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Anti-barnacle Activity of Isocyanides Derived from Amino Acids Takuya Fukuda,a Hideki Wagatsuma,a Yoshifumi Kominami,a Yasuyuki Nogata,b Erina Yoshimura,c Kazuhiro Chiba,a and Yoshikazu Kitano*a a
Laboratory of Bio-organic Chemistry, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan, e-mail: [email protected] b Environmental Science Research Laboratory, Central Research Institute of Electric Power Industry, 1646 Abiko, Abikoshi, Chiba 270-1194, Japan c CERES Inc., 1-4-5 Midori, Abiko-shi, Chiba 270-1153, Japan Creation of new potent antifouling active compounds is important for the development of environmentally friendly antifouling agents. Fifteen isocyanide congeners derived from proteinogenic amino acids were synthesized, and the antifouling activity and toxicity of these compounds against cypris larvae of the barnacle Balanus amphitrite were investigated. All synthesized amino acid-isocyanides exhibited potent anti-barnacle activity with EC50 values of 0.07 – 10.34 lg/ml after 120 h exposure without significant toxicity. In addition, seven compounds showed more than 95% settlement inhibition of the cypris larvae at 10 lg/ml after 120 h exposure without any mortality observed. Considering their structure, these amino acid-isocyanides would eventually be biodegraded to their original nontoxic amino acids. These should be useful for further research focused on the development of environmentally friendly antifoulants. Keywords: Antifouling activity, Barnacle, Isocyanide, Amino acids, Structure–activity relationships.
Introduction Marine fouling organisms, such as hydroids, mussels, and barnacles, settle on the hulls of ships, aquaculture cages, and cooling systems of power plants, and cause serious problems for the maritime and aquaculture industries. Settlement of fouling organisms on a ship’s hull leads to an increase in fuel consumption of up to 40%. Moreover, it was estimated that fouling organisms cost the marine economy over $6.5 billion per year.[1][2] To solve these problems, a large number of antifouling (AF) paints have been developed and are widely used. Of these, organotin compounds, such as tributyl tin, exhibit high AF activity against most fouling organisms and have been widely used since the 1960s. However, organotins also show serious toxicity in aquatic environments because they act on both target and nontarget organisms and disrupt their endocrine systems.[3 – 6] For this reason, the International Maritime Organization banned the use of organotins as of September 2008. In recent years, copper-based marine coatings and various other booster biocides have been used as alternatives to organotins. However, it has been suggested that these compounds might also have a negative influence on the marine environment.[7 – 10] Therefore, development of
more environmentally benign AF compounds is urgently needed. Some marine organisms are known to prevent the settlement of fouling organisms by producing AF compounds. With a focus on the development of natural antifoulants, many AF compounds have been extracted from algae, fungi, nudibranchs, sea sponges, and other marine organisms.[11 – 23] Although many natural antifoulants exhibit high AF activity and no toxicity, the amount of compounds extracted from the organisms has thus far been extremely low. To overcome this problem, comprehensive investigations of detailed structure–activity relationships with respect to AF activity and toxicity are required in the development of new AF compounds. Recently, the authors’ research group conducted detailed structure–activity relationship studies focusing on the compound 3-isocyanotheonellin, a natural product isolated from nudibranch species.[24] Despite its simple structure, this natural product showed potent AF activity against larvae of the barnacle Balanus amphitrite.[12] Structure–activity relationships were determined by synthesizing various analogues in the laboratory. The results indicated that the isocyano functional group is an important component for the potent AF activity observed.[24 – 26] On the basis of
© 2016 Wiley-VHCA AG, Zurich, Switzerland Chem. Biodiversity 2016, 13, 1502 – 1510 DOI: 10.1002/cbdv.201600063
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these findings, various artificial isocyano compounds with AF activity were created. In particular, several isocyano cyclohexanes and simple linear alkyl isocyanides demonstrated potent anti-barnacle (AF) activity without significant toxicity.[26 – 28] However, the biodegradability and toxicity of the degradation products of these compounds could not be determined on the basis of their structures alone, suggesting that there is scope for improvement in creating new AF active compounds. Therefore, the focus of this study was on the structure of amino acids, because the amino group is easily converted to the isocyano group. There are three main properties of isocyanides derived from amino acids that make them suitable candidates as environmentally friendly AF agents. First, amino acids can be converted to the corresponding isocyanides (amino acid-isocyanides) in only a few steps. Second, amino acid-isocyanides are expected to show potent anti-barnacle activity without significant toxicity because they have an isocyano group. Finally, amino acid-isocyanides are expected to eventually biodegrade to their original non-toxic amino acids. In this paper, the anti-barnacle activity of 15 congeners of amino acid-isocyanides derived from proteinogenic amino acids is presented. To our knowledge, this is the first study to assay the AF activity of isocyanides derived from amino acids against barnacles.
Results and Discussion Synthesis of Amino Acid-Isocyanides Fifteen congeners of amino acid-isocyanides (1 – 15) were synthesized from proteinogenic amino acids. The procedures for the synthesis of amino acid-isocyanides from amino acids are described in the Supporting Information. In brief, the amino group of an amino acid was converted to an isocyano group via formylation and dehydration. All amino acid-isocyanides were prepared as enantiomeric mixtures, except for the Gly derivative 1, because epimerization of a-C-atoms was observed during the reaction. The yields were not optimized. The structures of the synthesized compounds are shown in Fig. 1 and 2. In this study, benzyl ester was constructed as an ester moiety because it is able to repress the volatility and odor of the compounds. The amino group was converted to an isocyano group, while the COOH group was converted to benzyl ester. Amino acid-isocyanides of Gly 1, Ala 2, Val 3, Leu 4, Ile 5, Met 8, Phe 10, and Trp 12 were synthesized as the original amino acid forms, whereas amino acid-isocyanides of Ser 6, Thr 7, Gln 9, Tyr 11, Asp 13, Glu 14, and Lys 15 were synthesized as modified forms. Isocyanides of Ser, Tyr, Asp, Glu, and Lys © 2016 Wiley-VHCA AG, Zurich, Switzerland
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were prepared as benzyl ether 6, acetate 11, benzyl esters 13 and 14, and acetamide 15, respectively, because the OH, COOH, and amino groups of the side chains of a-amino acids were unstable under the conditions of the dehydration reaction. In addition, Thr and Gln derivatives were obtained as an alkene (7) and a nitrile (9), respectively, because of the occurrence of dehydration of alcohol and carboxamide during the process of isonitrile preparation. However, synthesis of isocyanides derived from Cys, Asn, Arg, and His was difficult, and they were not prepared in this study. AF Activity of the Synthesized Amino Acid-Isocyanides The rates of settlement and mortality of compounds 1 – 15 against cypris larvae of B. amphitrite after 48 and 120 h are shown in Fig. 1 and 2, respectively. CuSO4 was used as a positive control. The EC50 and LC50 values after 120 h exposure are summarized in Table 1. As expected, the synthesized amino acidisocyanides showed effective anti-barnacle activity. After 48 h, almost all of the synthesized compounds showed a rate of settlement of cypris larvae below 50% at 0.01 lg/ml, and no significant difference was observed among compounds 1 – 15 (Fig. 1). Therefore, EC50 values were analyzed from the AF assay data obtained after 120 h. All of the synthesized isocyano compounds showed effective AF activity with EC50 values of 0.07 – 10.34 lg/ml. Above all, according to the EC50 values, isocyanides 7, 10, and 11 showed more potent AF activity than CuSO4, which showed a similar EC50 value as 3-isocayanotheonellin. However, isocyanides 1 – 5 and 15 exhibited relatively high AF activity (EC50 = 0.97 – 2.67 lg/ml), whereas isocyanides 6, 8, 9, 13, and 14 showed moderate AF activity (EC50 = 3.96 – 10.34 lg/ml). These results indicate that the aromatic ring of the a-side chain might be effective in improving anti-barnacle activity. After 120 h, all of the synthesized amino acid-isocyanides showed low mortality rates at high concentration, and the LC50 values of the amino acid-isocyanides were all greater than 100 lg/ml, with the exception of the Leu 4, Ile 5, Met 8, and Asp 13 derivatives. In our previous study, secondary alkyl isocyanides showed weaker AF activity compared to tertiary alkyl isocyanides.[27][28] By contrast, the data of the present study indicated that the secondary alkyl isocyanides derived from a-amino acids also had potent AF activity as well as the tertiary alkyl isocyanides. The therapeutic ratios (LC50/EC50) after 120 h exposure are also summarized in Table 1. The therapeutic ratio (LC50/EC50) is used to assess the safety range of AF compounds, and a compound with an LC50/EC50 www.cb.wiley.com
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Figure 1. Antifouling (AF) activity of compounds 1 – 15 and CuSO4 against cypris larvae of Balanus amphitrite after 48 h exposure. The rates of settlement (●) and mortality (■) at different concentrations are plotted. Data plotted are means SD of three replicates. Data points not joined to a line indicate the rate of settlement and mortality in filtered seawater diluted to 80% with deionized water (control).
ratio > 15 is generally considered as a nontoxic AF compound.[21][22] As shown in Table 1, 11 of the synthesized compounds showed nontoxic LC50/EC50 www.cb.wiley.com
values (> 15). In particular, the amino acid-isocyanides of Thr 7, Phe 10, and Tyr 11 had remarkably high LC50/EC50 values (> 500). © 2016 Wiley-VHCA AG, Zurich, Switzerland
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Figure 2. Antifouling (AF) activity of compounds 1 – 15 and CuSO4 against cypris larvae of Balanus amphitrite after 120 h exposure. The rates of settlement (●) and mortality (■) at different concentrations are plotted. Data plotted are means SD of three replicates. Data points not joined to a line indicate the rate of settlement and mortality in filtered seawater diluted to 80% with deionized water (control).
