Design, synthesis, and antifouling activity of glucosamine-based isocyanides

Article abstract of DOI:10.3390/md15070203

Biofouling, an undesirable accumulation of organisms on sea-immersed structures such as ship hulls and fishing nets, is a serious economic issue whose effects include oil wastage and clogged nets. Organotin compounds were utilized since the 1960s as an antifouling material; however, the use of such compounds was later banned by the International Maritime Organization (IMO) due to their high toxicity toward marine organisms, resulting in masculinization and imposex. Since the ban, there have been extensive efforts to develop environmentally benign antifoulants. Natural antifouling products obtained from marine creatures have been the subject of considerable attention due to their potent antifouling activity and low toxicity. These antifouling compounds often contain isocyano groups, which are well known to have natural antifouling properties. On the basis of our previous total synthesis of natural isocyanoterpenoids, we envisaged the installation of an isocyano functional group onto glucosamine to produce an environmentally friendly antifouling material. This paper describes an effective synthetic method for various glucosamine-based isocyanides and evaluation of their antifouling activity and toxicity against cypris larvae of the barnacle Amphibalanus amphitrite. Glucosamine isocyanides with an ether functionality at the anomeric position exhibited potent antifouling activity, with EC₅₀ values below 1 μg/mL, without detectable toxicity even at a high concentration of 10 μg/mL. Two isocyanides had EC₅₀ values of 0.23 and 0.25 μg/mL, comparable to that of CuSO₄, which is used as a fouling inhibitor (EC₅₀ = 0.27 μg/mL).

Full text of DOI:10.3390/md15070203

marine drugs
Article
Design, Synthesis, and Antifouling Activity of
Glucosamine-Based Isocyanides
Taiki Umezawa 1,
*
, Yuki Hasegawa 1, Ira S. Novita 1, Junya Suzuki 1, Tatsuya Morozumi 1,
Yasuyuki Nogata 2,*, Erina Yoshimura 3 and Fuyuhiko Matsuda 1
1
Division of Environmental Materials Science, Graduate School of Environmental Science,
Hokkaido University, N10W5, Sapporo 060-0810, Japan; djebelmybabumilesianpartholon@yahoo.co.jp (Y.H.);
ira.novita.s@mail.ugm.ac.id (I.S.N.); suzujun55@ees.hokudai.ac.jp (J.S.); moro@ees.hokudai.ac.jp (T.M.);
fmatsuda@ees.hokudai.ac.jp (F.M.)
2
Environmental Science Research Laboratory, Central Research Institute of Electric Power Industry,
1646 Abiko, Abiko, Chiba 270-1194, Japan
CERES, Inc., 1-4-5 Midori, Abiko, Chiba 270-1153, Japan; megabalanusrosa@yahoo.co.jp
3
*
Correspondence: umezawa@ees.hokudai.ac.jp (T.U.); noga@criepi.or.jp (Y.N.);
Tel.: +81-11-706-2358 (T.U.); +81-70-6980-5559 (Y.N.)
Received: 16 May 2017; Accepted: 21 June 2017; Published: 29 June 2017
Abstract: Biofouling, an undesirable accumulation of organisms on sea-immersed structures such as
ship hulls and fishing nets, is a serious economic issue whose effects include oil wastage and clogged
nets. Organotin compounds were utilized since the 1960s as an antifouling material; however, the use
of such compounds was later banned by the International Maritime Organization (IMO) due to their
high toxicity toward marine organisms, resulting in masculinization and imposex. Since the ban,
there have been extensive efforts to develop environmentally benign antifoulants. Natural antifouling
products obtained from marine creatures have been the subject of considerable attention due to their
potent antifouling activity and low toxicity. These antifouling compounds often contain isocyano
groups, which are well known to have natural antifouling properties. On the basis of our previous
total synthesis of natural isocyanoterpenoids, we envisaged the installation of an isocyano functional
group onto glucosamine to produce an environmentally friendly antifouling material. This paper
describes an effective synthetic method for various glucosamine-based isocyanides and evaluation of
their antifouling activity and toxicity against cypris larvae of the barnacle Amphibalanus amphitrite.
Glucosamine isocyanides with an ether functionality at the anomeric position exhibited potent
antifouling activity, with EC50 values below 1
µg/mL, without detectable toxicity even at a high
concentration of 10 g/mL. Two isocyanides had EC50 values of 0.23 and 0.25
µ
µg/mL, comparable to
that of CuSO4, which is used as a fouling inhibitor (EC50 = 0.27 µg/mL).
