WO1996008967A1 - Aquatic surface antifouling compositions - Google Patents

Abstract

Methods and compositions for preventing aquatic surface-fouling organisms from attaching to aquatic surfaces. The compositions include nitric oxide or a nitric oxide releasing, donating or transferring compound in combination with a coating material.

Description

AQUATIC SURFACE ANTIFOULING COMPOSITIONS

The present invention relates to compositions containing nitric oxide-releasing compounds as antifouling agents for surfaces exposed to aquatic environments, whether fresh or salt water. In particular, nitric oxide-releasing compounds include thionitrites, thionitrates, and other nitro(so) aromatic compounds. This invention also relates to antifouling compositions containing these compounds in combination with a coating substance.

A significant and widespread problem which confronts the maritime industry and results in billions of dollars in damage is the fouling of ships and other structures resulting from the attachment of aquatic organisms such as barnacles, hydroides, ascidians, mussels, oysters, algae and slime molds. For example, adherence of such organisms on the submerged hull of a ship increases the frictional resistance of the hull to passage through water, leading to increased fuel consumption and a reduction in the speed at which a ship is able to travel.

Other structures exposed to aquatic environments, such as beacons, buoys, piers, breakwaters, docks, pipelines, bridges, seaside industrial plants, and stationary fishing nets are also subject to damage caused by the adherence of such organisms. For example, adherence of such organisms causes extensive damage, such as corrosion, sinking due to the increased weight, loss of balance of the structure, and enormous expense incurred by necessary cleaning and repainting. In plants and power stations which use sea water for cooling or other purposes, marine organisms adhere to the sea water inlets and outlets, resulting in an increase in resistance to the liquid flow and impairment of function. Adhesion of aquatic organisms also significantly interferes with the cost-effective use of nets required for commercial fishing because of the significant labor and large expense necessary to maintain such nets.

Fresh water and marine organisms accumulate so rapidly on surfaces exposed to aquatic environments that the necessary remedy of frequent cleaning and repainting has been generally considered too costly. Thus, the alternative approach has been to limit the extent of fouling by applying paints or other types of coatings which incorporate antifouling agents to such structures. Such antifouling agents are generally biocides which are released from the surface of the paint over a period of time at a concentration which is lethal to surface adherent aquatic organisms.

A wide variety of antifouling agents have been used to prevent adherence of such organisms. For example, antifouling coatings have been used which contain heavy metal particles such as copper or a copper alloy (Layton U.S. Patent 4,410,642, October 18, 1983). Other compounds which have been used in antifouling paints include the No. 3- isothiazolone, polymers containing organosilyl groups, guanidine salts, dialkyldimathyl ammonium compounds and a quinolinyl or substituted quinolinyl ester of an unsaturated carboxylic acid (Thayer et al . , U.S. Patent No. 5,068,338, November 26, 1991/ Masuoka et al., U.S. Patent No. 5,116,61 1, May 26, 1992; Springal et al., U.S. Patent No. 3,915,918, October 28, 1975; Stovicek, U.S. Patent No. 5,173,110, December 22, 1992; and Sghibartz, U.S. Patent No. 4,426,464, January 17, 1984) .

A number of other antifouling compositions have contained organic tin compounds such as tributyltin hydroxide, triphenyltin hydroxide, tin-containing copolymers of such monomers as tributyltin (meth)acrylate, triphenyltin (meth)acrylate, bis (tributyltin) fumarate, tin-containing copolymers. While these are considered to be most desirable in terms of retention or efficacy and stability of effect, there has been a growing concern about the environmental effects caused by the use of organic tin biocides in antifouling compositions. It has been shown that the widespread use and subsequent leaching of tributyltin-type compounds has resulted in a level of pollution in the surrounding water which has been sufficient to cause the significant degradation of shellfish and other organisms.