From the perspective of the practical application of a compound as an antifoulant, it is more important to consider the concentration of complete inhibition © 2016 Wiley-VHCA AG, Zurich, Switzerland
than the EC50 value. The > 95% effective concentration for inhibition (EC95) values of the synthesized amino acid-isocyanides and CuSO4 are summarized in www.cb.wiley.com
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Table 1. Settlement inhibition, toxicity effects, and therapeutic ratios of the synthesized compounds 1 – 15 and CuSO4 (positive control) against cypris larvae of Balanus amphitrite after 120 h exposure. Antifouling activity and toxicity are indicated as EC50 and LC50 values, respectively Compound
EC50 [lg/ml]
LC50 [lg/ml]
LC50/EC50
CuSO4 1 (Gly) 2 (Ala) 3 (Val) 4 (Leu) 5 (Ile) 6 (Ser) 7 (Thr) 8 (Met) 9 (Gln) 10 (Phe) 11 (Tyr) 12 (Trp) 13 (Asp) 14 (Glu) 15 (Lys)
0.27 0.97 2.67 1.60 1.51 1.41 7.99 0.07 3.97 10.34 0.14 0.17 3.94 5.49 3.96 2.63
2.57 > 100 > 100 > 100 84.02 58.29 > 100 > 100 43.79 > 100 > 100 > 100 > 100 43.22 > 100 > 100
9.52 > 103.09 > 37.45 > 62.50 55.64 41.34 > 12.52 > 1428.57 11.03 > 9.67 > 714.29 > 588.24 > 25.38 7.87 > 25.25 > 38.02
Table 2. EC95 values of the synthesized amino acid-isocyanides were 3 – 100 lg/ml and 10 – 100 lg/ml after 48 and 120 h exposure, respectively. It is notable that almost all of the synthesized compounds showed a low mortality rate at their EC95 values. In particular, the mortality rates of the compounds at their EC95 values after 48 h were close to zero. However, more than 70% mortality of cypris larvae was observed after 48 h in CuSO4 at its EC95. Ten of the amino acid-isocyanides exhibited lower or equal EC95 values than that of CuSO4 (10 lg/ml) after 48 h exposure; isocyanide 11 derived from Tyr showed a particularly low EC95 value of 3 lg/ml. Moreover, the EC95 values of seven isocyanides (3, 4, 8, 10, 11, 12, and 14) were the same as that of CuSO4 (10 lg/ml) even after 120 h exposure. These results suggested that the synthesized amino acid-isocyanides are promising candidate antifoulants with low toxicity. Recently, we also investigated the mechanisms underlying the observed AF activity using fluorescently labeled AF compounds.[29][30] The results suggested that efficient AF activity might rely on the action of a compound in the oil cell region of barnacle cyprids. Oil cells function as a nutritional source for cypris larvae, which do not take in food during this stage of development.[31][32] On the basis of their structures, the synthesized amino acid-isocyanides are considered to be hydrophobic and lipophilic compounds, and are therefore expected to enter the oil cell region in barnacle cyprids through membranes. Together, this suggests that the action of amino acid-isocyanides in the oil cell www.cb.wiley.com
Table 2. The 95% effective concentrations for inhibition of cypris larvae (EC95) of compounds 1 – 15 and CuSO4 after 48 and 120 h exposure Compound
48 h [lg/ml]
120 h [lg/ml]
CuSO4 1 (Gly) 2 (Ala) 3 (Val) 4 (Leu) 5 (Ile) 6 (Ser) 7 (Thr) 8 (Met) 9 (Gln) 10 (Phe) 11 (Tyr) 12 (Trp) 13 (Asp) 14 (Glu) 15 (Lys)
10 100 30 10 10 10 100 30 10 30 10 3 10 10 10 10
10 100 30 10 10 30 100 30 10 100 10 10 10 30 10 100
region in barnacle cyprids may be a factor contributing to its potent anti-barnacle activity, although the detailed mechanism is currently unclear. In this work, the amino groups of amino acids were converted to isocyano groups and the COOH groups were converted to benzyl esters. It is wellknown that the ester moiety can be hydrolyzed to a COOH moiety. In addition, the isocyano group is easily hydrolyzed to an amino group under mild conditions.[33] Therefore, the synthesized amino acid-isocyanides show strong potential to eventually biodegrade to their original nontoxic amino acids in the seawater.
Conclusions Fifteen congeners of amino acid-isocyanides were synthesized and evaluated for their AF activity and toxicity against the cypris larvae of the barnacle B. amphitrite. The anti-barnacle activity of the synthesized isocyanides was in the EC50 range of 0.07 – 10.34 lg/ml, and the therapeutic ratios (LC50/ EC50) of the amino acid-isocyanides revealed that they are promising candidate AF agents with low toxicity. In addition, nearly all of the synthesized compounds showed more than 95% settlement inhibition with no mortality observed. Since these isocyanides are expected to eventually biodegrade to their original nontoxic amino acids, these results will be applied in further research focused on the development of environmentally friendly AF agents. © 2016 Wiley-VHCA AG, Zurich, Switzerland
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Experimental Section
General
Cypris Larvae
The isocyanides 1,[37] 2,[38][39] 3,[38][39] 4,[38][39] 5,[38] 10,[38] 13,[39] and 14[39] were prepared according to literature procedure. Column Chromatograpy (CC): SiO2 Kanto Chemical 60N (SiO2, 63 – 210 lm). IR Spectra: JEOL WINSPEC-50 IR spectrometer; ~v in cm1. NMR Spectra: JEOL ECA-600 or JEOL ECS-400 NMR spectrometer; in CDCl3. All 1H-NMR data: d in ppm rel. to Me4Si as an internal standard, J in Hz. All 13C-NMR data: d in ppm rel. to the central line of the triplet for CDCl3 at 77.0 ppm. ESI-MS: JEOL JMS-T100 mass spectrometer; in m/z. Benzyl 3-(Benzyloxy)-2-isocyanopropanoate (6). 10-Camphorsulfonic acid (499 mg, 2.15 mmol) was added to a soln. of O-benzyl-L-serine (351 mg, 1.80 mmol) in BnOH (2 ml). After the reaction mixture was stirred at 60 °C for 48 h, sat. NaHCO3 (50 ml) was added, and resultant mixture was extracted with AcOEt. The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 4:1 ? 2:1 ? 1:1 ? 1:4) to give amine 6a (383 mg, 1.34 mmol, 75%) as a colorless oil. Ac2O (970 ll, 10.3 mmol) was added to a soln. of amine 6a (979 mg, 3.43 mmol) in HCOOEt (5 ml). After the reaction mixture was stirred at r.t. for 25 h, sat. NaHCO3 (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 1:1) to give formamide 6b (1.05 g, 3.35 mmol, 97%) as a colorless oil. Pyridine (674 ll, 8.35 mmol) and PhOPOCl2 (388 ll, 2.59 mmol) were added successively to a soln. of formamide 6b (524 mg, 1.67 mmol) in CH2Cl2 (5 ml). After the reaction mixture was stirred at r.t. for 2 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt. The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 3:1) to give isocyanide 6 (18.3 mg, 62 lmol, 4%) as a slightly yellow oil. IR (neat): 3087, 3064, 3033, 2952, 2871, 2152, 1756, 1456, 1280, 1195, 1112, 738, 698. 1H-NMR (400 MHz, CDCl3): 7.42 – 7.21 (m, 10 H); 5.29 – 5.20 (m, 2 H); 4.63 – 4.54 (m, 2 H); 4.51 – 4.40 (m, 1 H); 3.91 – 3.81 (m, 2 H). 13C-NMR (100 MHz, CDCl3): 164.7; 161.5; 136.9; 134.6; 128.9; 128.8; 128.5; 128.2; 127.8; 73.7; 69.5; 68.5; 56.9. ESI-MS: 318.1104 ([M + Na]+, C18 H17 NNaOþ 3 ; calc. 318.1106). Benzyl 2-Isocyanocrotonate (7). Ac2O (14.8 ml, 157 mmol) was added to a soln. of L-threonine