Keywords: antifouling; glucosamine; isocyanide
1. Introduction
Many essential human activities, such as transportation and fishing, are carried out in the ocean,
and submarine structures—seawater intakes pipes for power plants, breeding nets, and so on—are also
widely used. Ocean-based activities such as offshore energy generation will continue to increase in the
future [
biofouling, that is, an undesirable accumulation of organisms such as barnacles and mussels. Biofouling
can have serious economic consequences [ ], including fuel waste—up to 40% [ ]—and clogging of
pipes and nets [ ]. Removal of the fouling organisms for annual maintenance must be done at huge
cost [ ]. Since the 1960s, organotin compounds such as tributyltin (TBT) and triphenyltin (TPT) were
widely employed to prevent fouling [
1]. Any structure immersed in the ocean, including ship hulls and fishing nets, will encounter
2
3
4
5
6]. However, organotin compounds were suspected to be toxic,
Mar. Drugs 2017, 15, 203; doi:10.3390/md15070203
www.mdpi.com/journal/marinedrugs

Mar. Drugs 2017, 15, 203
2 of 9
and biological consequences for marine organisms included growth inhibition, masculinization of
shellfish, abnormal shell development, brittle shells, poor weight gain, and a disorder known as
imposex in snails [
crustaceans [10 11], and particularly mollusks [12]. These unfavorable aspects drove the International
Maritime Organization (IMO) to ban the use of organotin compounds on ships worldwide in 2008 [13].
Alternative antifouling agents in current use mainly contain copper [ ], and there have been some
reports of negative consequences for the ocean environment and ecosystem [14 15]. The development
7]. Harmful effects of TBT were also reported for marine organisms such as fish [8,9],
,
6
,
of an environmentally friendly antifouling agent that is suitable for the ocean environment is therefore
a priority.
In current research on green antifouling agents [16], natural products obtained from marine
creatures have attracted much attention [17
examples of natural antifouling products. For example, 10-isocyano-4-cadinene (
nudibranchs of the family Phyllidiidae by Okino, shows strong antifouling activity (50% effective
concentration (EC50) = 0.14 g/mL) against cypris larvae of the barnacle Amphibalanus amphitrite,
comparable to CuSO4 (EC50 = 0.27 g/mL), along with low toxicity (50% lethal concentration
(LC50) >10 g/mL) [25], as shown in Figure 1. Our group achieved the first total synthesis
of and examined the biological activity of a synthetic sample against the same cypris larvae,
obtaining similar activities (EC50 = 0.06 g/mL, LC50 > 10 g/mL) compared to those of the natural
compound [26 28]. Alcohol and the nitrile , synthetic intermediates in the total synthesis of
were found to have less potency than . Other isocyanide compounds such as 3-isocyanotheonellin
) also exhibited potent antifouling profiles [29 33]. These results clearly indicate that the isocyano
group confers potent antifouling properties. We envisaged a compound in which the isocyano
functionality was installed on a glucosamine unit ( ), obtained from crustaceans such as crabs and
shrimp as a cheap and abundant biomass platform, through organic synthesis, because possesses
an amino group, which is essential for introduction of the isocyano group. In addition, the four
hydroxy groups in enable easy access to a wide variety of synthetic isocyanides. Furthermore,
the compound, which is derived from a sugar-based structure, was expected to have low toxicity.
In this study, glucosamine-based isocyanides ( ) were designed and synthesized in short reaction
sequences. The antifouling activities and toxicities of the synthetic compounds against the cypris
–24]. In particular, isocyano compounds are well-known
1), obtained from
µ
µ
µ
1
µ
µ
–
2
3
1,
1
(4
–
5
5
5
6
6
larvae of Amphibalanus amphitrite were evaluated to prove the feasibility of these compounds as
alternative environmentally friendly antifouling agents, including through investigation of substituent
effects at the C-1 and C-3, 4, and 6 positions.
Figure 1. Materials containing isocyano groups exhibit potent antifouling properties. (a) Previously
reported natural products and derivatives with antifouling properties, along with their antifouling
activities. (b) Synthetic plan for glucosamine-based isocyanides.

