl-(R)-carnitine (4). In- was dissolved in 1% AcOH–methanol (MeOH). The soluble terestingly, carnitine esters, including a cyclopropylcarboxylic fraction was chromatographed on alumina using 1% AcOH– acid ester, were also isolated from a Boletaceae mushroom.9) MeOH as the eluent, and the resulting eluate was chromato- We examined the toxicity of 4 in mouse by an intraperitoneal graphed on an ODS (octadecyl group-bonded silica) column route; however, no toxicity was detected. by stepwise elution with H2O and 50% MeOH–H2O. The 50% Cyclopropylacetyl-(R)-carnitine is specific to genuine MeOH fraction was concentrated in vacuo, and the residue R. subnigricans and sufficiently stable under ordinary ex- was found to be partitioned between H2O–EtOAc. The aque- perimental conditions. In addition, the upfield signals in the ous layer was concentrated in vacuo and the residue was chro- 1H-NMR spectrum corresponding to the cyclopropane core matographed on an ODS column, involving stepwise elution are easily recognizable in the 1H-NMR spectrum of crude with 20 and 50% MeOH–H2O. The 20% MeOH eluate was mixtures of fruiting bodies; therefore, it would be a useful further purified by ODS TLC (20% CH3CN–H2O) followed chemical marker for the identification of genuine R. subnigri- by HPLC (ODS, gradient elution with CH3CN–H2O), yielding cans (Fig. 3). colorless syrup. A number of identification manuals for mushrooms, includ- The molecular mass of the obtained compound was esti- ing several local manuals of exceptional species and traits, mated to be 243 by FAB-MS. The NMR data of the purified have been published in Japan. In these manuals, it is often am- cyclopropane-containing compound are listed in Table 1. biguously described that R. subnigricans is found in summer The 1H-NMR signals indicated the existence of 21 protons, and fall in broadleaf forests where chinquapin or oak trees including a monosubstituted cyclopropane unit and a trimeth- grow. We have examined several Russula species during the ylammonium group, while 10 signals were observed in the past ten years, and have concluded that descriptions of genu- 13C-NMR spectrum, including two carbonyl carbons and one ine R. subnigricans and unclassified Russula spp. are often overlapped carbon corresponding to the trimethylammonium unclear and/or contradictory. Presently, we can locate genuine group. Taking these data into account with the heteronuclear R. subnigricans only in chinquapin forests, specifically forests multiple bond correlation (HMBC) spectra, we inferred the of Castanopsis cuspidate. This tree is distributed in warm, structure to be carnitine ester 4, as shown above. western areas of Japan, and the fruiting bodies of R. subnigri- To confirm the structure of 4, the identical carnitine ester cans appear only in the summer months. In contrast, the two was synthesized by condensation of (R)-carnitine and cy- unclassified Russula spp., which were collected in Miyagi and clopropylacetic acid using an acid chloride method (see Ex- Saitama, are found in rainy seasons during the summer and perimental). The 1H- and 13C-NMR data of the natural and fall months in broadleaf forests including the Japanese oak synthetic samples were identical, and the absolute configura- Quercus serrata. tion was also determined to be R by comparing the specific Japanese oak grows in nearly all areas of Japan, including rotation of the synthetic compound and that of the natural one. forests of Kyoto, where the two unclassified Russula spp. in- Thus, the isolated cyclopropane-containing new compound dependently grow during the identical season. Since the kinds Vol. 64, No. 6 (2016) Chem. Pharm. Bull. 605

1 a) Fig. 3. H-NMR Spectra (500 MHz, D2O, TSP =0.00 ppm) (A) Authentic sample of cyclopropylacetyl-(R)-carnitine (4). (B) Crude water extract of R. subnigricans collected in Kyoto. (C) Crude water extract of Russula sp. col- lected in Miyagi. (D) Crude water extract of Russula sp. collected in Saitama. a) TSP=3-(trimethylsilyl)propionic-2,2,3,3-d4 acid sodium salt.

