Novel Betaines from a Micronesian Sponge Dysidea herbacea Ryuichi Sakai,* Katsuji Suzuki, Keiko Shimamoto,† and Hisao Kamiya School of Fisheries Sciences, Kitasato University, Sanriku-cho, Ofunato, Iwate 022-0101, Japan
[email protected] Received October 12, 2003
Three new betaines, dysibetaine PP (1), dysibetaine CPa (2), and dysibetaine CPb (3), were isolated from an aqueous extract of the marine sponge Dysidea herbacea collected in Yap state, Micronesia. The structure of 1 was determined by spectral methods as well as chemical degradation to be a novel dipeptide betaine, and those for 2 and 3 were determined to be unprecedented cyclopropane betaines. Compounds 2 and 3 showed weak affinity toward the N-methyl-D-aspartic acid-type and the kainic acid-type glutamate receptors, respectively, in a radioligand binding assay. Introduction Dysidea herbacea is one of the most common sponges in the South Pacific region and is known as a rich source of diverse classes of secondary metabolites. One of the representative classes includes the chlorinated amino acid derivatives.1 This class of lipophilic peptidyl metabolites has been thoroughly studied because of its intriguing structures, biosynthesis, and biological activities.2 However, except for mycosporines, the watersoluble amino acid derivatives have been little studied.3 Recently, however, we reported the isolation and structure determination of potent excitatory amino acids dysiherbaine (DH),4 neodysiherbaine-A (NDH-A),5 and a novel betaine, dysibetaine (DB),6 from an aqueous extract of the sponge (Chart 1). DH and NDH-A were proven to be potent agonists for mammalian glutamate receptors with unusual selectivity.7 These findings demonstrate that the aqueous extract of D. herbacea is an interesting source of amino acid-derived bioactive secondary metabolites. During our continuing effort to investigate the chemical constituents in the aqueous extracts of D. herbacea, * To whom correspondence should be addressed. Tel: +81-192-441922. Fax: 81-192-44-3932. † Suntory Institute for Bioorganic Research. (1) For example: (a) MacMillan, J. B.; Trousdale E. K.; Molinski T. F. Org Lett. 2000 2, 2721-3. (b) MacMillan, J. B.; Molinski, T. F. J. Nat. Prod. 2000, 63, 155-157. (3) Harrigan G. G.; Goetz, G. H.; Luesch H., Yang S. J. Nat. Prod. 2001, 64, 1133-1138. (2) For example: (a) Flowers A. E.; Garson M. J.; Webb R. I.; Dumdei E. J.; Charan R. D. Cell Tissue Res. 1998, 292, 597-607. (b) Unson, M. D.; Faulkner, D. J. Experientia 1993, 49, 349-353. (c) Stapleton, B. L.; Cameron G. M.; Garson M. J. Tetrahedron 2001, 57, 4603-4607. (3) Bandaranayake, W. M.; Bemis, J. E.; Bourne, D. J.Comp. Biochem. Physiol. 1996, 115, 281-286. (4) Sakai, R.; Kamiya, H.; Murata, M.; Shimamoto, K. J. Am. Chem. Soc. 1997, 119, 4112-4116. (5) Sakai, R.; Koike, T.; Sasaki, M.; Shimamoto, K.; Oiwa, C.; Yano, A.; Suzuki, K.; Tachibana, K. Org Lett. 2001, 3, 1479-1482. (6) Sakai, R.; Oiwa, C.; Takaishi, K.; Kamiya, H.; Tagawa. M. Tetrahedron Lett. 1999, 40, 6941-6944. (7) (a) 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. (b) Swanson, G. T.; Green, T.; Sakai, R.; Contractor, A.; Che, W.; Kamiya, H.; Heinemann, S. F. Neuron 2002, 16, 589-598.