Mar. Drugs 2017, 15, 203
3 of 9
2. Results
2.1. Compounds with Ester Groups at the C-1 Position
Initially, the tetraacetate 6a and the tetrapalmitate 6b were prepared from D-glucosamine
hydrochloride ( ) by a sequence of formamide formation with HCO2Me and NaOMe [34], the acylation
5
of hydroxy groups with Ac2O or palmitoyl chloride (PalCl), respectively, and the dehydration of
formamides 7a and 7b with POCl3 and Et3N [35] (Figure 2). The antifouling activities of 6a and 6b were
evaluated against the cypris larvae of Amphibalanus amphitrite after a 48 h incubation period, showing
moderate activity (EC50 = 5.54 and 6.36 µg/mL, respectively) and very low toxicity (lethal larva was
not observed at the concentration of 10 µg/mL).
Figure 2. Synthesis and biological activity of tetraacylated glucosamine-based isocyanides. (
a) Synthesis
of tetraacetate 6a. (b) Synthesis of tetrapalmitate 6b.
2.2. Compounds with Ether at the C-1 Position
Next, the effect of replacement of the C-1 ester groups of 6a and 6b with ether groups upon
antifouling activity and toxicity against Amphibalanus amphitrite was examined. Phenyl (Ph), benzyl
(CH2Ph), allyl (CH2CH = CH2), isopropyl (CH(CH3)2, iPr), and 2,2,2-trifluoroethyl (CH2CF3) groups
were chosen as the substituent R of the C-1 alkoxy group. Figure 3 summarizes the synthesis
and biological activities of isocyanides 6c
D-glucosamine derivative was employed as a starting compound because glycosidation reactions
between formamide 7a and alcohols such as isopropyl alcohol under various conditions did not
–g. For the synthesis of 6c–g, the commercially available
8
afford the desired glycosidation products. In glycosidation reactions of
8
promoted by BF3 OEt2
·
(Step A), two solvent systems (CH2Cl2/room temperature, and MeCN/80 ◦C) were employed for
optimization of chemical yield. Phenyl glycoside 9c and trifluoroethyl glycoside 9g were obtained in
higher yields by allowing
CH2Cl2. However, treatment of
MeCN produced benzyl glycoside 9d, allyl glycoside 9e, and isopropyl glycoside 9f in better yields.
During each of the glycosidation reactions, the -anomer was formed as the sole glycosidation product.
The -configurations at the anomeric position (C-1) of 9c were determined based on coupling
8
to react with phenol and 2,2,2-trifluoroethanol at room temperature in
8
with benzyl alcohol, allyl alcohol, and isopropyl alcohol at 80 ◦C in
β
β
–g
constant analysis. Thus, a large vicinal coupling constant, J12 = 9 Hz (axial/axial relationship between
the vicinal methine protons at C-1 and 2), was observed in all the 1H NMR spectra. The conversion
of 9c–g into formamides 7c–g was accomplished in the following three steps (Step B): (i) removal of
the phthaloyl and acetyl groups with ethylenediamine (EDA); (ii) a formamide formation reaction
with HCO2Me and NaOMe; and (iii) acetylation of the three resulting hydroxyl groups with Ac2O

Mar. Drugs 2017, 15, 203
4 of 9
and pyridine. Isocyanides 6c
–
g
were derived from 7c
–
g
using the same dehydration protocol with
POCl3 and Et3N (Step C) that was used in the preparation of 7a and 7b. The biological activities
of synthetic 6c against the cypris larvae of Amphibalanus amphitrite were then evaluated to reveal
enhanced potency for 6c (EC50 = 0.23–0.71 g/mL) relative to 6a and 6b (EC50 = 5.54 and 6.36 g/mL,
–
g
–
g
µ
µ
respectively). Furthermore, it was noteworthy that mortality among the cypris larvae was not observed
during the assay, even at a high concentration of 10 µg/mL.
Figure 3. Synthesis and biological activities of glucosamine-based isocyanides. (
a) Synthesis of
isocyanides 6c–g. (b) Table of reactions and biological activities.
2.3. Effects of Substituents at the C-3, 4, and 6 Positions
Further modifications were made by replacing the acetyl groups at C-3, 4, and 6 of the isopropyl
glycoside 6f with other substituents. As illustrated in Figure 4, after removal of the phthaloyl and acetyl
groups of 9f followed by formamide formation, octanoyl (COnC7H15), palmitoyl (COnC15H31), benzoyl
(COPh), and TBS (SiMe2tBu) groups were installed to afford isopropyl glycosides 7h
Each of 7h was transformed to the corresponding isocyanide (6h ) under dehydration conditions
that were the same as those used for the preparation of 6a . Isocyanides 6h were tested against
Amphibalanus amphitrite and exhibited similar antifouling activities (EC50 = 0.25–0.81 g/mL) to 6f
(EC50 = 0.23 µg/mL). Again, none of 6h–k showed detectable toxicity (LC50 > 10 µg/mL).