Like organic tin compounds, dithiocarbamates which are organic sulfur compounds, are also widely used as antifouling components. For example, Japanese Unexamined Patent Publication No. Sho 51-49227 discloses that adhesion of harmful marine organisms is prevented by coating fishnets with a composition obtained by combining manganese ethylenebisdithiocarbamate as an antifouling component with a vehicle. Also, Japanese Unexamined Patent Publication No. Sho 51-51517 discloses that adhesion of such harmful organisms is prevented by coating fishnets with an antifouling composition obtained by combining a heavy metal salt of ethylenebisdithiocarbamic acid, a cellulose resin and a vehicle. In terms of safety and freedom from environmental pollution, metal salts of dithiocarbamic acid are viewed as desirable ingredients for antifoulant compositions. Often, however, they are not satisfactory in terms of retention of efficacy and stability of effect. In an attempt to solve these problems, compositions containing a heavy metal salt of alkylenebisthiocarbamic acid in combination with an inorganic copper compound or some other antifouling component such as organic tin compounds have been used. This practice, however, has not achieved satisfactory results.

Therefore, a need exists for an effective aquatic antifouling composition which is able to retain its effect for a long duration, is minimally susceptible to physical or chemical deterioration when coated, and which does not cause environmental pollution.

Nitric oxide (NO*) is a diatomic, free radical which mediates very important functions in the brain, immune system, smooth muscle, including the vasculature, digestive tract and reproductive organs (O'Dell et al . , Proc. Natl. Acad. Sci. USA. 80:11285, 11289 (1991); Shuman and Madison, Science 254 : 1503-1506 (199 1); Hibbs eϋ al . , Science, 235:473-476 (1987); Desai et al. , Nature 351:477-479 (1991) ; and Rajfer et al . , New England J. Med. 326:90-94 (1992)) . In the gas phase and in liquids, NO* reactions follow a pathway that eventually leads to the production of nitrite (Pogrebanaya et al . , Chem. Abstr. 78:285, a7639k (1972)). Conversely, nitrite and sea water can be photolyzed back to NO* by sunlight. (Zafiriou and True, Marine Chem. 8:9-32 (1979)) .

In spite of the vast amount of information relating to the role of NO* in higher invertebrates there has been comparatively little study of the role of NO* in the function of invertebrate organisms. Recent research, however, suggests that nitric oxide has a role as a biological messenger or effector molecule in invertebrates. It has been observed that hemocytes from horseshoe crabs, Limulus polyphemus, produce NO* through a nitric oxide synthase (Radomski et al . , Philos Trans R Soc Lond B, 334:129-134 (1991) ) . In addition, it has been discovered that some temperate marine sponges produce a compound or compounds which retard and restrict the settlement of larvae of other species on the surface of the sponge. The marine sponge X. halichondroides was shown to produce or exude a chemical that had an anesthetic effect upon larvae, was reversible in its action and was diffusible from the sponge surface. The identity of the chemical or chemicals produced by the sponge was never determined (Ware, the Patterns and Mechanisms of Antifouling and Some Temperate Marine Sponges. Doctoral thesis. At Duke University, Durham, North Carolina (1984)).

The present invention is directed to a method for preventing the attachment of surface fouling aquatic organisms to a surface exposed to an aquatic environment by coating onto or otherwise exposing the surface to an antifouling effective amount of nitric oxide or one or more nitric oxide-releasing substances, wherein the surface fouling organisms are prevented from attaching to the surface. In a preferred embodiment of this aspect, the method comprises coating the marine surface with a nitric oxide- releasing, donating, or transferring compound.

The present invention is further directed toward a method for preventing the attachment of surface fouling organisms to a marine surface by coating onto the surface a composition containing an antifouling effective amount of one or more of the compounds discussed below. The treatment with nitric oxide encompasses the use of gaseous nitric oxide and/or the use of a compound which is capable of delivering nitric oxide.

It is a particularly preferred aspect of the present invention that nitric oxide, or a nitric oxide releasing, donating, or transferring substance may be used prophylactically to prevent the fouling of surfaces exposed to aquatic environments, such as ship's hulls and equipment that is used in submerged locations for extended periods, particularly using compositions that are environmentally compatible with the type of aqueous environment and non- fouling organisms in the immediate surroundings.