with a focus on the biosynthesis and distribution of DH and its related compounds,8 we found that D. herbacea collected in Yap State, Micronesia, contains various amino acid derivatives. We thus conducted the isolation and chemical and biological characterization of the minor components, with an eye toward the potential biomedical interest and found three new betaines, dysibetaine PP (1), dysibetaine CPa (2), and dysibetaine CPb (3). Herein, the structure determinations and some of the biological activities of these structurally unique compounds are described. Results and Discussion An aqueous extract of Dysidea herbacea collected in Yap State, Micronesia, was separated first by a Sephadex LH20 column. Plural ninhydrin-positive spots on the TLC as well as the NMR spectra suggested that the bioactive fractions which contain DH or DB included some other amino acids as minor components. Thus, we carefully examined these fractions to isolate new amino acid derived compounds. As described in the Experimental Section, further separation using a combination of gel filtration (Biogel P-2 and HW-40) chromatographies followed by reversed-phase HPLC purification afforded compounds 1, 2, and 3 (Chart 1). The HRFABMS and NMR data established the molecular formula of 1 to be C12H18N2O3, which has five degrees of unsaturations. 13C NMR showed all 12 carbons, including two carbonyl carbons resonating at δ 176.5 and 170.3 (Table 1). The presence of the carboxylate and the lactam carbonyl groups was also evident from the IR absorptions at 1710 and 1609 cm-1, respectively. Since no other sp2 carbons were observed in the 13C NMR spectra, the remaining unsaturation accounted for three rings. The COSY and HMBC spectrum suggested the presence of an N-methylproline (N-MePro) moiety in 1 (Table 1). A treatment of 1 with 6 N HCl at 115 °C for 12 h afforded N-MePro, which was confirmed by the comparison with (8) These results will be published elsewhere. 10.1021/jo0355045 CCC: $27.50 © 2004 American Chemical Society
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Published on Web 01/22/2004
Novel Betaines from a Micronesian Sponge Dysidea herbacea CHART 1
TABLE 1. NMR Data for Compound 1 position 1 2 3 4 5 6 8 9 10 11 12 NCH3 a
1H
(δ, mult, J (Hz))a
4.48, d, 6.4 a 2.13, brd, 13.9 b 1.63, m a 1.95, dt, 10.5, 3.2 b 1.35, brq, 13.6 a 2.24, m b 1.68, m 5.20, dd, 11.5, 4.2 4.71, dd, 9.2, 2.8 a 2.5, m b 2.4, m a 2.3, m b 2.3, m a 3.71, brq, 9.5 b 3.54, m 3.27, s
SCHEME 1
13C
HMBC (H to C)
176.5 56.7 25.8
1, 3, 4, 6, 8
19.1 24.1 81.2 170.3 75.9 24.5
4 12, CH3 8, 9, 12 11, 8, 9
22.8 59.8 47.7
11 11 6, 9, 12
D2O, 293 K.
FIGURE 1. COSY and key HMBC for 1.
the authentic sample in TLC and LCMS.9 The nitrogen atom of N-MePro was connected to a methine carbon resonating at δ 81, which bore a rather downfield-shifted proton at δ 5.20, because the mutual HMBC cross-peaks between the methyl and the methine systems were observed. Tracing from this methine proton signal, interpretations of the COSY and HMBC data allowed us to construct the carbon framework of a 6-substituted pipecolic acid (ring A) (Figure 1). The presence of the N-MePro and 6-N-substituted pipecolate moiety in 1 was confirmed by the LCESI MS (9) Attempts to assign the absolute stereochemistry of N-MePro derived from 1 were unsuccessful, since the authentic D- and L-NMePro could not be resolved with the chiral GC or chiral HPLC.