Adult barnacles (Balanus amphitrite) attached to bamboo poles were procured from oyster farms in Lake Hamana, Shizuoka, Japan. Barnacles were maintained in an aquarium at 20 1 °C and were fed with brine shrimp (Artemia salina) nauplii. The barnacles were dried at r.t. overnight and were then immersed in seawater. After immersion, the broods released stage I – II nauplii. The nauplii thus obtained were collected and cultured in 2 l glass beakers at 25 °C at an initial density of 3 larvae/ml. Nauplii fed on the diatom Chaetoceros gracilis (initial concentration of 3.0 9 105 cells/ml). Cultures were maintained in filtered (0.2 lm pore filter) seawater diluted to 80% with deionized H2O (80% filtered seawater, salinity 2.8%), 30 lg/l streptomycin, and 20 mg/l penicillin G at 25 °C with mild aeration. The nauplii were cultured for 4 days, and C. gracilis was added daily (0.5 – 1.0 9 105 cells/ml). Larvae reached the cyprid stage in 5 days. The cyprids were stored in the dark at 5 °C until used. The day that the newly transformed cyprids were collected was designated as Day 0.[34]
AF Assay The isocyanides were dissolved in MeOH. The soln. was further diluted with MeOH at various concentrations: 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 lg/ml. A 2 ml soln. was pipetted into each well of 24-well polystyrene tissue culture plates and air-dried. A quantity of 2 ml of 80% filtered seawater and six 2or 3-day-old cyprids were added into the wells. Four wells were used for each experiment. The plates were maintained in the dark at 25 °C. After 48 and 120 h, the numbers of larvae that attached, metamorphosed, died, or did not settle were counted under a microscope.[35][36] Cyprids that did not move, had extended appendages, and did not respond after a light touch with a metal probe were regarded as dead. The experiment was performed three times with different batches of larvae. The same assay was carried out simultaneously with CuSO4 as a positive control. The median effective and lethal concentrations (EC50 and LC50, resp.) were calculated by probit analysis. However, since probit analysis could not be applied to calculate the EC50 values of isocyanide from Met 8, Glu 9, Trp 12, Asp 13, Glu 14, and Lys 15, these values were estimated by linear graphical interpolation. © 2016 Wiley-VHCA AG, Zurich, Switzerland
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(6.24 ml, 52.3 mmol) in HCOOH (25 ml). After the reaction mixture was stirred at r.t. for 48 h, the mixture was concentrated under reduced pressure. The residue was recrystallized with MeOH and AcOEt to give formamide 7a (2.62 g, 17.8 mmol, 34%). K2CO3 (7.37 g, 53.3 mmol) and BnBr (3.18 ml, 26.8 mmol) were added successively to a soln. of formamide 7a (2.63 g, 17.9 mmol) in DMF (25 ml). After the reaction mixture was stirred at 70 °C for 5 days, H2O (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 1:4) to give formamide 7b (762 mg, 3.21 mmol, 18%) as a yellow oil. Pyridine (380 ll, 4.71 mmol) was added to a soln. of formamide 7b (749 mg, 3.16 mmol) in Ac2O (6 ml). After the reaction mixture was stirred at r.t. for 6 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), H2O (20 ml), and brine (20 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt = 1:1) to give formamide 7c (785 mg, 2.81 mmol, 89%) as a white solid. Pyridine (330 ll, 4.09 mmol) and PhOPOCl2 (85.0 ll, 0.568 mmol) were added successively to a soln. of formamide 7c (132 mg, 0.47 mmol) in CH2Cl2 (6 ml). After the reaction mixture was stirred at r.t. for 6 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/ AcOEt 9:1) to give isocyanide 8 (56.3 mg, 280 lmol, 59%) as a slightly yellow oil. IR (neat): 3324, 2068, 2027, 2981, 2966, 2940, 2123, 1724, 1452, 1270, 1211, 754, 703. 1 H-NMR (400 MHz, CDCl3): 7.62 – 7.31 (m, 5 H); 7.16 – 7.07 (m, 1 H); 5.27 (s, 2 H); 2.05 (d, J = 7.2, 3 H). 13 C-NMR (100 MHz, CDCl3): 170.3; 160.1; 141.9; 135.0; 128.8; 128.7; 128.4; 120.7; 67.9; 15.1. ESI-MS: 224.0690 ([M + Na]+, C12H11NNaO+; calc. 224.0687). Benzyl 2-Isocyano-4-(methylthio)butanoate (8). Ac2O (10.8 ml, 114 mmol) was added to a soln. of L-methionine (5.23 g, 35.1 mmol) in HCOOH (25 ml). After the reaction mixture was stirred at r.t. for 48 h, the mixture was concentrated under reduced pressure. The residue was recrystallized with MeOH and AcOEt to give formamide 8a (4.39 g, 24.8 mmol, 71%) as a white solid. K2CO3 (5.49 g, 39.7 mmol) and BnBr (2.35 ml, 19.8 mmol) were added successively to a soln. of formamide 8a (2.34 g, 13.2 mmol) in DMF (20 ml). After the reaction mixture was stirred at r.t. for 24 h, H2O www.cb.wiley.com
(50 ml) was added, and resultant mixture was extracted with AcOEt. The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt = 1:1 ? 2:1) to give formamide 8b (1.48 g, 5.54 mmol, 42%) as a yellow oil. Pyridine (1.11 ml, 13.8 mmol) and PhOPOCl2 (740 ll, 4.95 mmol) were added successively to a soln. of formamide 8b (735 mg, 2.75 mmol) in CH2Cl2 (2 ml). After the reaction mixture was stirred at r.t. for 2.5 h, H2O (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), H2O (20 ml) and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 3:1) to give isocyanide 8 (174 mg, 0.697 mmol, 25%) as a slightly yellow green oil. IR (neat): 3089, 3066, 3033, 2969, 2917, 2148, 1756, 1456, 1440, 1272, 1211, 752, 698. 1H-NMR (600 MHz, CDCl3): 7.37 – 7.30 (m, 5 H); 5.23 – 5.17 (m, 2 H); 4.54 – 4.50 (m, 1 H); 2.65 – 2.57 (m, 2 H); 2.19 – 2.10 (m, 2 H); 2.03 (s, 3 H). 13C-NMR (150 MHz, CDCl3): 166.1; 160.8; 134.4; 128.6; 1283.5; 128.3; 68.1; 55.0; 31.7; 29.4; 15.2. ESI-MS: 272.0721 ([M + Na]+, C13H15NNaO2S+; calc. 272.0712). Benzyl 4-Cyano-2-isocyanobutanoate (9). Ac2O (4.06 ml, 43.0 mmol) was added to a soln. of L-glutamine (2.08 g, 14.2 mmol) in HCOOH (25 ml). After the reaction mixture was stirred at r.t. for 23 h, the mixture was concentrated under reduced pressure. The residue was recrystallized with MeOH and AcOEt to give formamide 9a (1.61 g, 9.26 mmol, 65%) as a white solid. K2CO3 (3.79 g, 27.4 mmol) and BnBr (1.63 ml, 13.7 mmol) were added successively to a soln. of formamide 9a (1.60 g, 9.17 mmol) in DMF (20 ml). After the reaction mixture was stirred at 60 °C for 3 h, H2O (50 ml) was added, resultant mixture was extract with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was recrystallized with AcOEt to give formamide 9b (453 mg, 1.71 mmol, 19%) as a white solid. Pyridine (609 μl, 7.55 mmol) and PhOPOCl2 (197 ll, 1.32 mmol) were added successively to a soln. of formamide 9b (232 mg, 0.89 mmol) in CH2Cl2 (10 ml). After the reaction mixture was stirred at r.t. for 2.5 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt = 2:1) to give isocyanide 9 (53.8 mg, 0.220 mmol, 25%) as a slightly yellow oil. IR (neat): 3089, 3064, 3035, 2956, 2250, 2152, 1754, 1456, 1214, © 2016 Wiley-VHCA AG, Zurich, Switzerland