Compounds contemplated for use in the invention are nitric oxide and compounds that release nitric oxide or otherwise directly or indirectly deliver or transfer nitric oxide to a site of its activity, such as on a cell membrane, in vivo. As used here, the term "nitric oxide" encompasses uncharged nitric oxide(NO*) and charged nitric oxide species, particularly including nitrosonium ion(NO⁺) and nitroxyl ion(NO) . The nitric oxide releasing, delivering or transferring compounds, having the structure X-NO wherein X is a nitric oxide releasing, delivering or transferring moiety, include any and all such compounds which provide nitric oxide to its intended site of action in a form active for their intended purpose. As used here, the term "NO adducts" encompasses any of such nitric oxide releasing, delivering or transferring compounds.

One group of such NO adducts is the S-nitrosothiols, which are compounds that include at least one -S-NO group. Such compounds include S-nitroso-polypeptides (the term "polypeptide" includes proteins and also polyamino acids that do not possess an ascertained biological function, and derivatives thereof); S-nitrosylated amino acids(including natural and synthetic amino acids and their stereoisomers and racemic mixtures and derivatives thereof) ; S-nitrosated sugars, S-nitrosated-modified and unmodified oligonucleotides (preferably of at least 5, and more particularly 5-200, nucleotides) ; and an S-nitrosated hydrocarbon where the hydrocarbon can be a branched or unbranched, and saturated or unsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon; S-nitroso hydrocarbons having one or more substituent groups in addition to the S-nitroso group; and heterocyclic compounds. S-nitrosothiols and the methods for preparing them are described in U.S. Patent Application No. 07/943,834, filed September 14, 1992, Oae et al . , Org. Prep. Proc. Int., 15(3) :165-198 (1983); Loscalzo et al . , J. Pharmacol. Exp. Ther. , 24£(3):726729 (1989) and Kowaluk et al . , J. Pharmacol. Exp. Ther., 256:1256-1264 (1990), all of which are incorporated in their entirety by reference.

One particularly preferred embodiment of this aspect relates to S-nitroso amino acids where the nitroso group is linked to a sulfur group of a sulfur-containing amino acid or derivative thereof. For example, such compounds include the following: S-nitroso-N-acetylcysteine, S-nitroso-captopril, S-nitroso-homocysteine, S-nitroso-cysteine and S-nitroso- glutathione.

Suitable S-nitrosylated proteins include thiol- containing proteins(where the NO group is attached to one or more sulfur group on an amino acid or amino acid derivative thereof) from various functional classes including enzymes, such as tissue-type plasminogen activator(TPA) and cathepsin B; transport proteins, such as lipoproteins, heme proteins such as hemoglobin and serum albumin; and biologically protective proteins, such as the immunoglobulins and the cytokines. Such nitrosylated proteins are described in PCT Publ. Applic. No. WO 93/09806, published may 27, 1993. Further examples of suitable S-nitrosothiols include those having the structures:

(i) CH₃(CH₂).SNO wherein x equals 2 to 20;

(ii) HStCH^SNO wherein x equals 2 to 20; and

(iii) ONS(CH,)-Y wherein x equals 2 to 20 and Y is selected from the group consisting of fluoro, C,-C« alkoxy, cyano, carboxamido, C^C* cycloalkyl, aralkoxy, Cj-Cj alkylsulfinyl, arylthio, Ci-C* alkylamino, Cj-Cij dialkylamino, hydroxy, carbamoyl, C,-Cβ N- alkylcarbamoyl, Cj-Cis N,N-dialkylcarbamoyl, amino, hydroxyl, carboxyl, hydrogen, nitro and aryl; wherein aryl includes benzyl, naphthyl, and anthracenyl groups.

Other suitable S-nitrosothiols that are S-nitroso- angiotensin converting enzyme inhibitors (hereinafter referred to as S-nitroso-ACE inhibitors) are described in Loscalzo, U.S. Patent No. 5,002,964 (1991) and Loβcalzo et al . , U.S. Patent No. 5,025,001 (1991) both of which are incorporated in their entirety by reference. Examples of such S-nitroso-ACE inhibitors include compounds having structure (1) :

(1)

wherein

R is hydroxy, NH₂, NHR⁴, NR⁴R³, or C,-C₇ alkoxy, wherein R⁴ and R^s are C,-C₄ alkyl, or phenyl, or C,-C₄ alkyl substituted by phenyl;

R¹ is hydrogen, -C, alkyl, or -C, alkyl substituted by phenyl, amino, guanidino, NHR⁶, NR'R⁷, wherein R⁶ and R⁷ are methyl or C,-C₄ alkanoyl;

R² is hydrogen, hydroxy, Cj-C₄ alkoxy, phenoxy, or

C.-C, alkyl;

R³ is hydrogen, C,-C or C₁-C₇ alkyl substituted by phenyl; m is 1 to 3; and n is 0 to 2.