–k, respectively.
–
k
–k
–
g
–k
µ

Mar. Drugs 2017, 15, 203
5 of 9
Figure 4. Synthesis and biological activity of glycosylated glucosamine-based isocyanides. (
a) Synthesis
of isocyanides 6h–k. (b) Table of reactions and biological activities.
3. Discussion
These procedures are short syntheses (up to 5 steps from commercially available materials)
allowing access to various glucosamine-based isocyanides using inexpensive reagents. In contrast,
27 steps were required for the total synthesis of 1. Although the chemical yields should be improved
by further optimization, the synthetic scheme seems to be a reliable one for the synthesis of various
isocyanides with latent antifouling activity. All of the samples obtained in the current study showed
not only antifouling profiles but also extremely low toxicity against the cypris larvae of Amphibalanus
amphitrite. Compounds 6a and 6b with the ester group at the C-1 position had EC50 values above
5
µ
g/mL, but 6c
below 1 g/mL. Compounds 6f and 6k exhibited EC50 values of 0.23 and 0.25
to that of CuSO4, which is used as a fouling inhibitor (EC50 = 0.27 g/mL) [27]. Substituent effects
at the C-1 position of glucosamine were clearly evident in the higher EC50 values of 6a and 6b
compared to 6c , suggesting that more detailed investigations of ether substituents at this position
may enable the discovery of more potent compounds, and that substituents at the C-1 position affect
the antifouling mechanism through steric or electronic effects [36 37] (Figures 2 and 3). On the other
–
g
with the ether group at the C-1 position were more active, with EC50 values
µ
µg/mL, comparable
µ
–
g
,
hand, no significant effect on antifouling activity was obtained by changing the substituents attached to
the three hydroxy groups at C-3, 4, and 6 (Figure 4). This result is advantageous from the point of view
of the development of nontoxic and environmentally benign antifouling agents through the synthesis
of functionalized glucosamine derivatives—for example, monomers for the synthesis of polymers that
can be used in combination with paint—since it is possible to introduce various functional groups at
the C-3, 4, and 6 positions without significant loss of antifouling activity.
The results obtained in this study are promising in terms of the development of green antifouling
agents for practical use. Further preparation of a wide variety of glucosamine-based isocyanides along
with relevant field assay and biodegradability test will be reported in our laboratory.

Mar. Drugs 2017, 15, 203
6 of 9
4. Materials and Methods
Optical rotations were obtained using a Horiba SEPA-300 instrument (Horiba, Kyoto, Japan).
IR spectra were recorded on a JASCO FT/IR 4100 spectrometer using an NaCl cell. 1H and 13C
NMR spectra were recorded using a JNM-EX 400 (400 MHz and 100 MHz) spectrometer (see
Supplementary Materials). N,N-dimethylformamide (DMF), methanol (MeOH), and acetonitrile
(MeCN) were purchased from Kanto Chemical Co. Inc. (Tokyo, Japan). Dichloromethane (CH2Cl2)
was distilled from CaH2. All commercially available reagents were employed as received. Analytical
TLC was carried out using pre-coated silica gel plates (TLC silica gel 60 F254). The silica gel used for
column chromatography was Wakogel 60N 63–212 µm.
4.1. General Procedure for the Preparation of Formamides 7a and 7b
To a solution of D-glucosamine hydrochloride (5) (1.0 equiv) in MeOH (0.40 M) and HCO2Me
(1.0 M), NaOMe (1.4 equiv) and Et3N (1.0 equiv) was added at room temperature under an Ar
atmosphere. The mixture was stirred at room temperature overnight and then concentrated in vacuo.
To a solution of the crude formamide in pyridine (15 equiv), Ac2O (6.0 equiv) for 7a or palmitoyl
chloride (6.0 equiv) for 7b was added at room temperature under an Ar atmosphere. After stirring
◦
at room temperature overnight, MeOH (6.0 equiv) was added at 0 C. The mixture was extracted
with EtOAc, washed successively with 1.0 M NaOH, 3.0 M HCl, and brine, dried over Na2SO4,
filtered, and concentrated in vacuo. The residue was purified by silica gel column chromatography
(EtOAc/hexane = 40:60) to yield 7a or 7b, respectively.