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750, 698. 1H-NMR (600 MHz, CDCl3): 7.43 – 7.32 (m, 5 H); 5.25 (s, 2 H); 4.49 – 4.42 (m, 1 H); 2.65 – 2.74 (m, 2 H); 2.38 – 2.17 (m, 2 H). 13C-NMR (150 MHz, CDCl3): 165.0; 162.9; 134.2; 129.2; 128.9; 128.7; 69.0; 55.0; 28.5; 13.7. ESI-MS: 251.0797 ([M + Na]+, C13 H12 N2 NaOþ 2 ; calc. 251.0797). Benzyl 3-(4-Acetoxyphenyl)-2-isocyanopropanoate (11). Ac2O (5.60 ml, 59.2 mmol) was added to a soln. of L-tyrosine (3.56 g, 19.6 mmol) in HCOOH (20 ml). After the reaction mixture was stirred at r.t. for 24 h, the mixture was concentrated under reduced pressure. The residue was recrystallized with MeOH and AcOEt to give formamide 11a (985 mg, 4.71 mmol, 24%). K2CO3 (1.91 g, 13.8 mmol) and BnBr (820 ll, 6.90 mmol) were added successively to a soln. of formamide 11a (968 mg, 4.62 mmol) in DMF (20 ml). After the reaction mixture was stirred at 70 °C for 47 h, H2O (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/ AcOEt = 1:1 ? 2:3) to give formamide 11b (290 mg, 0.970 mmol, 21%) as a yellow oil. Pyridine (116 ll, 1.43 mmol) was added to a soln. of formamide 11b (288 mg, 0.960 mmol) in Ac2O (2 ml). After the reaction mixture was stirred at r.t. for 18 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), H2O (20 ml) and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 3:1) to give formamide 11c (321 mg, 940 lmol, 98%) as a white solid. Pyridine (160 ll, 1.98 mmol) and PhOPOCl2 (73.0 ll, 488 lmol) were added successively to a soln. of formamide 11c (138 mg, 410 lmol) in CH2Cl2 (5 ml). After the reaction mixture was stirred at r.t. for 4 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 2:1) to give isocyanide 11 (16.2 mg, 50 lmol, 12%) as a slightly yellow oil. IR (neat): 3091, 3066, 3035, 2939, 2148, 1756, 1454, 1220, 752, 700. 1H-NMR (400 MHz, CDCl3): 7.44 – 7.28 (m, 5 H); 7.22 – 6.98 (m, 4 H); 5.20 (s, 2 H); 4.50 – 4.43 (m, 1 H); 3.27 – 3.09 (m, 2 H); 2.29 (s, 3 H). 13 C-NMR (100 MHz, CDCl3): 169.4; 165.9; 1661.3; 150.4; 134.4; 131.8; 130.5; 128.9; 128.8; 128.7; 122.1; 68.5; 58.0; 38.3; 21.3. ESI-MS: 346.1055 ([M + Na]+, C19 H17 NNaOþ 4 ; calc. 346.1055). © 2016 Wiley-VHCA AG, Zurich, Switzerland
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Benzyl 3-(1H-Indol-3-yl)-2-isocyanopropanoate (12). 10-Camphorsulfonic acid (5.00 g, 2.15 mmol) was added to a soln. of L-tryptophan (4.01 g, 19.6 mmol) in BnOH (4 ml). After the reaction mixture was stirred at 60 °C for 3 days, sat. NaHCO3 (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/ AcOEt 5:1 ? 3:1 ? 1:1 ? 1:2 ? 1:4) to give amine 12a (600 mg, 2.04 mmol, 10%) as a colorless oil. p-Toluenesufonic acid (35.3 mg, 205 lmol) was added to a soln. of amine 12a (547 mg, 1.86 mmol) in HCOOEt (30 ml). After the reaction mixture was stirred at 60 °C for 48 h, sat. NaHCO3 (50 ml) was added to the reaction mixture, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 1:1 ? 1:2) to give formamide 12b (492 mg, 1.53 mmol, 82%) as a red oil. Pyridine (995 ll, 7.18 mmol) and PhOPOCl2 (322 ll, 2.15 mmol) were added successively to a soln. of formamide 12b (463 mg, 1.43 mmol) in CH2Cl2 (5 ml). After the reaction mixture was stirred at r.t. for 2 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/ AcOEt = 4:1 ? 3:1) to give isocyanide 12 (113 mg, 370 lmol, 26%) as a slightly yellow oil. IR (neat): 3417, 3062, 3035, 2954, 2929, 2150, 1749, 1457, 1213, 744, 698. 1 H-NMR (400 MHz, CDCl3): 8.07 (s, 1 H); 7.58 – 7.53 (m, 1 H); 7.46 – 7.30 (m, 5 H); 7.24 – 7.10 (m, 4 H); 5.18 – 5.10 (m, 2 H); 4.60 – 4.54 (m, 1 H); 3.51 – 3.32 (m, 2 H). 13CNMR (100 MHz, CDCl3): 166.5; 160.4; 136.2; 134.7; 128.8; 128.8; 128.5; 126.9; 123.8; 122.6; 120.0; 118.3; 111.6; 108.6; 68.3; 57.6; 29.7. ESI-MS: 327.1107 ([M + Na]+, C19 H16 N2 NaOþ 2 ; calc. 327.1110). Benzyl 6-Acetamido-2-isocyanohexanoate (15). 10-Camphorsulfonic acid (2.59 g, 11.1 mmol) was added to a soln. of e-N-acetyllysine (703 mg, 3.74 mmol) in BnOH (2 ml). After the reaction mixture was stirred at 60 °C for 4 days, sat. NaHCO3 (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/ AcOEt 5:1 ? 3:1 ? 1:1 ? 1:2 ? 1:4) to give amine 15a (250 mg, 900 lmol, 24%) as a colorless oil. Ac2O (510 ll, 5.40 mmol) was added to a soln. of amine 15a (250 mg, 900 lmol) in HCOOEt (5 ml). After the reaction www.cb.wiley.com
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mixture was stirred at r.t. for 7 days, sat. NaHCO3 (50 ml) was added to the reaction mixture, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 1:1 ? 1:4) to give formamide 15b (161 mg, 530 lmol, 58%) as a colorless oil. Pyridine (108 ll, 1.33 mmol) and PhOPOCl2 (62 ll, 414 lmol) were added successively to a soln. of formamide 15b (84.3 mg, 280 lmol) in CH2Cl2 (5 ml). After the reaction mixture was stirred at r.t. for 3 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 2:7 ? 1:8) to give isocyanide 15 (23.5 mg, 82 lmol, 30%) as a colorless oil. IR (neat): 3303, 3087, 3068, 3035, 2937, 2865, 2148, 1753, 1654, 1456, 1197, 750, 698. 1H-NMR (600 MHz, CDCl3): 7.41 – 7.31 (m, 5 H); 5.22 (s, 2 H); 4.32 – 4.28 (m, 1 H); 3.26 – 3.15 (m, 2 H); 2.01 – 1.87 (m, 5 H); 1.57 – 1.41 (m, 4 H). 13C-NMR (100 MHz, CDCl3): 170.2; 166.5; 160.5; 134.7; 128.9; 128.8; 128.5; 68.3; 56.6; 39.2; 32.3; 28.7; 23.4; 22.6. ESI-MS: 311.1372 ([M + Na]+, C16 H20 N2 NaOþ 3 ; calc. 311.1372).
Supplementary Material Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbdv.201600063.
Acknowledgements The authors thank Prof. N. Fusetani of Hokkaido University for his helpful suggestions. The authors also gratefully acknowledge Associate Prof. K. Kikuchi and H. Suetake of the University of Tokyo, and Mr. T. Ookawara of the Maisaka Oyster Cultivation Union of Lake Hamana for providing the barnacles. This work was partially supported by JST A-STEP AS232Z01296D and JSPS KAKENHI 25660087.
References [1] P. Bhadury, P. C. Wright, Planta 2004, 219, 561. [2] D. M. Yabra, S. Kiil, K. D. Johansen, Prog. Org. Coat. 2004, 50, 75. [3] C. Alzieu, Sci. Total. Environ. 2000, 258, 99. [4] S. M. Evans, G. J. Nicholson, Sci. Total. Environ. 2000, 258, 73. [5] M. Ramon, M. J. Amor, Mar. Environ. Res. 2001, 52, 463. [6] Y. Vishwakiran, A. C. Anil, K. Venkat, S. S. Sawant, Chemosphere 2006, 62, 1718.
