Gamakamide C and D as Two New Analogues of Bitter-Tasting Cyclic Peptide with Hydantoin Structure from Oyster Crassostrea gigas

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    Two new bitter-tasting cyclic peptides comprising six amino acids, namely gamakamide C and D, were isolated from cultured oysters Crassostrea gigas. Dimethylaminoazobenzene sulfonyl–amino acid analysis detected Val and Leu in gamakamide C and Ile and Leu in gamakamide D. The molecular formula of gamakamide C was determined as C43H60N7O8S by high-resolution fast atom bombardment mass spectroscopy (HR FAB-MS) ([M + H]+ m/z 822.4200 Δ –2.4 mmu), and that of gamakamide D was determined as C43H62N7O8S by HR FAB-MS ([M + H]+ m/z 836.4379 Δ –2.0 mmu). Comparison of amino acid analyses and fragment ions by MS/MS among gamakamide C, D, and E (known), the structures of gamakamide C and D were confirmed as –ʟ-Val-ʟ-Met(SO)-ʟ-NMe-Phe-ʟ-Leu-ᴅ-Lys-ʟ-Phe– and –ʟ-Ile-ʟ-Met(SO)-ʟ-NMe-Phe-ʟ-Leu-ᴅ-Lys-ʟ-Phe–, respectively.


    Gamakamide C , Gamakamide D , Cyclic peptide , DABS-amino acid analysis , Bitter taste , Oyster

  • Introduction

    The oyster Crassostrea gigas is a common favorite seafood and is used as a minor ingredient in kimchi (traditional fermented Korean pickle) and various processed products, such as sauces and oyster extracts (Oyster, 2015). This, it is an important bivalve in the shellfish aquaculture industry in South Korea.

    Oysters take up and accumulate nutrients from plankton and organic matter in the water by filter feeding. Therefore, oyster quality depends on the cultivation conditions, such as the quantity and species of plankton available, degree of pollution, and water quality. The discoloration of oyster meat, such as greening of the intestine by chlorophyll (Kimura, 1969), red coloration (Hata et al., 1982), and yellowish coloration (Hata et al., 1987), occurs due to the plankton on which the bivalves feed. Hatano et al. (1990) also reported an unacceptable taste and coloring of cultured oysters caused by some plankton in Japan. Furthermore, Parry et al. (1989) reported a bitter taste in cultured oysters during a bloom outbreak of the diatom Rhizosolenia chunii in southern Australia. However, they were unable to detect the compound(s) responsible for the bitter taste.

    There have been no previous reports regarding the deterioration of oyster quality due to plankton in the natural setting, but shellfish farmers commonly believe that sudden changes in flavor or color are due to the feed plankton. Deterioration of the quality of cultured shellfish, including oysters, occurs sporadically every year. Therefore, it is necessary to monitor and control the feed plankton and water quality in culture farms during the harvest season for maintenance of both good quality and good food hygiene.

    Lee (1995) first reported bitter taste compounds contained in oysters cultured in Gamak Bay located on the southwestern coast of Korea. All of the oysters harvested at that time from Gamak Bay had a very bitter flavor, and even when smoked, boiled, and canned, these oysters remained bitter. Although no reports of illness followed the consumption of these bitter oysters, the unpleasant flavor adversely affected local oyster processing companies.

    Five bitter peptidic compounds, tentatively named gamakamides A to E (named after Gamak Bay), were isolated from the bitter-tasting oysters collected at Gamak Bay (Lee, 1995). The structure of gamakamide E was subsequently determined by spectrometry (Lee et al., 2012). We herein report the structures of two new analogues, gamakamides C and D.

    Materials and Methods

      >  Materials

    Bitter taste compounds were separated and isolated from the side fractions of cultured oyster Crassostrea gigas extract as reported previously (Lee et al., 2012). Repeated chromatography using an octadecyl-silica column with aqueous methanol as a solvent (Lee et al., 2012) resulted in isolation of two new analogues, gamakamide C (3.8 mg) and D (3.2 mg), as well as the known gamakamide E (2.5 mg) as bitter taste compounds.