Other suitable S-nitroso-ACE inhibitors include N- acetyl-S-nitroso-D-cysteinyl-L-proline, N-acetyl-S-nitroso- D,L-cysteinyl-L-proline, 1- (4-amino-2-S- nitroso)mercaptomethylbutanoyl) -L-proline, 1- [2-hexanoyl] -L- proline, 1- [5-guanidino-2- (S-nitroso) mercaptomethyl- pentanoyl] -L-proline, 1- [5-amino-2- (S-nitroso) mercaptomethyl-pentanoyl] -4-hydroxy-L-proline, 1- [5- guanidino-2- (S-nitroso)mercaptomethyl-pentanoyl] -4-hydroxy-L- proline, 1- [2-aminomethyl-3 (S-nitroso) -mercaptomethy 1 - pentanoyl -L-proline, and S-nitroso-L-cysteinyl-L-proline. Additional suitable S-nitroso-ACE inhibitors include those having structures (2-3) :

wherein

X is oxygen or sulfur; -A,,-A₂- is CH-NH or -C=N-;

R₃ 0 A is ON-S-CH₂-CH-C; R is selected from hydrogen, lower (C,-C*) alkyl, benzyl, benzhydryl, and salt forming ion;

R_t and Rj are independently selected from hydrogen, halogen, lower alkyl, lower alkoxy, halo substituted lower alkyl, nitro, and SOjNHj,-

0 0 0

Z is -C- or -S-

R₃ iβ hydrogen, lower alkyl, halo substituted lower alkyl, phenyl, benzyl, phenethyl, or cycloalkyl; and

₄ is hydrogen, lower alkyl, halo substituted lower alkyl, hydroxy substituted lower alkyl, -(CH₂)_q-N (lower alkyl)₂ or -(GH₂),-NH₂ and q is one, two, three or four. Additional suitable compounds include those having structures (4-11) :

TP

(8) (9)

(!•) (II) The S-nitroso-ACE inhibitors can be prepared by various methods of synthesis. In general, the thiol precursor is prepared first, then converted to the S-nitrosothiol derivative by nitrosation of the thiol group with NaN0₂ under acidic conditions (pH = 1 to 5) which yields the S-nitroso derivative. Acids which may be used for this purpose include aqueous sulfuric, acetic and hydrochloric acids. Thiol precursors are prepared as described in the following: U.S. Pat. NOS. 4,046,889 (1977); 4,052,511; 4,053,651; 4,113,751, 4,154,840, 4129,571 (1978), and 4,154,960 (1979) to Ondetti et al.; U.S. Pat. No. 4,626,545 (1986) to Taub; and U.S. Pat. Nos. 4,692,458 (1987) and 4,692,459 (1987) to Ryan et al . , Quadro, U.S. Pat. No. 4,447,419 (1984); Haugwitz et al . ,- U.S. Pat. No. 4,681,886 (1987), Bush et al., U.S. Pat. No. 4,568,675 (1986), Bennion et al . , U.S. Pat. No. 4,748,160 (1988), Portlock, U.S. Pat. No. 4,461,896 (1984), Hoefle et al . , European Patent Application Publication No. 0 088 341 (1983), Huange et al . , U.S. Pat. No. 4,585,758 (1986), European Patent application Publication No. 0 237 239, European Patent application Publication No. 0 174 162, published in 1986, European Patent application Publication No. 0 257 485, published in 1988, all of which are incorporated by reference herein.

In a particular aspect of the invention, preferred nitric oxide-releasing compounds include lipophilic thionitrites, organic nitrates, and nitro(so) aromatic compounds. Examples of thionitrites include S-nitrosothiol alkanes, such as, for example, propylthionitrite, butylthionitrite, pentylthionitrite, dodecathionitrite and benzenylthionitrite. Organic nitrates and nitro(so) aromatic compounds include compounds which contain nitric oxide in combination with carbon, sulfur, nitrogen, oxygen, a redox metal or an amino acid or acids. Especially preferred nitro(so) aromatic compounds include nitrosated indols, and in particular, nitrosated tryptophan and N and C derivatives of nitrosated tryptophan.