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[7] A. P. Negri, L. D. Smith, N. S. Webster, A. J. Heyward, Mar. Pollut. Bull. 2002, 44, 111. [8] I. Omae, Appl. Organomet. Chem. 2003, 17, 81. [9] I. K. Konstantinou, T. A. Albanis, Environ. Int. 2004, 30, 235. [10] K. V. Thomas, S. Brooks, Biofouling 2010, 26, 73. [11] T. Okino, E. Yoshimura, H. Hirota, N. Fusetani, Tetrahedron Lett. 1995, 36, 8637. [12] T. Okino, E. Yoshimura, H. Hirota, N. Fusetani, Tetrahedron 1996, 52, 9447. [13] H. Hirota, Y. Tomono, N. Fusetani, Tetrahedron 1996, 52, 2359. [14] S. Tsukamoto, H. Kato, H. Hirota, N. Fusetani, Tetrahedron Lett. 1996, 37, 1439. [15] N. Fusetani, Nat. Prod. Rep. 2004, 21, 94. [16] C. Hellio, J. P. Marechal, B. Veron, G. Bremer, A. S. Crare, Y. L. Gal, Mar. Biotechnol. 2004, 6, 67. [17] ‘Antifouling Compounds’, Eds. N. Fusetani and A. S. Clare, Springer-Verlag, Berlin; New York, 2006. [18] ‘Advances in Marine Antifouling Coatings and Technologies’, Eds. C. Hellio and D. M. Yebra, Woodhead Publishing Ltd., Cambridge, 2009. [19] S. H. Qi, Y. Xu, H. R. Xiong, P. Y. Qian, S. Zhang, World J. Microbiol. Biotechnol. 2009, 25, 399. [20] Y. Xu, H. Li, X. Li, X. Xiao, P. Y. Qian, Mar. Biotechnol. 2009, 11, 495. [21] P. Y. Qian, Y. Xu, N. Fusetani, Biofouling 2010, 26, 223. [22] P. Y. Qian, Z. Li, Y. Xu, Y. Li, N. Fusetani, Biofouling 2015, 31, 101. [23] S. Dobretsov, R. Abed, M. Teplitski, Biofouling 2013, 29, 423. [24] Y. Kitano, T. Ito, T. Suzuki, Y. Nogata, K. Shinshima, E. Yoshimura, K. Chiba, M. Tada, I. Sakaguchi, J. Chem. Soc., Perkin Trans. 1 2002, 2251. [25] Y. Kitano, A. Yokoyama, Y. Nogata, K. Shinshima, E. Yoshimura, K. Chiba, M. Tada, I. Sakaguchi, Biofouling 2003, 19, 187. [26] Y. Kitano, Y. Nogata, K. Shinshima, E. Yoshimura, K. Chiba, M. Tada, I. Sakaguchi, Biofouling 2004, 20, 93. [27] Y. Kitano, C. Akima, E. Yoshimura, Y. Nogata, Biofouling 2011, 27, 201. [28] Y. Nogata, Y. Kitano, E. Yoshimura, K. Shinshima, I. Sakaguchi, Biofouling 2004, 20, 87. [29] Y. Kitano, Y. Nogata, K. Matsumura, E. Yoshimura, K. Chiba, M. Tada, I. Sakaguchi, Tetrahedron 2005, 61, 9969. [30] S. Fujiwara, C. Akima, Y. Nogata, E. Yoshimura, K. Chiba, Y. Kitano, J. Exp. Mar. Bio. Ecol. 2013, 445, 88. [31] L. J. Walley, Philos. Trans. R. Soc. B, 1969, 256, 237. [32] D. L. Holland, G. Walker, ICES. J. Mar. Sci. 1975, 36, 162. [33] I. Ugi, ‘Isonitrile Chemistry’, Academic Press, New York, 1971. [34] C. G. Satuito, K. Shimuzu, K. Natoyama, M. Yamazaki, N. Fusetani, Mar. Biol. 1996, 127, 125. [35] D. Rittschof, A. S. Clare, D. J. Gerhart, S. A. Mary, J. Bonaventura, Biofouling 1992, 6, 115. [36] N. Fusetani, H. Hirota, T. Okino, Y. Tomono, E. Yosimura, J. Nat. Toxins. 1996, 5, 249. [37] S. B. Gomes, P. T. Gomes, M. T. Duarte, R. E. Di Paolo, A. L. Macßanita, M. J. Calhorda, Inorg. Chem. 2009, 48, 11176. [38] H. P. Hemantha, V. V. Sureshbabu, J. Pept. Sci. 2010, 16, 644. [39] M. Wang, X. Liu, P. He, L. Lin, X. Feng, Chem. Comm. 2013, 49, 2572. Received February 22, 2016 Accepted June 10, 2016
© 2016 Wiley-VHCA AG, Zurich, Switzerland
Anti-barnacle Activity of Isocyanides Derived from Amino Acids Takuya Fukuda,a Hideki Wagatsuma,a Yoshifumi Kominami,a Yasuyuki Nogata,b Erina Yoshimura,c Kazuhiro Chiba,a and Yoshikazu Kitano*a a
Laboratory of Bio-organic Chemistry, Tokyo University of Agriculture and Technology, 3-5-8 Saiwai-cho, Fuchu-shi, Tokyo 183-8509, Japan, e-mail: [email protected] b Environmental Science Research Laboratory, Central Research Institute of Electric Power Industry, 1646 Abiko, Abikoshi, Chiba 270-1194, Japan c CERES Inc., 1-4-5 Midori, Abiko-shi, Chiba 270-1153, Japan Creation of new potent antifouling active compounds is important for the development of environmentally friendly antifouling agents. Fifteen isocyanide congeners derived from proteinogenic amino acids were synthesized, and the antifouling activity and toxicity of these compounds against cypris larvae of the barnacle Balanus amphitrite were investigated. All synthesized amino acid-isocyanides exhibited potent anti-barnacle activity with EC50 values of 0.07 – 10.34 lg/ml after 120 h exposure without significant toxicity. In addition, seven compounds showed more than 95% settlement inhibition of the cypris larvae at 10 lg/ml after 120 h exposure without any mortality observed. Considering their structure, these amino acid-isocyanides would eventually be biodegraded to their original nontoxic amino acids. These should be useful for further research focused on the development of environmentally friendly antifoulants. Keywords: Antifouling activity, Barnacle, Isocyanide, Amino acids, Structure–activity relationships.
Introduction Marine fouling organisms, such as hydroids, mussels, and barnacles, settle on the hulls of ships, aquaculture cages, and cooling systems of power plants, and cause serious problems for the maritime and aquaculture industries. Settlement of fouling organisms on a ship’s hull leads to an increase in fuel consumption of up to 40%. Moreover, it was estimated that fouling organisms cost the marine economy over $6.5 billion per year.[1][2] To solve these problems, a large number of antifouling (AF) paints have been developed and are widely used. Of these, organotin compounds, such as tributyl tin, exhibit high AF activity against most fouling organisms and have been widely used since the 1960s. However, organotins also show serious toxicity in aquatic environments because they act on both target and nontarget organisms and disrupt their endocrine systems.[3 – 6] For this reason, the International Maritime Organization banned the use of organotins as of September 2008. In recent years, copper-based marine coatings and various other booster biocides have been used as alternatives to organotins. However, it has been suggested that these compounds might also have a negative influence on the marine environment.[7 – 10] Therefore, development of
more environmentally benign AF compounds is urgently needed. Some marine organisms are known to prevent the settlement of fouling organisms by producing AF compounds. With a focus on the development of natural antifoulants, many AF compounds have been extracted from algae, fungi, nudibranchs, sea sponges, and other marine organisms.[11 – 23] Although many natural antifoulants exhibit high AF activity and no toxicity, the amount of compounds extracted from the organisms has thus far been extremely low. To overcome this problem, comprehensive investigations of detailed structure–activity relationships with respect to AF activity and toxicity are required in the development of new AF compounds. Recently, the authors’ research group conducted detailed structure–activity relationship studies focusing on the compound 3-isocyanotheonellin, a natural product isolated from nudibranch species.[24] Despite its simple structure, this natural product showed potent AF activity against larvae of the barnacle Balanus amphitrite.[12] Structure–activity relationships were determined by synthesizing various analogues in the laboratory. The results indicated that the isocyano functional group is an important component for the potent AF activity observed.[24 – 26] On the basis of
© 2016 Wiley-VHCA AG, Zurich, Switzerland Chem. Biodiversity 2016, 13, 1502 – 1510 DOI: 10.1002/cbdv.201600063
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these findings, various artificial isocyano compounds with AF activity were created. In particular, several isocyano cyclohexanes and simple linear alkyl isocyanides demonstrated potent anti-barnacle (AF) activity without significant toxicity.[26 – 28] However, the biodegradability and toxicity of the degradation products of these compounds could not be determined on the basis of their structures alone, suggesting that there is scope for improvement in creating new AF active compounds. Therefore, the focus of this study was on the structure of amino acids, because the amino group is easily converted to the isocyano group. There are three main properties of isocyanides derived from amino acids that make them suitable candidates as environmentally friendly AF agents. First, amino acids can be converted to the corresponding isocyanides (amino acid-isocyanides) in only a few steps. Second, amino acid-isocyanides are expected to show potent anti-barnacle activity without significant toxicity because they have an isocyano group. Finally, amino acid-isocyanides are expected to eventually biodegrade to their original non-toxic amino acids. In this paper, the anti-barnacle activity of 15 congeners of amino acid-isocyanides derived from proteinogenic amino acids is presented. To our knowledge, this is the first study to assay the AF activity of isocyanides derived from amino acids against barnacles.