4.2. General Procedure for the Preparation of Isocyanides 6a–k
To a solution of each of the series of formamides 7a◦
–k (1.0 equiv) in CH2Cl2 (0.20 M), Et3N
(9.0 equiv) and POCl3 (3.0 equiv) were added dropwise at 0 C under an Ar atmosphere. After stirring
at 0 ◦C for 10 min, the mixture was warmed to room temperature, stirred at room temperature for 1 h,
quenched with saturated NaHCO3, and extracted with EtOAc. The combined organic extracts were
washed with brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified
by silica gel column chromatography (EtOAc/hexane = 10:90) to yield isocyanides 6a
–k, respectively.
4.3. General Procedure for the Preparation of Glycosides 9d–f
To a solution of
8 (1.0 equiv) in MeCN (0.25 M), benzyl alcohol, allyl alcohol, or isopropyl alcohol
(1.0 equiv) and BF3
·
OEt2 (3.◦0 equiv) were added at room temperature under an Ar atmosphere.
The mixture was stirred at 80 C overnight, quenched with saturated aqueous NaHCO3, and extracted
with EtOAc. The combined organic extracts were washed with brine, dried over Na2SO4, filtered,
and concentrated in vacuo. The residue was then purified by silica gel column chromatography
(EtOAc/hexane = 20:80) to yield 9d–f, respectively.
4.4. General Procedure for the Preparation of Glycosides 9c and 9g
To a solution of
8 (1.0 equiv) in CH2Cl2 (0.20 M), phenol or 2,2,2-trifluoroethanol (2.0 equiv) and
BF3
·
OEt2 (2.0 equiv) were added at room temperature under an Ar atmosphere. The mixture was
stirred at room temperature overnight, quenched with saturated aqueous NaHCO3, and extracted with
AcOEt. The combined extracts were washed with brine, dried over Na2SO4, filtered, and concentrated
in vacuo. The residue was then purified by silica gel column chromatography (EtOAc/hexane = 30:70)
to yield 9c or 9g, respectively.
4.5. General Procedure for the Preparation of Formamides 7c–g from Glycosides 9c–g
To a solution of each of 9c–g (1.0 equiv) in MeCN (0.20 M), EDA (4.7 equiv) was added at room
temperature under an Ar atmosphere. The mixture was stirred at 80 ◦C overnight and concentrated
in vacuo. To a solution of the crude amine in MeOH (0.40 M) and HCO2Me (0.40 M), NaOMe (1.2 equiv)

Mar. Drugs 2017, 15, 203
7 of 9
was added at room temperature under an Ar atmosphere. After stirring at room temperature overnight,
DOWEX was added for neutralization. The mixture was filtered through a celite pad and concentrated
in vacuo. To a solution of the crude formamide in pyridine (30 equiv), Ac2O (25 equiv) was added
at room temperature under◦an Ar atmosphere. After stirring at room temperature overnight, MeOH
(25 equiv) was added at 0 C. The mixture was extracted with EtOAc, washed successively with
1.0 M NaOH (25 equiv), 3.0 M HCl, and brine, dried over Na2SO4, filtered, and concentrated in
vacuo. The residue was purified by silica gel column chromatography (EtOAc/hexane = 40:60) to yield
7c–g, respectively.
4.6. Synthesis of 7h
To a solution of crude formamide in pyridine (50 equiv) and DMAP (0.50 equiv), obtained from
9f in two steps, octanoyl chloride (6.0 equiv) was added at room temperature under an Ar atmosphere.
The mixture was stirred for 24 h, quenched with 3.0 M HCl (60 equiv), diluted with AcOEt, washed
with brine, dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica
gel column chromatography (EtOAc:Hexane = 5:95) to yield 7h (24% over three steps) as a white solid.
4.7. Synthesis of 7i
To a solution of crude formamide obtained from 9f in two steps, DMF (0.050 M), palmitic acid
(5.0 equiv), DMAP (0.50 equiv), and EDCI (5.0 equiv) were added at room temperature under an
Ar atmosphere. The mixture was stirred for 20 h, supplemented with EtOH (2.0 equiv) and EDCI
(2.0 equiv), quenched with saturated aqueous NH4Cl, diluted with AcOEt, washed with brine, dried
over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel column
chromatography (EtOAc:hexane = 10:90) to yield 7i (16% over three steps) as a white solid.