In another aspect, the invention relates to a method for preventing the attachment of surface-fouling aquatic organisms to an aquatic surface by coating onto such a surface a composition containing an antifouling effective amount of one or more lipophilic compounds selected from the group consisting of: (i) a compound having the fomula

(X_iΛUO) _a) ₀, wherein X, is selected from the group consisting of 0, C, N, S, N0₂, N0₃, Al, Fe, Cr, Cu, an amine, an amino acid and a peptide comprising at least two amino acids linked by peptide bonds; m is 1 or 2; and n is any integer; (ii) a compound having the formula X₂R,S-NO wherein X₂ is selected from the group consisting of H, HS and NO; and R_t is selected from the group consisting of H, C₂-C_2o alkyl, C^ aryl, C₃-C₃ cycloalkyl and benzyl; and (Hi) a compound having the formula

ONSRjY wherein R₂ is a -C^, alkyl and Y is selected from the group consisting of fluoro, -C alkoxy, cyano, carboximido, C_ϊ-C₃ cycloalkyl, arylalkoxy, Cj-Cg alkylsulfinyl, arylthio, C,- Cj alkylamino,

dialkylamino, hydroxy, carbamoyl, -Cβ N- alkylcarbomoyl, Cj-C_u N,N-dialkylcarbamoyl, amino, hydroxyl, carboxyl, hydrogen, nitro, and aryl.

The amino acid or peptide can include amino acids selected from the group consisting of cysteine, homocysteine, N-acetylcysteine, methionine, cystine, serine, threonine, phenylalanine, tyrosine, tryptophan, thyroxine, arginine, lysine, histidine, hydroxylysine, glycine, alanine, valine, leucine, and isoleucine, proline, hydroxyproline, aspartic acid, asparagine, glutamic acid and glutamine.

As a result of their lipophilic character, the above- described nitric oxide-releasing compounds are especially suitable for use in coating substances. In the alternative, these compounds may be chemically modified to make them hydrophilic or amphiphilic (having both hydrophilic and lipophilic character) , so as to render them suitable for use in aqueous-based or both aqueous-based or organically-based coating substances.

Another group of such NO adducts are compounds that include at least one -0-NO group. Such compounds include O- nitroso-polypeptides (the term "polypeptide" includes proteins and also polyamino acids that do not possess an ascertained biological function, and derivatives thereof) ; O-nitrosylated amino acids (including natural and synthetic amino acids and their stereoisomers and racemic mixtures and derivatives thereof) ; O-nitrosated sugars,- 0-nitrosated-modified and unmodified oligonucleotides (preferably of at least 5, and more particularly 5-200, nucleotides) ; and an O-nitrosated hydrocarbon where the hydrocarbon can be a branched or unbranched, saturated or unsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon; 0-nitroso hydrocarbons having one or more substituent groups in addition to the O-nitroso group; and heterocyclic compounds.

Another group of such NO adducts is the nitrites which have an -O-NO group wherein R is a protein, polypeptide, amino acid, branched or unbranched and saturated or unsaturated alkyl, aryl or a heterocyclic. A preferred example is the nitosylated form of isosorbide. Compounds in this group form S-nitrosothiol intermediates in vivo in the recipient human or other animal to be treated and can therefore include any structurally analogous precursor R-O-NO of the S-nitrosothiols described above.

Another group of such NO adducts is the N-nitrosoamines, which are compounds that include at least one -N-NO group. Such compounds include N-nitroso-polypeptides (the term "polypeptide" includes proteins and also polyamino acids that do not possess an ascertained biological function, and derivatives thereof) ; N-nitrosylated amino acids (including natural and synthetic amino acids and their stereoisomers and racemic mixtures) ; N-nitrosated sugars; N-nitrosated-modified and unmodified oligonucleotides (preferably of at least 5, and more particularly 5-200, nucleotides) ; and an N- nitrosated hydrocarbon where the hydrocarbon can be a branched or unbranched, and saturated or unsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon; N-nitroso hydrocarbons having one or more substituent groups in addition to the N-nitroso group; and heterocyclic compounds.