Results and Discussion Synthesis of Amino Acid-Isocyanides Fifteen congeners of amino acid-isocyanides (1 – 15) were synthesized from proteinogenic amino acids. The procedures for the synthesis of amino acid-isocyanides from amino acids are described in the Supporting Information. In brief, the amino group of an amino acid was converted to an isocyano group via formylation and dehydration. All amino acid-isocyanides were prepared as enantiomeric mixtures, except for the Gly derivative 1, because epimerization of a-C-atoms was observed during the reaction. The yields were not optimized. The structures of the synthesized compounds are shown in Fig. 1 and 2. In this study, benzyl ester was constructed as an ester moiety because it is able to repress the volatility and odor of the compounds. The amino group was converted to an isocyano group, while the COOH group was converted to benzyl ester. Amino acid-isocyanides of Gly 1, Ala 2, Val 3, Leu 4, Ile 5, Met 8, Phe 10, and Trp 12 were synthesized as the original amino acid forms, whereas amino acid-isocyanides of Ser 6, Thr 7, Gln 9, Tyr 11, Asp 13, Glu 14, and Lys 15 were synthesized as modified forms. Isocyanides of Ser, Tyr, Asp, Glu, and Lys © 2016 Wiley-VHCA AG, Zurich, Switzerland
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were prepared as benzyl ether 6, acetate 11, benzyl esters 13 and 14, and acetamide 15, respectively, because the OH, COOH, and amino groups of the side chains of a-amino acids were unstable under the conditions of the dehydration reaction. In addition, Thr and Gln derivatives were obtained as an alkene (7) and a nitrile (9), respectively, because of the occurrence of dehydration of alcohol and carboxamide during the process of isonitrile preparation. However, synthesis of isocyanides derived from Cys, Asn, Arg, and His was difficult, and they were not prepared in this study. AF Activity of the Synthesized Amino Acid-Isocyanides The rates of settlement and mortality of compounds 1 – 15 against cypris larvae of B. amphitrite after 48 and 120 h are shown in Fig. 1 and 2, respectively. CuSO4 was used as a positive control. The EC50 and LC50 values after 120 h exposure are summarized in Table 1. As expected, the synthesized amino acidisocyanides showed effective anti-barnacle activity. After 48 h, almost all of the synthesized compounds showed a rate of settlement of cypris larvae below 50% at 0.01 lg/ml, and no significant difference was observed among compounds 1 – 15 (Fig. 1). Therefore, EC50 values were analyzed from the AF assay data obtained after 120 h. All of the synthesized isocyano compounds showed effective AF activity with EC50 values of 0.07 – 10.34 lg/ml. Above all, according to the EC50 values, isocyanides 7, 10, and 11 showed more potent AF activity than CuSO4, which showed a similar EC50 value as 3-isocayanotheonellin. However, isocyanides 1 – 5 and 15 exhibited relatively high AF activity (EC50 = 0.97 – 2.67 lg/ml), whereas isocyanides 6, 8, 9, 13, and 14 showed moderate AF activity (EC50 = 3.96 – 10.34 lg/ml). These results indicate that the aromatic ring of the a-side chain might be effective in improving anti-barnacle activity. After 120 h, all of the synthesized amino acid-isocyanides showed low mortality rates at high concentration, and the LC50 values of the amino acid-isocyanides were all greater than 100 lg/ml, with the exception of the Leu 4, Ile 5, Met 8, and Asp 13 derivatives. In our previous study, secondary alkyl isocyanides showed weaker AF activity compared to tertiary alkyl isocyanides.[27][28] By contrast, the data of the present study indicated that the secondary alkyl isocyanides derived from a-amino acids also had potent AF activity as well as the tertiary alkyl isocyanides. The therapeutic ratios (LC50/EC50) after 120 h exposure are also summarized in Table 1. The therapeutic ratio (LC50/EC50) is used to assess the safety range of AF compounds, and a compound with an LC50/EC50 www.cb.wiley.com
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Figure 1. Antifouling (AF) activity of compounds 1 – 15 and CuSO4 against cypris larvae of Balanus amphitrite after 48 h exposure. The rates of settlement (●) and mortality (■) at different concentrations are plotted. Data plotted are means SD of three replicates. Data points not joined to a line indicate the rate of settlement and mortality in filtered seawater diluted to 80% with deionized water (control).
ratio > 15 is generally considered as a nontoxic AF compound.[21][22] As shown in Table 1, 11 of the synthesized compounds showed nontoxic LC50/EC50 www.cb.wiley.com
values (> 15). In particular, the amino acid-isocyanides of Thr 7, Phe 10, and Tyr 11 had remarkably high LC50/EC50 values (> 500). © 2016 Wiley-VHCA AG, Zurich, Switzerland
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Figure 2. Antifouling (AF) activity of compounds 1 – 15 and CuSO4 against cypris larvae of Balanus amphitrite after 120 h exposure. The rates of settlement (●) and mortality (■) at different concentrations are plotted. Data plotted are means SD of three replicates. Data points not joined to a line indicate the rate of settlement and mortality in filtered seawater diluted to 80% with deionized water (control).
From the perspective of the practical application of a compound as an antifoulant, it is more important to consider the concentration of complete inhibition © 2016 Wiley-VHCA AG, Zurich, Switzerland
than the EC50 value. The > 95% effective concentration for inhibition (EC95) values of the synthesized amino acid-isocyanides and CuSO4 are summarized in www.cb.wiley.com
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Table 1. Settlement inhibition, toxicity effects, and therapeutic ratios of the synthesized compounds 1 – 15 and CuSO4 (positive control) against cypris larvae of Balanus amphitrite after 120 h exposure. Antifouling activity and toxicity are indicated as EC50 and LC50 values, respectively Compound
EC50 [lg/ml]
LC50 [lg/ml]
LC50/EC50
CuSO4 1 (Gly) 2 (Ala) 3 (Val) 4 (Leu) 5 (Ile) 6 (Ser) 7 (Thr) 8 (Met) 9 (Gln) 10 (Phe) 11 (Tyr) 12 (Trp) 13 (Asp) 14 (Glu) 15 (Lys)
0.27 0.97 2.67 1.60 1.51 1.41 7.99 0.07 3.97 10.34 0.14 0.17 3.94 5.49 3.96 2.63
2.57 > 100 > 100 > 100 84.02 58.29 > 100 > 100 43.79 > 100 > 100 > 100 > 100 43.22 > 100 > 100
9.52 > 103.09 > 37.45 > 62.50 55.64 41.34 > 12.52 > 1428.57 11.03 > 9.67 > 714.29 > 588.24 > 25.38 7.87 > 25.25 > 38.02
Table 2. EC95 values of the synthesized amino acid-isocyanides were 3 – 100 lg/ml and 10 – 100 lg/ml after 48 and 120 h exposure, respectively. It is notable that almost all of the synthesized compounds showed a low mortality rate at their EC95 values. In particular, the mortality rates of the compounds at their EC95 values after 48 h were close to zero. However, more than 70% mortality of cypris larvae was observed after 48 h in CuSO4 at its EC95. Ten of the amino acid-isocyanides exhibited lower or equal EC95 values than that of CuSO4 (10 lg/ml) after 48 h exposure; isocyanide 11 derived from Tyr showed a particularly low EC95 value of 3 lg/ml. Moreover, the EC95 values of seven isocyanides (3, 4, 8, 10, 11, 12, and 14) were the same as that of CuSO4 (10 lg/ml) even after 120 h exposure. These results suggested that the synthesized amino acid-isocyanides are promising candidate antifoulants with low toxicity. Recently, we also investigated the mechanisms underlying the observed AF activity using fluorescently labeled AF compounds.[29][30] The results suggested that efficient AF activity might rely on the action of a compound in the oil cell region of barnacle cyprids. Oil cells function as a nutritional source for cypris larvae, which do not take in food during this stage of development.[31][32] On the basis of their structures, the synthesized amino acid-isocyanides are considered to be hydrophobic and lipophilic compounds, and are therefore expected to enter the oil cell region in barnacle cyprids through membranes. Together, this suggests that the action of amino acid-isocyanides in the oil cell www.cb.wiley.com
Table 2. The 95% effective concentrations for inhibition of cypris larvae (EC95) of compounds 1 – 15 and CuSO4 after 48 and 120 h exposure Compound
48 h [lg/ml]
120 h [lg/ml]
CuSO4 1 (Gly) 2 (Ala) 3 (Val) 4 (Leu) 5 (Ile) 6 (Ser) 7 (Thr) 8 (Met) 9 (Gln) 10 (Phe) 11 (Tyr) 12 (Trp) 13 (Asp) 14 (Glu) 15 (Lys)
10 100 30 10 10 10 100 30 10 30 10 3 10 10 10 10
10 100 30 10 10 30 100 30 10 100 10 10 10 30 10 100
region in barnacle cyprids may be a factor contributing to its potent anti-barnacle activity, although the detailed mechanism is currently unclear. In this work, the amino groups of amino acids were converted to isocyano groups and the COOH groups were converted to benzyl esters. It is wellknown that the ester moiety can be hydrolyzed to a COOH moiety. In addition, the isocyano group is easily hydrolyzed to an amino group under mild conditions.[33] Therefore, the synthesized amino acid-isocyanides show strong potential to eventually biodegrade to their original nontoxic amino acids in the seawater.