4.8. Synthesis of 7j
To the solution of crude formamide in pyridine (15 equiv) and DMAP (0.50 equiv), obtained from
◦
9f in two steps, benzoyl chloride (5.0 equiv) was added at 60 C under an Ar atmosphere. The mixture
was stirred for 24 h, quenched with 3.0 M HCl (60 equiv), diluted with AcOEt, washed with brine,
dried over Na2SO4, filtered and concentrated in vacuo. The residue was purified by silica gel column
chromatography (EtOAc:hexane = 30:70) to yield 7j (46% over three steps) as a white solid.
4.9. Synthesis of 7k
To the solution of crude formamide, obtained from 9f in two steps, in DMF (0.050 M) and
◦
2,6-Lutidine (15 equiv), TBSOTf (20 equiv) was added at 120 C under an Ar atmosphere. The mixture
was stirred for 1.5 h, quenched with saturated aqueous NaHCO3, diluted with AcOEt, washed with
brine, dried over Na2SO4, filtered, and concentrated in vacuo. The residue was purified by silica gel
column chromatography (EtOAc:hexane = 10:90) to yield 7k (43% over three steps) as a white solid.
4.10. Antifouling Assay
Adult barnacles (Amphibalanus amphitrite) procured from oyster farms at Lake Hamana and a pier
◦
at Shimizu bay, Shizuoka, were kept in an aquarium at 20 C and fed on Artemia salina nauplii.
Broods were released as I–II stage nauplii upon immersion in seawater after drying overnight.
The nauplii thus obtained were cultured in filtered natural seawater (salinity 28) containing penicillin
G (20 µg/mL) and streptomycin sulfate (30 µg/mL) and were fed on the diatom Chaetoceros gracilis
at concentrations of 40
×
104 cells/mL. Larvae reached the cyprid stage in 5 days. The cyprids were
collected and stored at 4 ◦C until use (0 days old).
The test compounds were dissolved in ethanol and aliquots of the solution were transferred to
wells of a 24-well polystyrene culture plate and air-dried. Four wells were used for each concentration.
To each well, filtered seawater (2.0 mL, salinity 28) and six 2-day-old cyprids were added. The plates

Mar. Drugs 2017, 15, 203
8 of 9
◦
were kept in the dark at 25 C for 48 h. The numbers of cyprids that attached, metamorphosed,
died, and did not settle were counted under a microscope. Three or four trials were done for each
concentration. Probit analysis was used to calculate the EC50 values.
Supplementary Materials: The following are available online at www.mdpi.com/1660-3397/15/7/203/s1;
characterization of all compounds and 1H and 13C NMR data are available.
Acknowledgments: This research was supported by the Environment Research and Technology Development
Fund (1RF-1601) Environmental Restoration and Conservation Agency of Japan.
Author Contributions: Y.H., I.S.N., J.S., and T.M. prepared compounds; Y.N. and E.Y. evaluated biological
activities. T.U. conceived and designed the research and analyzed the experimental data. T.U., Y.N., and F.M.
wrote the paper. All of the authors reviewed and approved the manuscript.
Conflicts of Interest: The authors declare no conflict of interest.
References
1.
Global Wind Energy Council. Global Wind Report Annual Market Update 2015; GWEC: Brussels, Belgium, 2015.
Available online: http://www.gwec.net/wp-content/uploads/vip/GWEC-Global-Wind-2015-Report_
April-2016_22_04.pdf (accessed on 20 April 2017).
2.
3.
Fitridge, I.; Dempster, T.; Guenther, J.; de Nys, R. The impact and control of biofouling in marine aquaculture:
A review. Biofouling 2012, 28, 649–669. [CrossRef] [PubMed]
Champ, M.A. A review of organotin regulatory strategies, pending actions, related costs and benefits.
Sci. Total Environ. 2000, 258, 21–71. [CrossRef]
4.
5.
Flemming, H.-C. Reverse osmosis membrane biofouling. Exp. Therm. Fluid Sci. 1997, 14, 382–391. [CrossRef]
Bhadury, P.; Wright, P.C. Exploitation of marine algae: Biogenic compounds for potential antifouling
applications. Planta 2004, 219, 561–578. [CrossRef] [PubMed]
6.
7.
Brooks, S.J.; Waldock, M. Copper Biocides in the Marine Environment. In Ecotoxicology of Antifouling Biocides;
Arai, T., Harino, H., Ohji, M., Langston, W.J., Eds.; Springer: Tokyo, Japan, 2009; pp. 413–430.