      >  Amino acid analysis

    Constituent amino acids of gamakamide were evaluated by dimethylaminoazobenzene sulfonyl (DABS)-amino acid analysis after acidic hydrolysis (Syu et al., 2008). Aliquots of 20 μL of methanolic gamakamide C, D, and E solutions (50 μg/mL) were added to separate sample tubes and dried under a stream of nitrogen gas. The tubes were set on a hydrolysis rack after addition of 700 μL of 6 N HCl solution, and the samples were hydrolyzed for 24 hours at 110℃. Chlorine was removed under vacuum, and 20 μL of sodium hydrogen carbonate buffer (NaHCO3, pH 9.0) was added to the hydrolysate and mixed with 40 μL of DABS (1 mg/mL in acetonitrile) reagent and reacted for 12 minutes at 70℃. The reaction was stopped in an ice bath, and the solutions were diluted with 440 μL of sodium hydrogen phosphate buffer (pH 9).

    DABS-amino acids were analyzed by high-performance liquid chromatography using an Ultrasphere DABS column (5 μm, 250 × 4.6 mm; Beckman Coulter, Fullerton, CA) at a wavelength of 436 nm. Solvent elution was A: 4% DMF in 10 mM citric acid buffer (pH 6.5) and B:A (3): 4% DMF in acetonitrile (7) ratio. Gradient elution (flow rate, 1.4 mL/min) was carried out starting with 25% B to 56% B solution over 17.2 minutes, followed by gradient elution of 86% B for 23.2 minutes, with a final change to 100% B solution after 29.2 minutes.

      >  Mass analysis

    Low-resolution fast atom bombardment-mass spectrometry (FAB-MS) and high-resolution FAB-MS (HR FAB-MS) of gamakamides were performed in positive ion mode using glycerol as the matrix on a JEOL JMS 700 MS spectrophotometer (JEOL, Tokyo, Japan).

    MS/MS measurements of gamakamides were based on collision-induced dissociation occurring in the collision cell (Q2) of the triple quadrupole at a collision energy of 40 eV using an API2000 mass spectrometer (MDS SCIEX; Applied Biosystems, Foster City, CA) equipped with an electrospray ionization source in positive-ion mode. Electrospray ionization was evoked by a spray voltage of +5.5 kV, and the heated capillary temperature was maintained at 500℃. Nitrogen was used as the target gas. Gamakamide D (5 μL in methanol) was introduced into the mass spectrometer by flow injection with methanol at a flow rate of 0.2 mL/min.

    Results and Discussion

      >  Constituent amino acids

    The amino acid constituents of the gamakamides were investigated by highly sensitive DABS-amino acid analysis after hydrolysis of amino acids with HCl and comparison with the retention times of authentic amino acids. DABS-Val (17.2 min) and DABS-Leu (19.3 min) were detected at a ratio of 1:1 in gamakamide C (Fig. 1A). Gamakamide D showed DAB-Ile (18.9 min) and DAB-Leu (19.3 min) at a ratio of about 1:1 (Fig. 1B), while only a large peak of DAB-Leu (19.3 min) was detected in gamakamide E (Fig. 1C). These results indicate that in contrast to gamakamide E, which contains only two Val residues, gamakamide C contains Val and Leu while gamakamide D contains Ile and Leu.

    Val was also confirmed in gamakamide C on the mass spectrum. On FAB-MS measured in positive-ion mode, [M + H]+ ion peaks were detected at m/z 822 and m/z 822.4200 (obsd. –2.4 mmu) on HRFAB-MS (molecular formula, C42H60N7O8S) in gamakamide C, which showed 14 less mass units than gamakamide E containing Leu (m/z 836).

    In gamakamide D and E, [M + H]+ ion peaks were detected at the same mass unit, m/z 836. However, on HRFAB-MS, gamakamide D showed an [M + H]+ ion peak at m/z 836.4379 (obsd. –0.2 mmu), while in gamakamide E, the [M + H]+ ion appeared at m/z 836.4361 (obsd. –0.2 mmu). Although these mass units differed slightly on HR FAB-MS, they gave the same molecular formula of C43H62N7O8S due to Leu and Ile as isomers with the same molecular weight. The difference between gamakamide D and E on MS was ambiguous, but Ile and Leu were easily distinguishable by amino acid analysis.