Conclusions Fifteen congeners of amino acid-isocyanides were synthesized and evaluated for their AF activity and toxicity against the cypris larvae of the barnacle B. amphitrite. The anti-barnacle activity of the synthesized isocyanides was in the EC50 range of 0.07 – 10.34 lg/ml, and the therapeutic ratios (LC50/ EC50) of the amino acid-isocyanides revealed that they are promising candidate AF agents with low toxicity. In addition, nearly all of the synthesized compounds showed more than 95% settlement inhibition with no mortality observed. Since these isocyanides are expected to eventually biodegrade to their original nontoxic amino acids, these results will be applied in further research focused on the development of environmentally friendly AF agents. © 2016 Wiley-VHCA AG, Zurich, Switzerland
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Experimental Section
General
Cypris Larvae
The isocyanides 1,[37] 2,[38][39] 3,[38][39] 4,[38][39] 5,[38] 10,[38] 13,[39] and 14[39] were prepared according to literature procedure. Column Chromatograpy (CC): SiO2 Kanto Chemical 60N (SiO2, 63 – 210 lm). IR Spectra: JEOL WINSPEC-50 IR spectrometer; ~v in cm1. NMR Spectra: JEOL ECA-600 or JEOL ECS-400 NMR spectrometer; in CDCl3. All 1H-NMR data: d in ppm rel. to Me4Si as an internal standard, J in Hz. All 13C-NMR data: d in ppm rel. to the central line of the triplet for CDCl3 at 77.0 ppm. ESI-MS: JEOL JMS-T100 mass spectrometer; in m/z. Benzyl 3-(Benzyloxy)-2-isocyanopropanoate (6). 10-Camphorsulfonic acid (499 mg, 2.15 mmol) was added to a soln. of O-benzyl-L-serine (351 mg, 1.80 mmol) in BnOH (2 ml). After the reaction mixture was stirred at 60 °C for 48 h, sat. NaHCO3 (50 ml) was added, and resultant mixture was extracted with AcOEt. The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 4:1 ? 2:1 ? 1:1 ? 1:4) to give amine 6a (383 mg, 1.34 mmol, 75%) as a colorless oil. Ac2O (970 ll, 10.3 mmol) was added to a soln. of amine 6a (979 mg, 3.43 mmol) in HCOOEt (5 ml). After the reaction mixture was stirred at r.t. for 25 h, sat. NaHCO3 (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 1:1) to give formamide 6b (1.05 g, 3.35 mmol, 97%) as a colorless oil. Pyridine (674 ll, 8.35 mmol) and PhOPOCl2 (388 ll, 2.59 mmol) were added successively to a soln. of formamide 6b (524 mg, 1.67 mmol) in CH2Cl2 (5 ml). After the reaction mixture was stirred at r.t. for 2 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt. The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 3:1) to give isocyanide 6 (18.3 mg, 62 lmol, 4%) as a slightly yellow oil. IR (neat): 3087, 3064, 3033, 2952, 2871, 2152, 1756, 1456, 1280, 1195, 1112, 738, 698. 1H-NMR (400 MHz, CDCl3): 7.42 – 7.21 (m, 10 H); 5.29 – 5.20 (m, 2 H); 4.63 – 4.54 (m, 2 H); 4.51 – 4.40 (m, 1 H); 3.91 – 3.81 (m, 2 H). 13C-NMR (100 MHz, CDCl3): 164.7; 161.5; 136.9; 134.6; 128.9; 128.8; 128.5; 128.2; 127.8; 73.7; 69.5; 68.5; 56.9. ESI-MS: 318.1104 ([M + Na]+, C18 H17 NNaOþ 3 ; calc. 318.1106). Benzyl 2-Isocyanocrotonate (7). Ac2O (14.8 ml, 157 mmol) was added to a soln. of L-threonine
Adult barnacles (Balanus amphitrite) attached to bamboo poles were procured from oyster farms in Lake Hamana, Shizuoka, Japan. Barnacles were maintained in an aquarium at 20 1 °C and were fed with brine shrimp (Artemia salina) nauplii. The barnacles were dried at r.t. overnight and were then immersed in seawater. After immersion, the broods released stage I – II nauplii. The nauplii thus obtained were collected and cultured in 2 l glass beakers at 25 °C at an initial density of 3 larvae/ml. Nauplii fed on the diatom Chaetoceros gracilis (initial concentration of 3.0 9 105 cells/ml). Cultures were maintained in filtered (0.2 lm pore filter) seawater diluted to 80% with deionized H2O (80% filtered seawater, salinity 2.8%), 30 lg/l streptomycin, and 20 mg/l penicillin G at 25 °C with mild aeration. The nauplii were cultured for 4 days, and C. gracilis was added daily (0.5 – 1.0 9 105 cells/ml). Larvae reached the cyprid stage in 5 days. The cyprids were stored in the dark at 5 °C until used. The day that the newly transformed cyprids were collected was designated as Day 0.[34]
AF Assay The isocyanides were dissolved in MeOH. The soln. was further diluted with MeOH at various concentrations: 0.01, 0.03, 0.1, 0.3, 1, 3, 10, 30, and 100 lg/ml. A 2 ml soln. was pipetted into each well of 24-well polystyrene tissue culture plates and air-dried. A quantity of 2 ml of 80% filtered seawater and six 2or 3-day-old cyprids were added into the wells. Four wells were used for each experiment. The plates were maintained in the dark at 25 °C. After 48 and 120 h, the numbers of larvae that attached, metamorphosed, died, or did not settle were counted under a microscope.[35][36] Cyprids that did not move, had extended appendages, and did not respond after a light touch with a metal probe were regarded as dead. The experiment was performed three times with different batches of larvae. The same assay was carried out simultaneously with CuSO4 as a positive control. The median effective and lethal concentrations (EC50 and LC50, resp.) were calculated by probit analysis. However, since probit analysis could not be applied to calculate the EC50 values of isocyanide from Met 8, Glu 9, Trp 12, Asp 13, Glu 14, and Lys 15, these values were estimated by linear graphical interpolation. © 2016 Wiley-VHCA AG, Zurich, Switzerland
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(6.24 ml, 52.3 mmol) in HCOOH (25 ml). After the reaction mixture was stirred at r.t. for 48 h, the mixture was concentrated under reduced pressure. The residue was recrystallized with MeOH and AcOEt to give formamide 7a (2.62 g, 17.8 mmol, 34%). K2CO3 (7.37 g, 53.3 mmol) and BnBr (3.18 ml, 26.8 mmol) were added successively to a soln. of formamide 7a (2.63 g, 17.9 mmol) in DMF (25 ml). After the reaction mixture was stirred at 70 °C for 5 days, H2O (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 1:4) to give formamide 7b (762 mg, 3.21 mmol, 18%) as a yellow oil. Pyridine (380 ll, 4.71 mmol) was added to a soln. of formamide 7b (749 mg, 3.16 mmol) in Ac2O (6 ml). After the reaction mixture was stirred at r.t. for 6 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), H2O (20 ml), and brine (20 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt = 1:1) to give formamide 7c (785 mg, 2.81 mmol, 89%) as a white solid. Pyridine (330 ll, 4.09 mmol) and PhOPOCl2 (85.0 ll, 0.568 mmol) were added successively to a soln. of formamide 7c (132 mg, 0.47 mmol) in CH2Cl2 (6 ml). After the reaction mixture was stirred at r.t. for 6 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/ AcOEt 9:1) to give isocyanide 8 (56.3 mg, 280 lmol, 59%) as a slightly yellow oil. IR (neat): 3324, 2068, 2027, 2981, 2966, 2940, 2123, 1724, 1452, 1270, 1211, 754, 703. 1 H-NMR (400 MHz, CDCl3): 7.62 – 7.31 (m, 5 H); 7.16 – 7.07 (m, 1 H); 5.27 (s, 2 H); 2.05 (d, J = 7.2, 3 H). 13 C-NMR (100 MHz, CDCl3): 170.3; 160.1; 141.9; 135.0; 128.8; 128.7; 128.4; 120.7; 67.9; 15.1. ESI-MS: 224.0690 ([M + Na]+, C12H11NNaO+; calc. 224.0687). Benzyl 2-Isocyano-4-(methylthio)butanoate (8). Ac2O (10.8 ml, 114 mmol) was added to a soln. of L-methionine (5.23 g, 35.1 mmol) in HCOOH (25 ml). After the reaction mixture was stirred at r.t. for 48 h, the mixture was concentrated under reduced pressure. The residue was recrystallized with MeOH and AcOEt to give formamide 8a (4.39 g, 24.8 mmol, 71%) as a white solid. K2CO3 (5.49 g, 39.7 mmol) and BnBr (2.35 ml, 19.8 mmol) were added successively to a soln. of formamide 8a (2.34 g, 13.2 mmol) in DMF (20 ml). After the reaction mixture was stirred at r.t. for 24 h, H2O www.cb.wiley.com