Horiguchi, T.; Shiraishi, H.; Shimizu, M.; Yamazaki, S.; Morita, M. Imposex in Japanese gastropods
(Neogastropoda and Mesogastropoda): Effects of tributyltin and triphenyltin from antifouling paints.
Mar. Pollut. Bull. 1995, 31, 402–405. [CrossRef]
8.
9.
Shimasaki, Y.; Kitano, T.; Oshima, Y.; Inoue, S.; Imada, N.; Honjo, T. Tributyltin causes masculinization in
fish. Environ. Toxicol. Chem. 2003, 22, 141–144. [CrossRef] [PubMed]
McAllister, B.G.; Kime, D.E. Early life exposure to environmental levels of the aromatase inhibitor tributyltin
causes masculinisation and irreversible sperm damage in zebrafish (Danio rerio). Aquat. Toxicol. 2003, 65,
309–316. [CrossRef]
10. Weis, J.S.; Perlmutter, J. Effects of tributyltin on activity and burrowing behavior of the fiddler crab,
Uca pugilator. Estuaries 1987, 10, 342–346. [CrossRef]
11. Weis, J.S.; Kim, K. Tributyltin is a teratogen in producing deformities in limbs of the fiddler crab, Uca pugilator.
Arch. Environ. Contam. Toxicol. 1988, 17, 583–587. [CrossRef] [PubMed]
12. Terlizzi, A.; Delos, A.L.; Garaventa, F.; Faimali, M.; Gerace, S. Limited effectiveness of marine protected
areas: Imposex in Hexaplex trunculus (Gastropoda, Muricidae) populations from Italian marine reserves.
Mar. Pollut. Bull. 2004, 48, 188–192. [CrossRef] [PubMed]
13. Evans, S.M. TBT or not TBT?: That is the question. Biofouling 1999, 14, 117–129. [CrossRef]
14. Gibbs, P.E.; Bryan, G.W.J. Reproductive failure in populations of the dog-whelk, Nucella lapillus, caused by
imposex induced by tributyltin from antifouling paints. Mar. Biol. Assoc. UK 1986, 66, 767–777. [CrossRef]
15. Gibbs, P.E.; Bryan, G.W. TBT-induced imposex in neogastropod snails: Masculinization to mass extinction.
In Tributyltin: Case Study of an Environmental Contaminant; de Mora, S.J., Ed.; Cambridge University Press:
Cambridge, UK, 1996; pp. 212–236.
16. Chambers, L.D.; Stokes, K.R.; Walsh, F.C.; Wood, R.J.K. Modern approaches to marine antifouling coatings.
Surf. Coat. Technol. 2006, 201, 3642–3652. [CrossRef]
17. Steinberg, P.D.; de Nys, R.; Kjelleberg, S. Chemical cues for surface colonization. J. Chem. Ecol. 2002, 28,
1935–1951. [CrossRef] [PubMed]
18. Fusetani, N. Biofouling and antifouling. Nat. Prod. Rep. 2004, 21, 94–104. [CrossRef] [PubMed]

Mar. Drugs 2017, 15, 203
9 of 9
19. Raveendran, T.V.; Limna Mol, V.P. Natural product antifoulants. Curr. Sci. 2009, 97, 508–520.
20. Maréchal, J.-P.; Hellio, C. Challenges for the development of new non-toxic antifouling solutions. Int. J.
Mol. Sci. 2009, 10, 4623–4637. [CrossRef] [PubMed]
21. Qian, P.-Y.; Xu, Y.; Fusetani, N. Natural products as antifouling compounds: Recent progress and future
perspectives. Biofouling 2009, 26, 223–234. [CrossRef] [PubMed]
22. Fusetani, N. Antifouling marine natural products. Nat. Prod. Rep. 2011, 28, 400–410. [CrossRef] [PubMed]
23. White, D.E.; Stewart, I.C.; Grubbs, R.H.; Stoltz, B.M. The catalytic asymmetric total synthesis of elatol. J. Am.
Chem. Soc. 2008, 130, 810–811. [CrossRef] [PubMed]
24. Umezawa, T.; Oguri, Y.; Matsuura, H.; Yamazaki, S.; Suzuki, M.; Yoshimura, E.; Furuta, T.; Nogata, Y.;
Serisawa, Y.; Matsuyama-Serisawa, K.; et al. Omaezallene from red alga Laurencia sp.: Structure elucidation,
total synthesis, and antifouling activity. Angew. Chem. Int. Ed. 2014, 53, 3909–3912. [CrossRef] [PubMed]
25. Okino, T.; Yoshimura, E.; Hirota, H.; Fusetani, N. New antifouling sesquiterpenes from four nudibranchs of
the family Phyllidiidae. Tetrahedron 1996, 52, 9447–9454. [CrossRef]
26. Nishikawa, K.; Nakahara, H.; Shirokura, Y.; Nogata, Y.; Yoshimura, E.; Umezawa, T.; Okino, T.; Matsuda, F.
Total synthesis of 10-isocyano-4-cadinene and determination of and its absolute configuration. Org. Lett.