      >  Arrangement of amino acids

    Two Leu residues were connected at positions 1 and 4 in gamakamide E, as shown in Fig. 3 (Gamakamide E), which was confirmed by nuclear magnetic resonance and MS analyses (Lee et al., 2012). Comparing the MS and MS/MS data of these gamakamides and the arrangement of amino acids in gamakamide E, structural differences among gamakamide C, D, and E were presumed to only exist at position 1 or 4 (Val in gamakamide C and Ile in gamakamide D). Therefore, gamakamide C possesses Val and Leu at position 1 or 4, respectively, while gamakamide D possesses Ile and Leu at these positions.

    Fig. 2 shows a comparison of the MS/MS spectra of gamakamide C and D. The [M – S(O)CH3]+ ion peak showed m/z at 758.7 and 772.6 in gamakamide C and D, respectively. In addition, the fragment ion [M – NMePhe-Leu + H]+ was observed at m/z 548.0 and 562.5 in gamakamide C and D, respectively. In gamakamide C, a series of fragment ions [M – Val-Phe-Lys-Leu + H]+ was observed at m/z 309.5. Therefore, the position of Val in gamakamide C was confirmed as position 4 (Fig. 3). In gamakamide D, a series of fragment ions [M – Ile-Phe-Lys-Leu + H]+ was found at m/z 309.5. Therefore, it was concluded that Ile was present at position 4 in gamakamide D (Fig. 3).

    The stereochemistries of Val in gamakamide C and Ile in gamakamide D were presumed to be of the l-type because of confirmation of a Lys in the gamakamide E peptide as l-type by Marfey’s method (Lee et al., 2012). Based on these results, the structure of gamakamide C was presumed to be a six-membered cyclic peptide, –ʟ-Val-ʟ-Met(SO)-ʟ-NMePhe-ʟ-Leu-ᴅ-Lys-ʟ-Phe–, containing a hydantoin ring between Lys and Phe, while gamakamide D is –ʟ-Ile-ʟ-Met(SO)-ʟ-NMePhe-ʟ-Leu-ᴅ-Lys-ʟ-Phe–.

    Hydrophobic or branched amino acids, such as Phe, Tyr, Arg, Leu, Ile, Val, and Met, are known to give a bitter taste (Food, 2015). Therefore, it was presumed that Phe, Val, Leu, and Ile, alone or in some combination, in gamakamides produce a bitter taste. Enzymatic hydrolysis or processing of protein using endopeptidase frequently leads to the production of an undesirable bitter taste due to the production of hydrophobic oligopeptides, including the bitter amino acid residues described above (Kim, 2013).

    Separate from these changes during metabolism, bitter compounds were presumed to accumulate in the digestive organs of oysters because of their consumption of plankton present in seawater. Therefore, it is necessary to identify the plankton responsible for the bitter taste of the oyster meat.

    Many cyclic peptides with strong and varied biological activities have been isolated from sponges and tunicates (Fusetani and Matsunaga, 1993) but rarely from shellfish (Reese et al., 1996). Although gamakamides C, D, and E have yet to be found to have any biological activity except bitter taste, they are expected to have some activity due to their structure. Hydantoin, which is similar to gamakamide, has a unique imidazolidine 2,4-dione with biological activity. Many chemical synthesis methods for hydantoin groups have been used for production of pharmaceuticals and pesticides (Hydantoin, 2015).

    The structure of gamakamides is similar to that of keramamides, anabaenopeptins, and oscillamides isolated from sponges and cyanobacteria with a ureido carbon and ᴅ-Lys in the molecule (Harada et al., 1885; Nagasawa et al., 1991; Marsh et al., 1997). However, little is known about the biosynthesis of the hydantoin ring in nature. Further studies using living cells are required to determine the mechanism of synthesis of the hydantoin structure of gamakamides, with a terminal carboxyl group and amino group of Phe and amino group of ᴅ-Lys.