analysis for the acid hydrolysate of 1, which showed ions at m/z 128, 130, and 146 (M + H)+. The ion at m/z 130 was identified as the molecular ion for N-MePro and the LCESI MS/MS spectrum for the trace at m/z 130 afforded a product ion at m/z 84 (Scheme 1a). On the other hand, the ions at m/z 146 and 128 could be assigned as 6-hydroxypipecolic acid (4) and the imine (5), the plausible hydrolysis products from the 6-N-substituted pipecolate moiety. In the LCESI MS/MS spectrum, the product ions from m/z 146 were detected at m/z 128, 100, and 82, which corresponded well to the possible fragmentation ions for 6-hydroxypipecolic acid (4) (Scheme 1b). Moreover, a treatment of hydrolysate with NaBH4 provided pipecolic acid, which was identical to the authentic sample on TLC. The reaction product was further derivatized with butanol acidified with an addition of 10% AcCl, followed by reacting with a mixture of TFA and TFAA. A chiral GC (Chirasyl Val-III) analysis of the reaction products gave a 1:1 set of peaks corresponding to the same derivative from DL-pipecolic acid. J. Org. Chem, Vol. 69, No. 4, 2004 1181
Sakai et al.
FIGURE 3. Partial ball-and-stick models for 1 (ring C region) SCHEME 2
generated by computer software ChemDraw after the MM2 energy minimization. Some long-range couplings observed in the COSY for 1 are indicated.
Although no information on the absolute stereochemistry was obtained, this result supported the presence of the 6-hydroxypipecolic acid (4) as a hydrolysis intermediate which could be subjected to racemization via equilibration to imine 5 under the reaction conditions employed (Scheme 2). Furthermore, the unique N,N-aminal linkage in compound 1 was supported by the NOE between the H-6 and N-methyl group shown in the NOESY spectra (Figure 2). The nitrogen atom of the pipecolic acid moiety was shown to be connected to the carbonyl group of the N-MePro moiety based on the HMBC cross-peak between H-2 and C-8. Taken together, we assigned a unique tricyclic dipeptidyl betaine structure as a planar structure of 1. This compound was designated as dysibetaine PP after its amino acid components (proline and pipecolate). It should be noted that H-9, the R-proton of the N-MePro moiety resonated at δ 4.65, was slowly exchanged to a deuterium atom while the NMR spectrum was taken in D2O under ambient conditions. This unusual acidic nature of H-9 is probably due to a sum of the electronwithdrawing property of the amide carbonyl and the ammonium nitrogen. We next examined the relative configuration of 1. Some stereochemical information for 1 was provided by 3JH-H analyses, long-range couplings, and NOEs. Long-range COSY cross-peaks between H-4a (δ 1.95) and H-2, and between H-3a and H-5a, indicated that all of these protons were oriented equatorially to the piperidine ring. Furthermore, the coupling pattern of H-6 (dd, J ) 11.5, 4.2 Hz) and a NOESY cross-peak between H-6 and H-4b