2010, 12, 904–907. [CrossRef] [PubMed]
27. Nishikawa, K.; Nakahara, H.; Shirokura, Y.; Nogata, Y.; Yoshimura, E.; Umezawa, T.; Okino, T.; Matsuda, F.
Total synthesis of 10-isocyano-4-cadinene and its stereoisomers and evaluations of antifouling activities.
J. Org. Chem. 2011, 76, 6558–6573. [CrossRef] [PubMed]
28. Nishikawa, K.; Umezawa, T.; Garson, M.J.; Matsuda, F. Confirmation of the configuration of
10-isothiocyanato-4-cadinene diastereomers through synthesis. J. Nat. Prod. 2012, 75, 2232–2235. [CrossRef]
[PubMed]
29. Gulavita, N.K.; De Silva, E.D.; Hagadone, M.R.; Karuso, P.; Scheuer, P.J.; Van Duyne, G.D.; Clardy, J.
Nitrogenous bisabolene sesquiterpenes from marine invertebrates. J. Org. Chem. 1986, 51, 5136–5139. [CrossRef]
30. Kassühlke, K.E.; Potts, B.C.M.; Faulkner, D.J. New nitrogenous sesquiterpenes from two Philippine
nudibranchs, Phyllidia pustulosa and P. varicosa, and from a Palauan sponge, Halichondria cf. lendenfeldi.
J. Org. Chem. 1991, 56, 3747–3750. [CrossRef]
31. Kitano, Y.; Ito, T.; Suzuki, T.; Nogata, Y.; Shinshima, K.; Yoshimura, E.; Chiba, K.; Tada, M.; Sakaguchi, I.
Synthesis and antifouling activity of 3-isocyanotheonellin and its analogues. J. Chem. Soc. Perkin Trans. 1
2002, 2251–2255. [CrossRef]
32. Nogata, Y.; Kitano, Y.; Yoshimura, E.; Shinshima, K.; Sakaguchi, I. Antifouling activity of simple synthetic
isocyanides against larvae of the barnacle Balanus amphitrite. Biofouling 2004, 20, 87–91. [CrossRef] [PubMed]
33. Fukuda, T.; Wagatsuma, H.; Kominami, Y.; Nogata, Y.; Yoshimura, E.; Chiba, K.; Kitano, Y. Anti-barnacle
activity of isocyanides derived from amino acids. Chem. Biodivers. 2016, 13, 1502–1510. [CrossRef] [PubMed]
34. Grenouillat, N.; Vauzeilles, B.; Beau, J.-M. Lipid analogs of the nodulation factors using the Ugi/Passerini
multicomponent reactions: Preliminary studies on the carbohydrate monomer. Heterocycles 2007, 73, 891–901.
35. Tavecchia, P.; Trumtel, M.; Veyrières, A.; Sinay, P. Glycosidations with N-formylamino sugar: A new approach
0
to 2 -deoxy-β-disaccharides. Tetrahedron Lett. 1989, 43, 2533–2536. [CrossRef]
36. Kitano, Y.; Nogata, Y.; Matsumura, K.; Yoshimura, E.; Chiba, K.; Tada, M.; Sakaguchi, I. Design and synthesis
of anti-barnacle active fluorescence-labeled probe compounds and direct observation of the target region in
barnacle cypris larvae for dimethyl-isocyanoalkyl compounds. Tetrahedron 2005, 61, 9969–9973. [CrossRef]
37. Fujiwara, S.; Akima, C.; Nogata, Y.; Yoshimura, E.; Chiba, K.; Kitano, Y. Bio-organic and anti-barnacle
studies of fluorescence-labeled probe compounds against cyprids of barnacles. J. Exp. Mar. Biol. Ecol. 2013
445, 88–92. [CrossRef]
,
©
2017 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
(CC BY) license (http://creativecommons.org/licenses/by/4.0/).