(50 ml) was added, and resultant mixture was extracted with AcOEt. The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt = 1:1 ? 2:1) to give formamide 8b (1.48 g, 5.54 mmol, 42%) as a yellow oil. Pyridine (1.11 ml, 13.8 mmol) and PhOPOCl2 (740 ll, 4.95 mmol) were added successively to a soln. of formamide 8b (735 mg, 2.75 mmol) in CH2Cl2 (2 ml). After the reaction mixture was stirred at r.t. for 2.5 h, H2O (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), H2O (20 ml) and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 3:1) to give isocyanide 8 (174 mg, 0.697 mmol, 25%) as a slightly yellow green oil. IR (neat): 3089, 3066, 3033, 2969, 2917, 2148, 1756, 1456, 1440, 1272, 1211, 752, 698. 1H-NMR (600 MHz, CDCl3): 7.37 – 7.30 (m, 5 H); 5.23 – 5.17 (m, 2 H); 4.54 – 4.50 (m, 1 H); 2.65 – 2.57 (m, 2 H); 2.19 – 2.10 (m, 2 H); 2.03 (s, 3 H). 13C-NMR (150 MHz, CDCl3): 166.1; 160.8; 134.4; 128.6; 1283.5; 128.3; 68.1; 55.0; 31.7; 29.4; 15.2. ESI-MS: 272.0721 ([M + Na]+, C13H15NNaO2S+; calc. 272.0712). Benzyl 4-Cyano-2-isocyanobutanoate (9). Ac2O (4.06 ml, 43.0 mmol) was added to a soln. of L-glutamine (2.08 g, 14.2 mmol) in HCOOH (25 ml). After the reaction mixture was stirred at r.t. for 23 h, the mixture was concentrated under reduced pressure. The residue was recrystallized with MeOH and AcOEt to give formamide 9a (1.61 g, 9.26 mmol, 65%) as a white solid. K2CO3 (3.79 g, 27.4 mmol) and BnBr (1.63 ml, 13.7 mmol) were added successively to a soln. of formamide 9a (1.60 g, 9.17 mmol) in DMF (20 ml). After the reaction mixture was stirred at 60 °C for 3 h, H2O (50 ml) was added, resultant mixture was extract with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was recrystallized with AcOEt to give formamide 9b (453 mg, 1.71 mmol, 19%) as a white solid. Pyridine (609 μl, 7.55 mmol) and PhOPOCl2 (197 ll, 1.32 mmol) were added successively to a soln. of formamide 9b (232 mg, 0.89 mmol) in CH2Cl2 (10 ml). After the reaction mixture was stirred at r.t. for 2.5 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt = 2:1) to give isocyanide 9 (53.8 mg, 0.220 mmol, 25%) as a slightly yellow oil. IR (neat): 3089, 3064, 3035, 2956, 2250, 2152, 1754, 1456, 1214, © 2016 Wiley-VHCA AG, Zurich, Switzerland
Chem. Biodiversity 2016, 13, 1502 – 1510
750, 698. 1H-NMR (600 MHz, CDCl3): 7.43 – 7.32 (m, 5 H); 5.25 (s, 2 H); 4.49 – 4.42 (m, 1 H); 2.65 – 2.74 (m, 2 H); 2.38 – 2.17 (m, 2 H). 13C-NMR (150 MHz, CDCl3): 165.0; 162.9; 134.2; 129.2; 128.9; 128.7; 69.0; 55.0; 28.5; 13.7. ESI-MS: 251.0797 ([M + Na]+, C13 H12 N2 NaOþ 2 ; calc. 251.0797). Benzyl 3-(4-Acetoxyphenyl)-2-isocyanopropanoate (11). Ac2O (5.60 ml, 59.2 mmol) was added to a soln. of L-tyrosine (3.56 g, 19.6 mmol) in HCOOH (20 ml). After the reaction mixture was stirred at r.t. for 24 h, the mixture was concentrated under reduced pressure. The residue was recrystallized with MeOH and AcOEt to give formamide 11a (985 mg, 4.71 mmol, 24%). K2CO3 (1.91 g, 13.8 mmol) and BnBr (820 ll, 6.90 mmol) were added successively to a soln. of formamide 11a (968 mg, 4.62 mmol) in DMF (20 ml). After the reaction mixture was stirred at 70 °C for 47 h, H2O (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/ AcOEt = 1:1 ? 2:3) to give formamide 11b (290 mg, 0.970 mmol, 21%) as a yellow oil. Pyridine (116 ll, 1.43 mmol) was added to a soln. of formamide 11b (288 mg, 0.960 mmol) in Ac2O (2 ml). After the reaction mixture was stirred at r.t. for 18 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), H2O (20 ml) and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 3:1) to give formamide 11c (321 mg, 940 lmol, 98%) as a white solid. Pyridine (160 ll, 1.98 mmol) and PhOPOCl2 (73.0 ll, 488 lmol) were added successively to a soln. of formamide 11c (138 mg, 410 lmol) in CH2Cl2 (5 ml). After the reaction mixture was stirred at r.t. for 4 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 2:1) to give isocyanide 11 (16.2 mg, 50 lmol, 12%) as a slightly yellow oil. IR (neat): 3091, 3066, 3035, 2939, 2148, 1756, 1454, 1220, 752, 700. 1H-NMR (400 MHz, CDCl3): 7.44 – 7.28 (m, 5 H); 7.22 – 6.98 (m, 4 H); 5.20 (s, 2 H); 4.50 – 4.43 (m, 1 H); 3.27 – 3.09 (m, 2 H); 2.29 (s, 3 H). 13 C-NMR (100 MHz, CDCl3): 169.4; 165.9; 1661.3; 150.4; 134.4; 131.8; 130.5; 128.9; 128.8; 128.7; 122.1; 68.5; 58.0; 38.3; 21.3. ESI-MS: 346.1055 ([M + Na]+, C19 H17 NNaOþ 4 ; calc. 346.1055). © 2016 Wiley-VHCA AG, Zurich, Switzerland
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Benzyl 3-(1H-Indol-3-yl)-2-isocyanopropanoate (12). 10-Camphorsulfonic acid (5.00 g, 2.15 mmol) was added to a soln. of L-tryptophan (4.01 g, 19.6 mmol) in BnOH (4 ml). After the reaction mixture was stirred at 60 °C for 3 days, sat. NaHCO3 (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/ AcOEt 5:1 ? 3:1 ? 1:1 ? 1:2 ? 1:4) to give amine 12a (600 mg, 2.04 mmol, 10%) as a colorless oil. p-Toluenesufonic acid (35.3 mg, 205 lmol) was added to a soln. of amine 12a (547 mg, 1.86 mmol) in HCOOEt (30 ml). After the reaction mixture was stirred at 60 °C for 48 h, sat. NaHCO3 (50 ml) was added to the reaction mixture, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 1:1 ? 1:2) to give formamide 12b (492 mg, 1.53 mmol, 82%) as a red oil. Pyridine (995 ll, 7.18 mmol) and PhOPOCl2 (322 ll, 2.15 mmol) were added successively to a soln. of formamide 12b (463 mg, 1.43 mmol) in CH2Cl2 (5 ml). After the reaction mixture was stirred at r.t. for 2 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/ AcOEt = 4:1 ? 3:1) to give isocyanide 12 (113 mg, 370 lmol, 26%) as a slightly yellow oil. IR (neat): 3417, 3062, 3035, 2954, 2929, 2150, 1749, 1457, 1213, 744, 698. 1 H-NMR (400 MHz, CDCl3): 8.07 (s, 1 H); 7.58 – 7.53 (m, 1 H); 7.46 – 7.30 (m, 5 H); 7.24 – 7.10 (m, 4 H); 5.18 – 5.10 (m, 2 H); 4.60 – 4.54 (m, 1 H); 3.51 – 3.32 (m, 2 H). 13CNMR (100 MHz, CDCl3): 166.5; 160.4; 136.2; 134.7; 128.8; 128.8; 128.5; 126.9; 123.8; 122.6; 120.0; 118.3; 111.6; 108.6; 68.3; 57.6; 29.7. ESI-MS: 327.1107 ([M + Na]+, C19 H16 N2 NaOþ 2 ; calc. 327.1110). Benzyl 6-Acetamido-2-isocyanohexanoate (15). 10-Camphorsulfonic acid (2.59 g, 11.1 mmol) was added to a soln. of e-N-acetyllysine (703 mg, 3.74 mmol) in BnOH (2 ml). After the reaction mixture was stirred at 60 °C for 4 days, sat. NaHCO3 (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/ AcOEt 5:1 ? 3:1 ? 1:1 ? 1:2 ? 1:4) to give amine 15a (250 mg, 900 lmol, 24%) as a colorless oil. Ac2O (510 ll, 5.40 mmol) was added to a soln. of amine 15a (250 mg, 900 lmol) in HCOOEt (5 ml). After the reaction www.cb.wiley.com
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mixture was stirred at r.t. for 7 days, sat. NaHCO3 (50 ml) was added to the reaction mixture, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 1:1 ? 1:4) to give formamide 15b (161 mg, 530 lmol, 58%) as a colorless oil. Pyridine (108 ll, 1.33 mmol) and PhOPOCl2 (62 ll, 414 lmol) were added successively to a soln. of formamide 15b (84.3 mg, 280 lmol) in CH2Cl2 (5 ml). After the reaction mixture was stirred at r.t. for 3 h, brine (50 ml) was added, and resultant mixture was extracted with AcOEt (120 ml). The org. phase was washed with 1M HCl (20 ml), sat. NaHCO3 (20 ml), and brine (40 ml), dried (MgSO4), and concentrated under reduced pressure. The residue was purified by performing SiO2 CC (hexane/AcOEt 2:7 ? 1:8) to give isocyanide 15 (23.5 mg, 82 lmol, 30%) as a colorless oil. IR (neat): 3303, 3087, 3068, 3035, 2937, 2865, 2148, 1753, 1654, 1456, 1197, 750, 698. 1H-NMR (600 MHz, CDCl3): 7.41 – 7.31 (m, 5 H); 5.22 (s, 2 H); 4.32 – 4.28 (m, 1 H); 3.26 – 3.15 (m, 2 H); 2.01 – 1.87 (m, 5 H); 1.57 – 1.41 (m, 4 H). 13C-NMR (100 MHz, CDCl3): 170.2; 166.5; 160.5; 134.7; 128.9; 128.8; 128.5; 68.3; 56.6; 39.2; 32.3; 28.7; 23.4; 22.6. ESI-MS: 311.1372 ([M + Na]+, C16 H20 N2 NaOþ 3 ; calc. 311.1372).
Supplementary Material Supporting information for this article is available on the WWW under http://dx.doi.org/10.1002/cbdv.201600063.
Acknowledgements The authors thank Prof. N. Fusetani of Hokkaido University for his helpful suggestions. The authors also gratefully acknowledge Associate Prof. K. Kikuchi and H. Suetake of the University of Tokyo, and Mr. T. Ookawara of the Maisaka Oyster Cultivation Union of Lake Hamana for providing the barnacles. This work was partially supported by JST A-STEP AS232Z01296D and JSPS KAKENHI 25660087.
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