Another group of such NO adducts is the C-nitroso compounds that include at least one -C-NO group. Such compounds include C-nitroso-polypeptides (the term "polypeptide" includes proteins and also polyamino acids that do not possess an ascertained biological function, and derivatives thereof) ; C-nitrosylated amino acids (including natural and synthetic amino acids and their stereoisomers and racemic mixtures) ; C-nitrosated sugars; C-nitrosated-modified and unmodified oligonucleotides (preferably of at least 5, and more particularly 5-200, nucleotides) ; and a C-nitrosated hydrocarbon where the hydrocarbon can be a branched or unbranched, and saturated or unsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon; C-nitroso hydrocarbons having one or more substituent groups in addition to the C-nitroso group; and heterocyclic compounds

Another group of such NO adducts is the nitrates which have at least one -0-N0₂ group. Such compounds include polypeptides (the term "polypeptide" includes proteins and also polyamino acids that do not possess an ascertained biological function, and derivatives thereof) ; amino acids (including natural and synthetic amino acids and their stereoisomers and racemic mixtures and derivatives thereof) ; sugars; modified and unmodified oligonucleotides (preferably of at least 5, and more particularly 5-200, nucleotides) ; and a hydrocarbon where the hydrocarbon can be a branched or unbranched, and saturated or unsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon; hydrocarbons having one or more substituent groups; and heterocyclic compounds. A preferred example is nitroglycerin.

Another group of such NO adducts is the nitroso-metal compounds which have the structure (R)_n-A-M- (NO)_x. R includes polypeptides (the term "polypeptide" includes proteins and also polyamino acids that do not possess an ascertained biological function, and derivatives thereof) ; amino acids (including natural and synthetic amino acids and their stereoisomers and racemic mixtures and derivatives thereof) ; sugars; modified and unmodified oligonucleotides (preferably of at least 5, and more particularly 5-200, nucleotides); and a hydrocarbon where the hydrocarbon can be a branched or unbranched, and saturated or unsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon; hydrocarbons having one or more substituent groups in addition to the A-nitroso group; and heterocyclic compounds. A is S, 0, or N, n and x are each integers independently selected from l, 2 and 3, and M is a metal, preferably a transition metal. Preferred metals include iron, copper, manganese, cobalt, selenium and luthidium. Also contemplated are N-nitrosylated metal centers such as nitroprusside.

Another group of such NO adducts is the N-oxo-N- nitrosoamines which have an R-N(0^"M⁺) -NO group or an R-NO-NO- group. R includes polypeptides (the term "polypeptide" includes proteins and also polyamino acids that do not possess an ascertained biological function, and derivatives thereof); amino acids(including natural and synthetic amino acids and their stereoisomers and racemic mixtures and derivatives thereof) ; sugars; modified and unmodified oligonucleotides (preferably of at least 5, and more particularly 5-200, nucleotides); and a hydrocarbon where the hydrocarbon can be a branched or unbranched, and saturated or unsaturated aliphatic hydrocarbon, or an aromatic hydrocarbon,- hydrocarbons having one or more substituent groups; and heterocyclic compounds. R is preferably a nucleophilic (basic) moiety. M+ is a metal cation, such as, for example, a Group I metal cation.

Another group of such NO adducts is the thionitrates which have the structure R-(S).-N0 wherein x is an integer of at least 2. R is as described above for the S-nitrosothiols. Preferred are the dithiols wherein x is 2. Particularly preferred are those compounds where R is a polypeptide or hydrocarbon and a pair or pairs of thiols are sufficiently structurally proximate, i.e. vicinal, that the pair of thiols will be reduced to a disulfide. Those compounds which form disulfide species release nitroxyl ion(NO) and uncharged nitric oxide(NO*) . Those compounds where the thiol groups are not sufficientl close to form disulfide bridges generally only provide nitric oxide as the NO^' form but not as the uncharged NO* form. Also, agents which stimulate endogenous NO synthesis will be suitable.

The present invention is also directed toward antifouling compositions containing an antifouling effective amount of nitric oxide or one or more nitric oxide-releasing, donating or transferring compounds, as discussed above, in combination with a coating substance.