Table 2. Comparative Data of Russula subnigricans and Two Similar Russula spp. Used in This Study

Russula sp. Russula sp. Russula subnigricans Collected in Miyagi Collected in Saitama Collected in Kyoto

Location of the spot where each E 140°49′N 38°15′ E 139°27′N 35°46′ E 135°47′N 35°00′ mushroom was collected (Aoba-yama) (Hachikoku-yama) (Koudaiji-san) Toxicity (p.o.)a) of the water extract Non toxic Non toxic Toxic Cycloprop-2-ene carboxylic acid (1) Absent Absent Present 3-Hydroxybaikiain (2) Present Absent Absent Russuphelins (3) Present Absent Absent Cyclopropylacetyl-(R)-carnitine (4) Absent Absent Present Color change of fresh White to reddish brownb) White to reddish brownc) White to reddish brownd) Odor Pungent like halogens Not special Not special Gills Distant Distant Distant Proposed host treese) Quercus serrata Quercus serrata Castanopsis cuspidata Growth Rainy seasons in summer and fall Rainy seasons in summer and fall Summer a) p.o.: per os (oral administration). b) Faint and extremely slow coloration to reddish brown on scratching, and mainly aging-dependent coloration. c) Fast and intense color- ation on scratching: Red to reddish brown or reddish gray, and then to black. d) Through reddish brown on scratching, finally to gray during about 1 h. e) The major tree in the forest where each mushroom was collected. of mushroom species that flourish and their respective colony season. Although more than one species may grow in these sizes depend on location and climate, it is expected, as in the locations, the exclusive appearance of apparently only one cases of Miyagi and Saitama, that only a single kind of Rus- type of species misled local mushroom collectors on the cor- sula spp. would be found in each forest during the identical rect identification of Russula spp. This situation led to differ- 606 Chem. Pharm. Bull. Vol. 64, No. 6 (2016) ing and unclear descriptions of R. subnigricans in numerous in Saitama). identification manuals, and also hampered the accuracy of 3-Hydroxybaikiain (2) previous chemical studies of this toxic mushroom.5–8) Isolation of 3-Hydroxybaikiain (2) from a Russula sp. In addition to physical appearance, it is also possible that Collected in Miyagi R. subnigricans can be discriminated from the two unclas- The frozen fresh fruiting bodies of the mushroom collected sified Russula spp. based on the proposed kind of host tree. in Miyagi Prefecture (22.2 g) were cut in pieces and extracted However, it is difficult to distinguish the host tree with which with MeOH (200 mL). The mixture was filtered and the filtrate the mushroom symbiotically lives in a mixed broadleaf forest. was concentrated in vacuo. The residue was partitioned be-

Therefore, our chemical method described herein can impart tween EtOAc (50 mL×3) and H2O and each layer was concen- useful information to aid in the accurate identification of R. trated to dryness to give the EtOAc extract (255 mg) as a red subnigricans. syrup and the water extract (173 mg) as a brown syrup. The comparative data of the two unclassified Russula spe- According to the Nozoe’s procedure,5) 3-hydroxybaikiain cies with those of genuine R. subnigricans are summarized in (2) was isolated from the water extract through two-step ion Table 2. Detailed data together with the dried materials of the exchange chromatography (Amberlite IRC-50, eluted with mushrooms used in this study were deposited in The Royal H2O, and then Amberlite IR-120-B, eluted with 4% aqueous Botanical Gardens, Kew, U.K. and classification of the two NH4OH) to give a mixture (33.8 mg) containing 2. Finally, Russula spp. collected in Miyagi and Saitama is currently in recrystallization from H2O–MeOH gave pure 2 (14.1 mg) as progress. colorless crystals. mp >300°C (lit.5) 300–302°C; HR-EI-MS + 36 m/z 143.0601 [M] ; Calcd for C6H9NO3, 143.0582; [α]D −322.7 5) 20 1 Experimental (H2O, c=1.19) (lit : [α]D −332.7 (H2O, c=0.3)). H-NMR, General Melting points were determined on a micro hot- 13C-NMR, and IR spectra of 2 were identical to those of the stage Yanaco MP-S3 and were uncorrected. Optical rotations reported data.5) were measured on a JASCO DIP-360 polarimeter. IR spectra Detection of 3-Hydroxybaikiain (2) in Other Two Russula were recorded on a JASCO FT-IR-200 spectrometer as a KBr spp. Collected in Saitama and Kyoto pellet. 1H- and 13C-NMR spectra were recorded on JEOL Using the same method as above, detection of 3-hydroxy- Lambda-300, JEOL ECA-500, or Varian MERCURY plus baikiain (2) was tried on the other two Russula spp. by 300 at ambient temperature and their chemical shifts were 1H-NMR. The MeOH extract of the Russula sp. collected in reported as values in ppm downfield or upfield from internal Saitama (fruiting bodies; 350 g/MeOH; 1.2 L×2) was parti- standard (noted before data). High and low-resolution electron tioned to give the EtOAc extract (1.43 g) and the water extract ionization (HR-EI and LR-EI) and FAB mass spectra were (6.00 g). On the other hand, the MeOH extract of the Russula recorded on JEOL GC mate mass spectrometer. sp. collected in Kyoto (fruiting bodies; 720 g/MeOH; 1.0 L×2) (R)-Carnitine was purchased from TCI (Tokyo Chemical was partitioned to give the EtOAc extract (2.68 g) and the Industry Co., Ltd., Tokyo, Japan) and cyclopropylacetic acid water extract (9.67 g). Each water extract was separated, re- from Wako (Wako Pure Chemical Industries, Ltd., Osaka, spectively, as mentioned in the section of “Isolation of 3-hy- Japan) and these were used without further purification. droxybaikiain (2) from a Russula sp. collected in Miyagi” Mushroom Material The mushrooms were collected in and the resulting aqueous ammonia eluate was checked by Kyoto in 2004–2007, Saitama in 2002–2003, and Miyagi in 1H-NMR. No 3-hydroxybaikiain (2) was detected in either ex-

