論文の公開元へStudies on cytotoxic cyclic peptides from marine sponges
概要
Cyclic peptides isolated from sponges have gained increasing attention recently due to their diverse bioactivities, in particular cytotoxicity. Some of them were shown to be produced by symbiotic microorganisms. Structurally, these peptides are composed of 5-14 amino acids, frequently including rare amino acid residues as well as residues of non-amino acid origin. In this work, bioassay-guided fraction of deep-sea marine sponges afforded three new cytotoxic microsclerodermins N-Q. They are hexapeptides possessing a 23-membered ring, and their differences arise from the structure of the polyhydroxylated long chain residue. The stereochemistry of the peptide was determined by analyzing NMR spectra and Marfey’s derivatives. A congener termed microsclerodermin Q was proposed as a diastereomer of dehydromicrosclerodermin B. A macrocyclic depsipeptide named cyclolithistide A was also isolated and characterized.
1. Microsclerodermins N (1) and O (2) from Pachestrella sp.
A marine sponge Pachastrella sp. was collected at Yakushinsone, at a depth of 190 m. The combined EtOH and MeOH extract of the sponge afforded microsclerodermins N (1) and O (2) (Fig. 1). The corresponding dehydration products, dehydromicrosclerodermins N (3) and O (4), which were considered as artifacts generated from 1 and 2, respectively, during the course of isolation, were also isolated (Fig. 2).
COSY and HSQC spectra showed the presence of three conservative amino acid residues in 1, which are γ-amino-β‐hydroxybutyric acid (GABOB), glycine (Gly), and sarcosine (Sar). Based on HMBC correlations related to two CH2CH units, 2-(3-amino-2-hydroxy-5-oxopyrrolidin-2-yl) acetic acid (Pyrr) and 2-carboxylated tryptophan (TrpC) residues were assigned. Interpretation of the HMBC spectrum in conjunction with the COSY spectrum permitted us to assign the remaining residue in 1 as 3-amino-12-(p-ethoxyphenyl)-2,4,5-trihydroxydodec-11-enoic acid (AETD). The amino acid sequence of the peptide was established mainly by HMBC and NOESY correlations to give the planar structure for 1.
The relative configurations of AETD were determined by coupling constant analysis together with NOE experiment. The absence of coupling between H-2 and H-3 suggested that the dihedral angle of these protons are ca 90°. A coupling constant of 10.2 Hz between H-3 and H-4 showed that these protons are anti-periplanar. In order to establish the relative configuration from C-4 and C-5, 3 was treated with 2,2- dimethoxypropane using pyridinium p-toluenesulfonate as catalyst to yield the acetonide of dehydromicrosclerodermin N (5, Fig. 2). NOE interactions were detected by NOE difference experiments. The prominent NOEs between H-2 and H-3, between H-2 and H-4, between H-3 and H-5, and between H- 4 and H-6 demonstrated that H-4 and H-5 were anti to each other within the acetonide ring and proximity of H-2 to H-4 and H-3 to H-5. These data demonstrated the (2S*, 3R*, 4S*, 5S*)-relative configuration.
Marfey's analysis of the acid hydrolyzate of 1 gave (3R)-GABOB. The vicinal diol at C-4 and C-5 in 1 was oxidized with NaIO4 followed by oxidative work-up to afford a truncated derivative with a carboxyl group at C-4, thereby converting the AETD residue into β‐hydroxyaspartic acid (OHAsp) residue. Marfey's analysis of the acid hydrolyzate of the NaIO4 oxidation product gave (2S, 3S)-OHAsp (Fig. 3). With the assigned relative configuration in mind, the absolute configuration of the AETD residue was determined as (2S, 3R, 4S, 5S). Ozonolysis of 3 followed by acid hydrolysis not exceeding 60 minutes generated D- Asp (Fig. 3). This short acid hydrolysis also afforded D-Asn which originate only from Pyrr to be observed as well (Fig. 3).
HRESIMS data indicated that 2 (Fig. 1) is 14 amu heavier than 1, and NMR analyses of 2 demonstrated that the only difference in the structures of 1 and 2 being in methyl substitution at C-6. The stereocenters of 2 were shown to be identical to those of 1 by employing same reactions and analysis (Fig. 2-3). Microsclerodermins N and O are cytotoxic against HeLa cells with IC50 values of 0.77 μM and 0.81 μM, respectively.
2. Microsclerodermins P (7) and Q (8) from the sponge designated S17502
Two congeners of microsclerodermins, microsclerodermins P (7) and Q (8) (Fig. 4) were isolated from the sponge designated S17502.
The planer structure of microsclerodermin Q (8) was elucidated to be identical with that of the known compound dehydromicrosclerodermin B (9) (Fig. 1). However, we found that the carbon chemical shifts of 8 did not coinside with those of dehydromicrosclerodermin B. Large discrepancies were observed in TrpC and Sar residues (Fig. 5), which indicated that 8 is a diastereomer of 9. To our surprise, analysis of the component amino acids showed their identity with those of 9. Because the chemical shift disagreements are notable, we suspected that we failed to assign the configuration of TrpC residue by the conventional method. We are now trying to establish a reliable method to analyse the configuration of TrpC residue. Microsclerodermin P (7) is a demethyl derivative of 8.
3. Cyclolithistide A (10) from an unidentified marine sponge S17363
Cyclolithistide A (10) was obtained from an unidentified marine sponge S17363. It was previously reported as a unique macrocyclic depsipeptide (Fig. 6) with cytotoxicity and antifungal bioactivity. Until now, nonproteinogenic Cl-Ile in 10 is still unassigned. Efforts to assign the absolute configuration of Cl-Ile is ongoing.
In summary, this is the first record of microsclerodermines from a marine sponge other than lithistid sponges. AETD, AMETD, and AEMD residues in 1-2, and 7are new amino acids which have not previously been reported. This study showed that structurally new and biologically active metabolites can be isolated from deep-sea sponges.
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