The present invention is also directed toward a method of reducing N0₂ ^' and N0₃ ^' in sea water using a coating substance reductant.

The present invention is also directed toward marine antifouling by photolysis using ultraviolet, visible or laser light to generate NO* from ambient sea water.

Figure l. UV spectra of 15 μl and 30 μl of 10² M DEA/NO in 10 mM NAOH in 1 ml of 100 kD CH₂0. Lambda maximum for the 15 μl sample is 246. Lambda maximum for the 30 μl sample is 244. Both spectra were taken immediately after addition and mixing within the cuvette.

Figure 2. UV spectra showing the conversion of the HbA to Met-Hb over a 55 minute period. HbA was added (0.5 ml) to a cuvette in which 15 μl of 10²M DEA/NO in 10 mM NaOH had been added to 1.5 mL 100 kD CH₂0 approximately 30 min after the DEA/NO was added.

In accordance with the present invention it has been discovered that exposure to nitric oxide immobilizes aquatic surface-fouling organisms including, for example, barnacles, thus preventing the organisms from adhering to such surfaces. Thus the invention provides compositions and methods whereby nitric oxide-releasing compounds may be coated onto the surface of a structure in order to prevent the adherence of such aquatic surface-fouling organisms to the structure, a method of reducing N0₂ ^~ and N0₃ ^" in sea water using a coating substance reductant and a method for marine antifouling by photolysis using ultraviolet, visible or laser light to generate NO* from ambient sea water.

The term "aquatic surface-fouling organism" is used to mean any aquatic organism which attaches to an aquatic surface as defined below. This includes, for example, barnacles (members of the class Salanus) such as B. eburneus, B. improvisus, B. balanoides, B. nubilus, and B. amphitri te; Chthamalus fragilis, Darwin (barnacle) , B. surpulae, sea mussels, Zebra mussels, hydroides, ectoprocts, tube-building amphipods; oysters, sea moss, mollusks, shellfish, ulba, enteromorpha, ectocorpus, ostrea, mytilus, ascidian or slime, seaweed, and algae such as sea lettuce, green laver, marine- spirogyra and bryozoan. The invention is contemplated to include any additional aquatic organisms whose attachment would be prevented. The term "aquatic" is contemplated to include fresh and salt water environments and organisms.

The term "aquatic surface" is used to mean any surface which is in contact with fresh, salt, estuarine, brakish, sea or other bodies of water including, for example, ship surfaces, deck surfaces, buoys, piers, fishing nets, cooling system surfaces, cooling water intake or discharge pipes, nautical beacons, floating beacons, floating breakwaters, docks, pipelines, tanks, water pipes in power stations, seaside industrial plants, fish preserving structures, aquatic constructions, port facilities, and bridges.

The term "antifouling effective amount" means an amount of the present nitric oxide-releasing compounds, sufficient to prevent the attachment of surface fouling organisms to an aquatic surface. The amount sufficient to prevent attachment of a surface-fouling organism to an aquatic surface, can be readily adjusted by one of ordinary skill in the art and will vary, depending upon the characteristics of the surface, including for example, porosity, material, i.e. wood, metal, plastic, surface contour, surface roughness, and the presence or absence of additional non-essential components of the composition, i.e., the presence or absence of components other than the present nitric oxidereleasing compounds.

The term "lipophilic" means nitric oxide-releasing compounds which are soluble in organic solvents.

The term "coating" means the application of the present composition to an aquatic surface by any means known to those of ordinary skill in the art to which the present invention pertains. Such means include, for example, painting or spraying the present composition onto a marine surface. The present composition is preferably applied to an aquatic surface in one or more layers.

The term "coating substances" means any substance which is suitable for coating a marine structure or surface and includes, for example, paints, lacquers, stains, resins, epoxys, and enamels.

In one embodiment, the invention relates to a method for preventing the attachment of surface-fouling organisms to a an aquatic surface by coating onto the surface, a composition containing an antifouling effective amount of one or more nitric oxide-releasing compounds. By coating the composition onto the surface, the continuous release of nitric oxide prevents the organisms from attaching to the surface. In another embodiment, the nitric oxide-releasing compound may include a nitrite-reducing compound, for example, redox dyes such as methylene blue or indium red and redox metals. These compounds are contained in the antifouling composition, and when applied to the aquatic surface, create a reducing surface, resulting in the conversion of nitrites or nitrates in the water to NO*.