2007 and stored in freezer at −30°C and used for the chemical tracts (5–10 mg in D2O (0.75 mL)). study. Exact locations for all collections were summarized in Russuphelins (3) Table 2. Isolation of Russuphelins (3) from a Russula sp. Collected The Russulaceae are a family of fungi in the order Agari- in Miyagi cales. The Russulaceae mushrooms have common character- The EtOAc extract (see Experimental of “Isolation of 3-hy- istic appearances which are easily recognizable as the species droxybaikiain (2) from a Russula sp. collected in Miyagi”) should be classified into the family; however, further identifi- was separated according to the Nozoe’s procedure6,7) using cation of each species is troublesome. Typical characteristics silica gel column chromatography (successive 4 : 1 hexane– of the Russulaceae are the followings; friable fruit bodies, EtOAc, 9 : 1 CHCl3–EtOAc, and 4 : 1 CHCl3–EtOAc), and the whitish to ocher spore print, spores with amyloid fibrils, no chemical components of each fraction were checked by TLC clamp connection in the hyphae of the fruit bodies. Friable and 1H-NMR spectra. Russuphelin-containing fractions (fr. 1:

flesh is due to its heteromerous structure which is constructed eluted with 9 : 1 CHCl3–EtOAc, 12.8 mg; fr. 2: eluted with 9 : 1 from filamentous hyphae and many spherical cells. The Rus- CHCl3–EtOAc, 6.0 mg; fr. 3: eluted with 4 : 1 CHCl3–EtOAc, sulaceae family is classified into two genera, Russula and 3.8 mg) were further separated by preparative TLC as follows. Lactarius. The former has no latex; on the other hand, the 4,4′-[(2,5-Dimethoxy-1,3-phenylene)bis(oxy)]bis[3,5- latter has it.10) dichlorophenol] (Russuphelin A, 3A) All of the Russula mushrooms used in this study meet the Above fr. 3 (3.8 mg) was separated by preparative TLC above requirements. Identification of each mushroom was per- (silica gel, 19 : 1 CHCl3–MeOH) to give russuphelin A (3A, formed by Shigeo Morimoto (Kansai Mycological Club). De- 1.3 mg) as a colorless powder. mp 294–297°C (not recrystal- tailed data together with the dried materials of the mushrooms lized) (lit.6) 293–294°C); HR-EI-MS m/z 489.9523 [M]+; Calcd 1 13 collected in 2011 were deposited in The Royal Botanical for C20H14O6Cl4, 489.9544. H-NMR, C-NMR, and IR spec- Gardens. The reference numbers are as follows: K(M) 173268 tra of 3A were identical to those of the reported data.6,7) for Russula subnigricans, K(M) 173269 for Russula sp1 (col- lected in Miyagi), and K(M) 173270 for Russula sp2 (collected Vol. 64, No. 6 (2016) Chem. Pharm. Bull. 607