indicated that H-6 is an axial proton. These data showed that the piperidine ring is in a chair form, and thus, the axial orientation of the carboxylate at C-2 and the equatorial orientation of the nitrogen atom of the NMePro at C-6 were readily assigned. A COSY long-range coupling between H-9 and H-12b (the R and δ protons of the N-MePro substructure, respectively) indicated that these protons are both pseudoequatorial rather than psueudoaxal to the pyrrolidine ring (ring C) (Figure 3). Similarly, a long-range coupling between the N-methyl protons and H-12a indicated that they share the same plane. These observations supported the fact that the pyrrolidine ring is in an envelope conformation, and that the orientation of both the Nmethyl group and the carbonyl group are pseudoaxial to the five-membered ring. Finally, a β-equatorial orientation of the amide bond, between N-7 and C-8, to the piperidine ring (ring A) was assigned on the basis of the following evidence. In a difference NOE experiment carried out at 278 K, an irradiation at H-9 resulted in an enhancement of H-6 as well as the N-methyl group and H-10a (Figure 2). The NOE between H-9 and H-6 indicated that both of these protons orient pseudoaxially to the imidazolidine ring (ring B). In addition, a NOESY cross-peak was observed between H-5b and H-12a. Only a trans-fused AB ring system can fulfill all the stereochemical requirements shown by the above data. These data allowed us to assign relative stereochemistries for C-2, C-6, C-9, N-7, and N-13, as shown in Figure 2. Compound 2 was isolated from a fraction absorbed to the anion-exchange resin, indicating the acidic nature of this compound. The molecular formula C9H15NO4 of 2 deduced by the HRFABMS data accounted for three degrees of unsaturation. Since two carbonyl groups, but no other sp2 carbons, were identified in the 13C NMR spectrum, a remaining unsaturation was accounted for by a cyclic structure of 2. The NMR (1H, 13C, HMQC) data taken in D2O identified, besides two carbonyl groups, five sets of spin systems: a heteroatom-substituted methylene, a trimethylammonium group, and three methines (Table 2). COSY and HMBC revealed H-H and C-H connectivity for 2 in that the trimethylammonium group was attached to the methylene carbon, which was also connected to a trisubstituted cyclopropane ring. One of the two carbonyl groups substituted on the cyclopropane ring must be a carboxylic acid, and the other a carboxylate, on the basis of the molecular formula. In the deuterium shift NMR experiment, the differences in the 13C chemical
FIGURE 2. Important NOEs for 1.
1182 J. Org. Chem., Vol. 69, No. 4, 2004
Novel Betaines from a Micronesian Sponge Dysidea herbacea TABLE 2. NMR Data for Compounds 2, DCG IV (6), and 3 6b
2 1Ha
position
(δ, mult, J (Hz))
13Cd
3.56 (2H), m
64.09
0.11
2
1.88, brquint, 7.6e
21.75
-0.01
28.24 28.29 174.85 175.92
i i 0.46 0.29 0.06
3 1.97, t, 5.0 4 2.15, dd, 5.0, 9.0 5 6 N(CH3)3 3.04, s a
b
HMBC (H to C)
∆13CH2O-D2Od
1
1H
(δ mult, J (Hz))
CH3, 2, 3, 4 3.72, d, 11.0 6
3c 1H
(δ, mult, J (Hz))
13Cd
a 3.3, dd, 6.8, 13.6 b 3.2, dd, 7.2, 13.6 2.11, brquint, 6.4e
1.99, ddd, 10.2, 9.6, 5.9 2.11, t, 5.5e 2.17, dd, 6.8, 9.6 2.23, dd, 4.9, 9.5 2.19, dd, 5.2, 9.6
1, 2, 5 6 1, 2
3.02, s c
∆13CH2O-D2Od
68.30
0.04
19.11
-0.04
30.82g 28.56f 175.49 173.99 54.02h
0.36 -0.03 0.33 0.10 -0.04
HMBC (H to C) CH3, 2, 3, 4 CH3, 2, 3, 4 4, 6 1, 2, 4, 6 1, 2, 3, 5 1, 2
d
Taken in D2O at 298 K. Recorded in D2O at 284 K. Taken in 2 mM TFA in D2O at 281 K. Recorded at 188 MHz. A signal for CD3CN at δ 120.16 was used as an internal reference. e Apparent value. f,g 2JCH value ) 168 Hz. h 2JCH value ) 140 Hz. i These peaks are too close to each other to define the deuterium shift.
