Design and synthesis of a new bioactive compound for marine antifouling inspired by natural products

Abstract A marine antifouling compound, N-octyl-2-hydroxybenzamide (OHBA), inspired by ceramide and paeonol molecules, was created. First, methyl salicylate was synthesized with salicylic acid and methanol, followed by n-octylamine through an ester-amine condensation reaction. Fourier transform infrared spectroscopy, nuclear magnetic resonance spectroscopy, and mass spectrometry confirmed the characteristic structure of the OHBA compound. Bioassays showed that OHBA inhibits the growth of typical marine fouling organisms, such as Vibrio azureus, Navicula subminuscula, Ulva pertusa, Mytilus edulis, and Amphibalanus amphitrite, indicating its broad-spectrum antifouling ability. A one-year marine real-sea test further demonstrated the excellent antifouling properties of OHBA. OHBA is also extremely biodegradable, with a half-life of 6.3 days, making it a less environmentally harmful replacement for widely-used heavy metal-containing antifoulants. GRAPHICAL ABSTRACT


Introduction
Marine biological fouling seriously harms the marine industry and environment by accelerating biological metal corrosion, increasing ship navigation resistance and fuel consumption, and causing species invasion (Sun et al. 2016;Chen et al. 2021;He et al. 2021;Tian et al. 2021).Biocides designed to kill or repel marine fouling organisms can effectively prevent the settlement of marine fouling organisms in seawater.Initially, simple lead and copper sheets were used to protect wooden boats, and heavy metals, including copper, arsenic, and mercury, were incorporated into the coatings.Later, the heavy metal-based biocide tributyltin (TBT), which is a highly efficient and broad-spectrum antifoulant, was synthesized and is currently widely used (Antizar-Ladislao 2008; Maan et al. 2020).Because heavy metal-based biocides seriously damage marine ecosystems, a global ban on TBT followed since 2008 (Evans et al. 2000;Liu et al. 2017).Therefore, the development of non-toxic and eco-friendly alternatives is imperative to replace the use of toxic biocides and reduce the environmental impacts of marine fouling.
Bioactive compounds extracted from natural products have been well studied because of their biodegradability and environmental benefits.Several bioactive compounds obtained from natural sources, including terpenoids, nitrogen compounds, phenolic compounds, and steroids, show good antifouling ability (Qian et al. 2015).For example, ceramide, a nitrogen-containing compound derived from sponges, exhibits antifouling activity, and its acyl residues are critical for the survival of marine organisms (Hattori et al. 1998).Paeonol, extracted from peony root by our laboratory, inhibits aquatic bacteria, diatoms, and Ulva zoospores.However, obtaining bioactive compounds in large quantities is challenging, resulting in the limited commercial applications of natural products.
Therefore, in this study, we designed and synthesized a bioactive compound, Noctyl-2-hydroxybenzamide (OHBA), based on natural products (ceramide and paeonol) that can be prepared in large quantities via chemical routes.The structural features were determined using Fourier transform infrared (FT-IR) spectroscopy, mass spectrometry (MS), and nuclear magnetic resonance spectroscopy 1 H/ 13 C-NMR.The antifouling properties were evaluated with bioassays, and real-sea tests were also discussed.

Synthesis and spectral analysis of the compound
The molecular structure of ceramide and paeonol inspired the design of OHBA (Figure 1a); the combination of the acyl residues of ceramide and 2-hydroxyphenyl ketone residues of paenol causes antifouling properties.The two-step synthesis procedure is illustrated in Figure 1b.An intermediate product, methyl salicylate, was prepared using salicylic acid and methanol, and was then used with n-octylamine under heating to synthesize the final compound (N-octyl-2-hydroxybenzamide).The functional groups of the compounds were observed using FT-IR spectroscopy (Figure S1).

Evaluation of marine antifouling performance
To identify the antifouling properties of OHBA, we evaluated its inhibitory effect on typical marine fouling organisms, including bacteria, diatoms, algae, mussels, and barnacle larvae, in the laboratory and in natural conditions (Figure S7A).A low effective concentration successfully inhibited the settlement of Vibrio azureus (MIC ¼ 0.11 mg/ L), Navicula subminuscula (EC 50 ¼ 1.05 mg/L), Ulva pertusa (EC 50 ¼ 0.12 mg/L), Mytilus edulis (EC 50 ¼ 0.52 mg/L) and Amphibalanus amphitrite (LC 50 ¼ 0.13 mg/L), showing the broad-spectrum antifouling capacity of OHBA.A real-sea test was performed to confirm the antifouling ability.After immersion in the sea for one year, the ceramidecontaining (Figure S7B-a) and paeonol-containing antifouling coatings (Figure S7B-c) failed; typical fouling organisms, such as sludge, diatoms, algae, barnacles, and ascidians were able to settle.In contrast, no fouling organisms attached to the OHBA-containing coating surface (Figure S7B-b).This result indicates that the OHBA coating has broad-spectrum and long-term antifouling effects.

Biodegradability of the OHBA
There are strict regulations on the environmental impacts of antifoulants, therefore, the biodegradability of OHBA directly determines the future application of OHBA in the field.The biodegradability of OHBA was assessed using the shake flask method (Nyholm and Kristensen 1992), and the results are shown in Figure S8.The OHBA compound begins to decompose after a stagnation period of about 12.8 days (t L ), and is decomposed by 50% after another 6.3 days; that is, OHBA has a half-life of 6.3 days and is highly biodegradable.OHBA as a carbon source may be decomposed by microorganisms into small molecules, such as carbonate, carbon dioxide, and water, which do not harm the environment.

Conclusion
In conclusion, we synthesized a bioactive compound for marine antifouling, inspired by natural products (ceramide and paeonol) extracted from sponges and peony roots.The compound exhibited broad-spectrum antifouling ability that was superior to natural products, which was verified by bioassays in the laboratory and real-sea tests.The compound also has a shorter half-life, so it is less harmful to the environment.The compound designed and synthesized in this study can be used in the future instead of the currently used heavy-metal-containing or toxic antifoulants.

Figure 1 .
Figure 1.Design ideas (a) and synthetic routes (b) of the OHBA compound.