4-(3-Chloro-2,5-dimethoxyphenoxy)-3,5-dichlorophenol (ODS ϕ6×250 mm, eluted with H2O for 10 min and then linear (Russuphelin D, 3D) gradient from H2O to 20% CH3CN for 50 min at a flow rate Above fr. 1 (12.8 mg) was separated by preparative TLC of 1.5 mL/min with monitoring at 210 nm) to give 4 (3.4 mg,

(silica gel, 19 : 1 CHCl3–MeOH) followed by further puri- tR=20.9 min) as colorless syrup. Rf=0.31 (ODS, 1 : 4 MeOH– fication by preparative TLC (silica gel, 4 : 1 CHCl3–EtOAc) H2O); IR νmax (KBr): 3735, 3468, 1732, 1594, 1398, 1188, −1 + + to give russuphelin D (3D, 2.3 mg) as colorless solids. mp 1054 cm ; LR-FAB-MS m/z 244 [M+H] , 162 [M−C5H6O] , 7) + 127–128°C (not recrystallized) (lit. 136–138°C); HR-EI-MS HR-FAB-MS m/z 244.1572 [M+H] ; Calcd for C12H22NO4, + 1 31 m/z 347.9720 [M] ; Calcd for C14H11O4Cl3, 347.9723. H-NMR, 244.1549; [α]D −14.5 (H 2O, c=0.96). 13C-NMR, and IR spectra of 3D were identical to those of the Detection of Cyclopropylacetyl-(R)-carnitine (4) in Three reported data.6,7) Russula spp. Collected in Kyoto, Miyagi, and Saitama 2-Chloro-6-(2,6-dichloro-4-methoxyphenoxy)-1,4- The water extract of R. subnigricans collected in Kyoto hydroquinone (Russuphelin G, 3G) (fruiting bodies; 7.42 g/H2O; 22.3 mL) was concentrated to Above fr. 2 (6.0 mg) was separated by preparative TLC dryness to give the crude extract (369 mg). A partial crude

(silica gel, 19 : 1 CHCl3–MeOH) to give 2-chloro-6-(2,6-di- extract (10 mg) was dissolved into D2O (0.75 mL) contain- chloro-4-methoxyphenoxy)-1,4-hydroquinone (3G, 2.9 mg) as ing TSP (0.77 mM) as an internal standard. Carnitine ester 4 colorless solids. mp 184–185°C (not recrystallized); Rf=0.39 was detected in 1H-NMR spectrum of the crude extract and

(1 : 19 MeOH–CHCl3); IR νmax (KBr): 3419, 1610, 1504, 1470, its content was estimated to be ca. 133 mg/kg fruiting bod- −1 1 1219 cm ; H-NMR (CDCl3, TMS=0.00) δ: 3.82 (3H, s, ies. Similarly, Saitama species (fruiting bodies; 6.96 g/H2O; 4′-OMe), 5.92 (1H, d, J=3.0 Hz, H-5), 6.56 (1H, d, J=3.0 Hz, 20.9 mL) and Miyagi species (fruiting bodies; 11.4 g/H2O; 1 H-3), 6.95 (2H, s, H-3′, 5′); H-NMR (CD3OD, solvent 34.2 mL) were extracted with H2O to give the crude extracts peak=3.31) δ: 3.84 (3H, s, 4′-OMe), 5.77 (1H, d, J=3.0 Hz, (83.0, 257 mg, respectively). A part of each water extract was 1 H-5), 6.44 (1H, d, J=3.0 Hz, H-3), 7.10 (2H, s, H-3′, 5′); NOE dissolved in D2O and this was checked by H-NMR. No cyclo- (increased by 5.1%) was observed at H-3′ and H-5′ overlapped propylacetyl-(R)-carnitine (4) was detected in either extracts 13 signals by irradiation of 4′-OMe; C-NMR (CD3OD, solvent (10 mg in D2O (0.75 mL)). peak=49.00) δ: 56.8 (4′-OMe), 101.5 (C5), 110.8 (C3), 116.0 Synthesis of Cyclopropylacetyl-(R)-carnitine (4) (C3′, 5′), 122.8 (C2), 131.0 (C2′, 6′), 137.0 (C1), 141.9 (C1′), To a stirred cyclopropylacetic acid (0.098 mL, 1.05 mmol) 147.9 (C6), 151.2 (C4), 158.9 (C4′); HR-EI-MS m/z 333.9547 was added thionyl chloride (0.080 mL, 1.10 mmol) and the + [M] ; Calcd for C13H9O4Cl3, 333.9567. mixture was stirred at room temperature (rt) for 1 h. To the Detection of Russuphelins (3) in Other Two Russula spp. crude acid chloride was directly added (R)-carnitine (86.0 mg, Collected in Saitama and Kyoto 0.533 mmol) and the mixture was stirred at rt for 1.5 h. After