shifts taken in 1:1 H2O-D2O and D2O (∆H-D) for carboxyl carbons at each of δ 174.8 (C-5) and 175.9 (C-6) was 0.46 and 0.29 ppm, respectively (Table 2). This result indicated that the carboxyl of C-5 is more likely in a protonated form than that of C-6. The orientation of these two carbonyl groups was assigned to be trans, because in a difference NOE experiment, an irradiation of methylene protons at δ 3.56 resulted in the enhancement of only one of the carbonyl-bearing methane protons (δ 1.97). It is feasible to assign that the carboxylate group at C-4 is cis, due to the fact that the cationic center and the carboxylic acid at C-3 is trans (Figure 4). These assignments were reinforced by a comparison of the 1H NMR spectrum for DCG-IV, (2S,2′R,3′R)-2(2′,3′-dicarboxycyclopropyl)glycine (6)10 with that for 2. DCG-IV is a cyclopropane-containing amino acid designed and synthesized as a conformationally restricted analogue of glutamate.11 Since the skeletal structure of DCG-IV is very similar to that of 2, similar spectral patterns for the protons on the cyclopropane ring between 2 and 6 were expected in the 1H NMR spectra. These protons for 2 and 6 did in fact correspond well to each other, both in the chemical shifts and coupling patterns, as shown in Table 2, confirming that the relative stereochemistries for the three substituents of 2 and 6 on the cyclopropane ring are the same. The molecular formula of 3, C9H16N2O3, was determined on the basis of its HRFABMS and NMR data. The overall NMR spectral pattern of 3 was very similar to that of 2, suggesting that 2 and 3 were structurally related to each other. The cyclopropane ring structure was supported by the 2JC-H values between C-3 and H-3,
and C-4 and H-4 (both 168 Hz) observed from the satellite peaks in the HMBC spectra.12 The difference in the formula between 2 and 3 suggested that the planar structure of 3 was a monoamide derivative of 2. The presence of the amide group was supported by the chromatographic behavior of 3. Compound 3 was not absorbed to the DE 52 anion exchange column in contrast to 2, indicating the neutral (i.e., zwitterionic) nature of this compound. Assignment of the stereochemistry of 3 from the 1H NMR spectrum in D2O (pH 7), however, was hampered because all of the three protons attached to the cyclopropane ring appeared at δ 2.0 ppm as a complex multiplet, and the NOE experiment or J analysis was not directly applicable. We thus measured 1H NMR for 3 in various pH’s (pH’s between 1 and 12) and found that H-3 and H-4 can be separated by the downfield shift in the acidic conditions (pH < 3). 1H NMR for 3 taken in 2 mM TFA showed that JH2-H3 and JH2-H4 were 6.8 and 5.2 Hz, respectively, while JH3-H4 was 9.6 Hz. Since it has been reported that 3Jcis is always larger than 3Jtrans in the cyclopropane system,13 the above data indicate that the relationship for H-3 and H-4 is cis, while those for both H-2 and H-3, and H-2 and H-4 are trans (Figure 4). A difference NOE experiment also supported these results, in that an irradiation of either H-1a or H-1b resulted in enhancements of H-3 and H-4 along with adjacent H-2 (Figure 4). The results from these experiments led to an unexpected conclusion: the orientation of the carboxylate and the amide groups of 3 is cis, whereas the corresponding functional groups of the 2 are trans. To confirm this, 3 was treated with LiOH to hydrolyze the amide to carboxylic acid. Although HRFABMS for the resulted product 7 provided the same molecular formula as 2, the 1H NMR was clearly different from that of 2. Therefore, the possibility that 3 as well as 7 are 2,3-trans isomers was excluded. Finally, in a deuterium shift experiment in the 13C NMR for 3, the carbon atom resonating at δ 175.49 (C-5) was shifted larger (∆δH-D 0.33) than that resonating at 173.99 (C-6, ∆δH-D 0.10). Consequently, C-5 was found to be an amide carbonyl. These data allowed us to assign the relative stereostructure of 3, as depicted in Chart 1. As both compounds 2 and 3 possess a cyclopropyl ring, we named them dysibetaine CPa and CPb, respectively.
(10) The carbons for 6 are numbered so that they correspond with those of 2 and 3 throughout this paper. (11) Ohfune, Y.; Shimamoto, K.; Ishida, M.; Shinozaki, H. Bioorg. Med. Chem. Lett. 1993, 13, 15-18.
(12) Unfortunately, no satellite peaks appeared in the HMBC spectrum for 2. (13) Pretsch, E.; Bu¨hlmann, P.; Affolter, C. Structure Determination of Organic Compounds; Springer-Verlag: Berlin, 2000; p 176.