  • 1. 2015 Food google
  • 2. Fusetani N, Matsunaga S 1993 Bioactive sponge peptides [Chem Rev] P.1793-1806 google
  • 3. Harada KI, Fujii K, Shimada T, Suzuki M, San H, Adachi K, Carmichael WW 1995 Two cyclic peptides, anabaenopeptins, a third group of bioactive compounds from the cyanobacterium Anabaena flos-aqua NRC-525-17 [Tetrahedron Lett] Vol.36 P.1511-1514 google doi
  • 4. Hata M, Nakamura K, Fujisawa H 1982 Brick-red coloration of oyster, Crassosterea gigas [Nippon Suisan Gakkaishi] Vol.48 P.975-979 google doi
  • 5. Hata M, Matsumoto I, Itoh M, Akashige S 1987 Yellowish coloration of oyster Crassostrea gigas [Nippon Suisan Gakkaishi] Vol.53 P.677-680 google doi
  • 6. Hatano M, Iida H, Seki K, Takahashi K 1990 Occurrence of unacceptable taste and coloring of giant Pacific oyster [Nippon Suisan Gakkaishi] Vol.56 P.1481-1484 google doi
  • 7. Horigome Y. 2000 Structure and synthesis of the bitter taste compound isolated from oyster, Master Thesis google
  • 8. 2015 Hydantoin google
  • 9. Kim MJ 2013 Fractionation of exopeptidase from common octopus hepatopancreas and its utilization as a food resources for debittering. Master Thesis google
  • 10. Kimura T 1969 Study on greening of canned oyster [Bull Jap Soc Sci Fish] Vol.35 P.67-76 google doi
  • 11. Lee J-S. 1995 Isolation and some properties of bitter taste compounds from cultured oyster, Crassostrea gigas [Bull Korean Fish Soc] Vol.28 P.98-104 google
  • 12. Lee JS, Satake M, Horigome Y, YOshima Y, Yasumoto T 2012 Gamakamide-E, a Strongly Bitter Tasting Cyclic Peptide with a Hydantoin Structure from Cultured Oysters Crassostrea gigas [Fish Aquat Sci] Vol.15 P.15-19 google
  • 13. Marsh IR, Bradly M, Teague SJ 1997 Total synthesis of oscilliamide Y and analogues [J Org Chem] Vol.62 P.6199-6203 google doi
  • 14. Nagasawa S, Nakamura T, Hirota H, Ohta T, Nozoe S 1991 Keramides B-D, Novel peptides from the Okinawan marine sponge, Theonella sp [J Am Chem Soc] Vol.113 P.7812-7813 google doi
  • 15. 2015 Oyster google
  • 16. Parry CD, Langdon JS, Huismann JM 1989 Toxic effects of a bloom of the diatom Rhizosolenia chunii on shellfish in Port of Phillip bay, Southern Australia [Mar Biol] Vol.102 P.25-41 google doi
  • 17. Reese MT, Gulavita NK, Nakao Y, Haean MT, Yoshida WY, Coval SJ, Scheuer PJ 1996 Kulolide: A cytotoxic depsipeptide from a Cephalaspidean Philinopsis speciosa [J Am Chem Soc] Vol.118 P.11081-11054 google doi
  • 18. Syu KY, Lin CL, Huang HC, Lin JK 2008 Determination of Theanine, GABA, and Other Amino Acids in Green, Oolong, Black, and Pearch Teas with Dabsylation and High-Performance Liquid Chromatography [J Agric Food Chem] Vol.56 P.7637-7643 google doi
  • [Fig. 1] HPLC Chromatograms of DABS-amino acid after acid hydrolysis. (A), (B) and (C) are for gamakamide C, D, and E, respectively.
    HPLC Chromatograms of DABS-amino acid after acid hydrolysis. (A), (B) and (C) are for gamakamide C, D, and E, respectively.
  • [Fig. 3.] Structures of gamakamide C, D and E.
    Structures of gamakamide C, D and E.
  • [Fig. 2.] FAB-MSMS (positive mode) spectra of gamakamide C (A) and gamakamide D (B).
    FAB-MSMS (positive mode) spectra of gamakamide C (A) and gamakamide D (B).