Each EtOAc extract (see Experimental of “Detection of evaporation, the residue was dissolved into H2O and the aque- 3-hydroxybaikiain (2) in other two Russula spp. collected in ous layer was washed with EtOAc. The aqueous layer was ap-

Saitama and Kyoto”) was dissolved in CDCl3 and checked by plied to ODS column chromatography (H2O). The eluate was 1 H-NMR. No russuphelins (3) were detected in either extracts neutralized by saturated aqueous NaHCO3 solution and then (5–10 mg in CDCl3 (0.75 m L)). concentrated to dryness. The resulting residue was extracted Cyclopropylacetyl-(R)-carnitine (4) with MeOH and it was filtered through Celite. The filtrate Isolation of Cyclopropylacetyl-(R)-carnitine (4) from the and washings were concentrated in vacuo to afford cyclopro- Russula subnigricans (Collected in Kyoto) pylacetyl-(R)-carnitine (4) (60.4 mg, 47% yield) as colorless

The fruiting bodies (500 g) of Russula subnigricans (col- foam. mp 180°C (decomp.); Rf=0.31 (ODS, 1 : 4 MeOH–H2O); −1 lected in Kyoto) were cut into pieces and soaked in aqueous IR νmax (KBr): 3735, 3433, 1734, 1592, 1392, 1179, 1034 cm ; 1 0.3% AcOH (1.5 L) at 4°C overnight. The extract was filtered H-NMR (D2O, HOD=4.79) δ: 0.15 (2H, m, H-4′, 5′), 0.52 through filter paper under suction and then the filtrate was (2H, m, H-4′, 5′), 0.99 (1H, m, H-3′), 2.28, (1H, dd, J=7.0, concentrated to about 100 mL under reduced pressure. The 16.0 Hz, H-2′), 2.36 (1H, dd, J=7.4, 16.0 Hz, H-2′), 2.49 (1H, concentrated solution was dialyzed (relative molecular mass dd, J=8.0, 16.0 Hz, H-2), 2.63 (1H, dd, J=5.6, 16.0 Hz, H-2), + (Mr) 14000) against aqueous 0.3% AcOH (2.0 L×2) overnight. 3.18 (9H, s, 4-N Me3), 3.61 (1H, d, J=14.0 Hz, H-4), 3.86 (1H, 13 The dialyzate was concentrated to dryness and lyophilized to dd, J=9.0, 14.0 Hz, H-4), 5.63 (1H, m, H-3); C-NMR (D2O, give a crude extract (27.1 g). A part (4.8 g) of the crude extract DSS=–2.04) δ: 4.2 (C4′, 5′), 4.4 (C4′, 5′), 6.7 (C3′), 39.7 (C2′), + was dissolved in 1% AcOH in MeOH (48 mL), and then the 40.9 (C2), 54.5 (4-N Me3), 67.5 (C3), 68.9 (C4), 175.7 (C1′), + + soluble part was applied to an alumina column (aluminium 177.2 (C1); LR-FAB-MS m/z 244 [M+H] , 162 [M−C5H6O] , + oxide 90 standardized, Merck, 32 g), which was eluted with HR-FAB-MS m/z 244.1555 [M+H] ; Calcd for C12H22NO4, 34 1% AcOH in MeOH (300 mL). The eluate was concentrated 244.1549; [α]D −16.6 (H 2O, c=0.67). to 5 mL, and this was diluted with aqueous 0.3% AcOH Toxicity on Mice (10 mL) and chromatographed on ODS (Cosmosil 140C18 Oral Injection