FIGURE 4. Coupling patterns (solid lines) and NOE (dotted line) for 2, 6,10 and 3.
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Sakai et al. SCHEME 3
as conformationally restricted GABA analogues with both extended and folded conformations in one. Thus, the biological activity of this compound on GABA receptors is of interest.20 To test this hypothesis and to define absolute stereochemistries, the synthetic studies of 2, 3, and their desmethyl analogues are in progress.
Experimental Section These compounds showed weak affinity toward glutamate receptors in the radioligand binding assay using rat cerebrocortical membrane. Specifically, compound 2 displaced [3H] kainate and [3H]CGP39653 (the NMDA-type receptor ligand) with IC50 values of 13 and 10 µM, respectively. On the other hand, compound 3 only inhibited the binding of [3H]kainate with an IC50 value of 4.9 ( 2.3 µM. Although the affinity of these compounds for GluR is lower than that of glutamate,14 the mechanism of binding to GluR is of interest, since both compounds lack a glutamate equivalent structure in the molecules. Compound 1 did not show any affinity toward GluRs at the highest concentration tested (100 µM). In the present study we demonstrated that the aqueous extract of the sponge D. herbacea is a rich source of structurally interesting amino acid derivatives. The structure of dysibetaine PP (1) is considerably unique. To the best of our knowledge, 1 is the first example of a simple peptide with an N,N-aminal linkage. Dysibetaine CPs are a new class of naturally occurring cyclopropane amino acids. Several cyclopropane-containing amino acids are known in nature, including 1-aminocyclopropane carboxylic acid, a precursor of ethylene in higher plants,15 and marine algae-derived carnosadines.16 cis- and trans-L-(carboxycyclopropyl)glycines (CCG), natural excitatory amino acids isolated from seeds of genus Aesclus and Blighia, are the closest example to the present compounds.17 All these cyclopropane amino acids, however, have only two substituents on the cyclopropane ring. Thus, the biosynthetic pathways of compounds 2 and 3, which have a fully substituted cyclopropane ring, are intriguing issues to be investigated. Incidentally, compound 2 possesses a skeletal structure similar to that of DCG-IV (6), which was designed and synthesized based on the structure of CCGs.11 CCGs and compound 6 are conformationally restricted analogues of glutamate, and display selective affinity toward glutamate receptor subtypes.18 Compound 6, which possesses both folded- and extended conformations of glutamate within one molecule, is a selective and highly potent agonist of the Group II metabotropic glutamate receptor.19 Likewise, the desmethyl derivatives of 2 could be envisioned (14) Glutamate replaced the [3H]CGP39653 and [3H] kainate binding with IC50 values of 69 ( 14 and 137 ( 23 nM, respectively. For the binding assay conditions, see ref 7a. (15) (a) Burroughs, L. F. Nature 1957, 179, 360-361. (b) Vahatalo, M. L.; Virtanen, A. I. Acta Chem. Scand. 1957, 11, 741-756. (c) Ichihara, A.; Shiraishi, K.; Sato, H.; Sakamura, S.; Nishiyama, K.; Sakai, R.; Furusaki, A.; Matsumoto, T. J. Am. Chem. Soc. 1977, 99, 636-637. (16) Wakamiya, T.; Nakamoto, H.; Shiba, T. Tetrahedron Lett. 1984, 39, 4411-4412. (17) Fowden, L.; Smith, A. Phytochemistry 1969, 8, 437-443. (18) (a) Shinozaki, H.; Ishida, M.; Shimamoto, K.; Ohfune, Y. Br. J. Pharmacol. 1989, 98, 1213-1224. (b) Shinozaki, H.; Ishida, M.; Shimamoto, K.; Ohfune, Y. Brain Res. 1989, 98, 355-359. (19) Hayashi, Y.; Momiyama, A.; Takahashi, T.; Ohishi, H.; Ogawameguro, R.; Shigemoto, R.; Mizuno, N.; Nakanishi, S. Nature 1993, 366, 687-690.