OPN, 16 g) which was eluted with H2O (300 mL) and 50% The water extract (200 mg) of the mushroom was dissolved aqueous MeOH (100 mL). After removal of MeOH from the in H2O (0.2 mL) and the solution was orally afforded to a 50% MeOH fraction, the aqueous solution was washed with mouse (ddy, female, 19–21 g body weight) using a catheter. EtOAc (100 mL×3). The aqueous layer was concentrated in Intraperitoneal Injection vacuo and then chromatographed on ODS by elution with Synthetic carnitine ester (4, 10 mg) was dissolved in saline 20% MeOH. The obtained fractions which contained a cyclo- (0.2 mL) and the solution was intraperitoneally injected to a propane derivative were concentrated (16.2 mg) and purified mouse (ddy, female, 9.5–10.5 g body weight). by preparative TLC (ODS, 20% CH3CN) followed by HPLC Within 24 h, when the mouse died, the extract was regarded 608 Chem. Pharm. Bull. Vol. 64, No. 6 (2016) as toxic. in collecting and identifying the Russula species. All animal experiments were performed with the approval of the Keio University School of Medicine Laboratory Animal Conflict of Interest The authors declare no conflict of Care and Use Committee. interest. The animals were maintained under constant environmental conditions and free access to food and water. References 1) Imazeki R., Hongo T., “Colored Illustrations of Mushrooms of Conclusion Japan,” Vol. II, Hoikusha, Osaka, 1989, pp. 47–48. Incidents of mushroom poisoning attributed to R. subni- 2) Lee P. T., Wu M. L., Tsai W. J., Ger J., Deng J. F., Chung H. M., gricans have recently increased in Japan; however, accurate Am. J. Kidney Dis., 38, E17 (2001). identification of the is difficult due to the existence of 3) “Poisonous Fungi in Japan,” Field Best Encyclopedia Vol. XIV, ed. by Nagasawa E., Gakken, Tokyo, 2009 (Revised Edition), p. 46. several similar, unidentified Russula species. Here, cyclopro- 4) Matsuura M., Saikawa Y., Inui K., Nakae K., Igarashi M., Hashi- pylacetyl-(R)-carnitine (4) was isolated as a new compound moto K., Nakata M., Nat. Chem. Biol., 5, 465–467 (2009). and turned out to be a useful marker specific to genuine R. 5) Kusano G., Ogawa H., Takahashi A., Nozoe S., Yokoyama K., 1 subnigricans. In the H-NMR spectrum of a crude water ex- Chem. Pharm. Bull., 35, 3482–3486 (1987). tract, the cyclopropane unit of the marker compound is easily 6) Takahashi A., Agatsuma T., Matsuda M., Ohta T., Nunozawa T., recognizable, since the signals appear in the upfield region Endo T., Nozoe S., Chem. Pharm. Bull., 40, 3185–3188 (1992). (0.15, 0.52 ppm), where other signals are rarely observed. It is 7) Takahashi A., Agatsuma T., Ohta T., Nunozawa T., Endo T., Nozoe expected that this unique marker compound will aid in future S., Chem. Pharm. Bull., 41, 1726–1729 (1993). classification of this toxic fungus. 8) Ohta T., Takahashi A., Matsuda M., Kamo S., Agatsuma T., Endo T., Nozoe S., Tetrahedron Lett., 36, 5223–5226 (1995). 9) Kawagishi H., Murakami H., Sakai S., Inoue S., Phytochemistry, 67, Acknowledgments We are grateful to Messrs. Shigeo 2676–2680 (2006). Morimoto and the late Toshiho Ueda (Kansai Mycological 10) Imazeki R., Hongo T., “Colored Illustrations of Mushrooms of Club), Takashi Suda (Gunma Mushroom Club), and Prof. Japan,” Vol. I, Hoikusha, Osaka, 1987, p. 20. Emeritus Kazumasa Yokoyama (Shiga University) for the help