1184 J. Org. Chem., Vol. 69, No. 4, 2004
Isolation Procedure. A specimen of D. herbacea was collected at reef flat in Yap State, Micronesia, in July 2000. A sponge specimen (1400 g) was homogenized with an equal amount of water, and 2-popanol was added followed by centrifugation. The organic solvent was removed in vacuo, and the supernatant was then lyophilized. The extract (69 g) was redissolved and chromatographed on Sephadex LH20 (5 × 60 cm, H2O, 4 mL/min, 4 g extract/load). The fraction containing dysiherbaine (DH) was collected (e.g., 330-490 mL of eluate) and lyophilized, and the active fraction (mouse assay)7a was further separated by Biogel P-2 (5 × 60 cm, H2O, 4 mL/min, 2.5 g/load). The DH-containing fractions were pooled, lyophilized, and subjected to further separations. The DH-containing fraction (2.95 g) was applied on an anion exchanger (DE52, 2.5 × 15 cm, H2O, H2O-0.05N ammonium acetate, 1 mL/min). The fractions eluted with water were pooled (fraction A), and the fraction eluted with ammonium acetate buffer were denoted as fraction B. Further separations were conducted by monitoring fractions with TLC (ninhydrin) and with 1H NMR. Dysibetaine PP (1). A portion of lyophilized fraction A (157 mg) was further separated by gel-filtration chromatography (HW-40, 2.5 × 120 cm, H2O, 0.5 mL/min). Fractions (360370 mL), which showed ninhydrin-positive spots on TLC, were combined. The 1H NMR of the combined mixture indicated the characteristic double-doublet at δ 5.2. This was further chromatographed on an HW-40 column (1.5 × 180 cm, H2O, 0.5 mL/min), then purified by HPLC to give 1 (1 × 20 cm C18 column, 0.2% aqueous acetic acid, 1 mL/min, tR 30.6 min, 3.6 mg): colorless amorphous; [R]18D -70.3 (c 0.24, H2O); IR (KBr) ν 3371, 1710, 1609, 1451, 1385, 1343, 1283, 1015 cm-1; 1H and 13 C NMR (see Table 1); HRFABMS m/z 239.1420 (M + H)+ (calcd for C12H19N2O3 239.1395). Acid Hydrolysis of 1. Compound 1 (50 µg) was dissolved in 6 N HCl (100 µL) in a sealed tube, and then the mixture was heated to 110 °C for 12 h. The solvent was removed under a stream of air. The residue was chromatographed on TLC (SiO2, 75:35:15:30, pyridine-EtOAc-acetic acid-water, ninhydrin) and compared with authentic N-methylproline (Aldrich). The hydrolysate gave two spots with Rf values each of 0.39 and 0.56, and the first spot corresponded to the authentic N-MePro. Next, the hydrolysate was treated with NaBH4. On the TLC of the product, the spot corresponding to N-MePro (Rf ) 0.39) remained unchanged. However, a new spot (Rf ) 0.79) which was identical with the authentic pipecolic acid appeared when the unidentified spot (Rf ) 0.56) disappeared. The LC-ESIMS analysis for the acid hydrolysate showed ions at m/z 127.7 (rel int 50), 129.8 (100), and 145.7 (12.5) (M + H)+. Selected ion chromatogram of the hydrolysate for the ion at m/z 130 gave peaks at tR of 2.4 min. The MS/CID/ MS for this ion gave a product ion at m/z 84. The authentic N-MePro gave the identical ion peak and product ion. The same analysis for the ion at m/z 146 indicated a peak at tR of 2.7 min and gave product ions at m/z 128, 100, 82, and 55. Chiral Gas Chromatographic Analysis. The above reaction product was treated with butanol acidified by adding AcCl (10%). After removal excess reagent, the product was further treated by TFAA (100 µL) and TFA (100 µL) at 110 °C for 30 min. This derivative was analyzed on a Chirasyl Val-III column (0.25 mm × 30 m, 60 °C initial for 5 min at 5 °C/min (20) Duke, R. K.; Chebib, M.; Balkar, V. J.; Allan, R. D.; Mewett, K. N.; Johnston, G. A. J. Neurochem. 2000, 75, 2602-2610.
Novel Betaines from a Micronesian Sponge Dysidea herbacea to 180 °C for 1 min). The GC derivatives from each D- and L-pipecolic acid gave a peak at tR 19.9 and 20.0 min, respectively. The analate gave about a 1:1 set of peaks which corresponded to DL-pipecolic acid. Dysibetaine CPb (3). Later fractions of the above HW-40 column (tR 1000-1200 min) were pooled, and subsequent HPLC purification afforded pure 3 (1 × 20 cm C18 column, 0.2% aqueous acetic acid, 1 mL/min, tR 7.85 min) as a colorless solid (2.7 mg): [R]18D +8.5 (c 0.13, H2O); CD (H2O) λext 204 (∆ 1.46), 217 (16.7); IR (KBr) ν 3404, 1679 br, 1604 (sh), 1477, 1420, 1204, 1131, 973, 939, 905, 834, 802, 721 cm-1; 1H and 13 C NMR (see Table 2); HRFABMS m/z 201.1186 (M + H)+ (calcd for C9H17N2O3 201.1239). Base Hydrolysis of 3. A mixture of 3 (200 µg) and LiOH (1 mg/mL, 72 µL, 3 equiv) was heated at 60 °C for 2 days. The reaction mixture was neutralized with HCl, and lyophilized to give 7: 1HNMR (D2O, 25 °C) δ 3.33 (2H, d, J ) 6.4 Hz), 3.08 (9H, s), 2.16-2.10 (3H, m); HRFABMS m/z 202.1080 (M + H)+ (calcd for C9H16NO4 202.1080). Dysibetaine CPa (2). Fraction B was further chromatographed on HW40 (1.5 × 160 cm, H2O, 0.5 mL/min). A pooled fraction (tR 680-700 min) was purified on reversed-phase HPLC (1 × 20 cm C18 column, 0.2% aqueous acetic acid, 1 mL/min, tR 16.81 min) to give pure 2 (2.2 mg) as an amorphous solid: [R]18D -8.1 (c 0.12, H2O); CD λext 199 (∆ -28.7), 221 (7.39); IR (KBr) ν 3435, 1702 (sh), 1682 br, 1478, 1427, 1204,
1137 cm-1; 1H and 13CNMR (see Table 2). HRFABMS m/z 202.1102 (M + H)+ (calcd for C9H16NO4 202.1079).
Acknowledgment. We would like to thank Mr. A. Tafileichig at the Marine Resources Management Division, Department of Resources & Development, Yap State Government, and Ms. M. Yasui for assisting with the sample collection. We are grateful to Dr. J. Hooper at the Queensland Museum for identifying the sponges, Prof. M. Satake at Tohoku University for recording the HMBC and HMQC spectra, Dr. K. Adachi at Marine Biotechnology Co. for measuring the 13C NMR spectra, and Dr. Y. Shigeri at the National Institute of Advanced Industrial Science and Technology for helping with the binding assay. The LCMS analysis was performed by Prof. S. Sato and Mr. Y. Kurisu at Kitasato University. A portion of this research was supported by a SUNBOR Grant. Supporting Information Available: 1H and 13C NMR and IR spectra for compounds 1-3, HMBC spectra of 1-3, HMQC spectra of 1, and 1H NMR of compound 7. This material is available free of charge via the Internet at http://pubs.acs.org. JO0355045
J. Org. Chem, Vol. 69, No. 4, 2004 1185