In another embodiment, NO is generated using a photoylsis method employing UV, visual or laser light to release NO from ambient nitrite in sea water. In another embodiment, a ship will release NO from gas tanks situated in the hull, the gas then protecting the ship as it moves.

In another aspect the invention includes antifouling compositions containing an antifouling effective amount of one or more nitric oxide releasing compounds in combination with a coating substance. The coating substances will contain, in addition to the antifouling compound, a liquid vehicle (solvent) for dissolving or suspending the active antifouling compounds, and an organic binder. The vehicle may typically contain at least one of a diluent, an emulsifier and a wetting agent. Selection of the most appropriate coating substance, along with the solvent and binder, may be accomplished using routine methods available to those of skill in the art depending on the particular antifouling compounds utilized.

Any conventional organic binder may be utilized in fresh water or marine antifouling coating substances incorporating the antifouling compounds of the present invention. Examples of trade-recognized binders are polyvinyl chloride resins in a solvent based system, chlorinated rubbers in a solvent based system, acrylic resins and methacrylate resins in solvent based or aqueous systems, vinyl chloride-vinyl acetate copolymer systems as aqueous dispersions or solvent based systems, butadiene copolymers such as butadiene-styrene rubbers, butadiene-acrylonitrile rubbers, and butadiene- styrene-acrylonitrile rubbers, drying oils such as linseed oil, alkyd resins, asphalt, epoxy resins, urethane resins, polyester resins, phenolic resins and the like.

The coating substances commonly may contain inorganic pigments, such as titanium dioxide, ferric oxide, silica, talc, or china clay, organic pigments such as carbon black or dyes water insoluble dyes, and may contain materials such as rosin to provide controlled release of the antifoulant, rosin being to a very slight extent water soluble. The coating substances may contain plasticizers, rheology characteristic modifiers and other conventional ingredients.

In still other aspects of the present invention, the compositions, particularly when formulated as coating substances, also are provided with other adjuvants conventionally employed in compositions used for protecting materials exposed to an aquatic environment such as additional fungicides, auxiliary solvents, processing additives such as defoamers, fixatives, plasticizers, UV- stabilizers or stability enhancers, water soluble or water insoluble dyes, color pigments, siccatives, corrosion inhibitors, thickeners or antisettlement agents such as carboxymethyl cellulose, polyacrylic acid or polymethacrylic acid, anti-skinning agents and the like. Additional fungicides used in the compositions are preferably soluble in the liquid vehicle.

Compositions of the present invention can be provided as a ready-for-use product in the form of aqueous solutions and dispersions, oil solutions and dispersions, emulsions, aerosol preparations and the like or as a concentrate. The concentrate can be used as is, for example as an additive for coating substance, or can be diluted prior to use with additional solvents or suspending agents.

When the compositions are supplied as a concentrate with the active ingredients dissolved or dispersed in a liquid vehicle or carrier material, the active biocidal ingredient or mixture of ingredients typically comprises from about 0.1 % by weight up to about 80 % by weight of the total composition. After formulation as a coating substance, the preparation typically will contain from about 0.1 % by weight up to about 40 % by weight, more generally from about 1.0 % by weight up to about 20 % by weight, and most often about 1 % to about 10% by weight of the active ingredient or mixture of active ingredients. A liquid vehicle normally comprises more than about 70% by weight, and more generally above about 90% by weight of the composition when it is formulated as a coating substance. In some concentrates, however, the liquid vehicle can constitute as little as 5 % by weight of the composition.

The liquid vehicle is not a critical aspect of the present invention and any liquid which does not interfere with the nitric oxide-releasing capacity of the active ingredient and which is compatible with the disclosed applications may be used. Suitable materials for the liquid vehicle include water and organic solvents including aliphatic and aromatic hydrocarbons having boiling points between 100° C and 320° C, preferably between 150° C and 230° C; high aromatic petroleum distillates, e.g., solvent naphtha, distilled tar oil and mixtures thereof; alcohols such as butanol, octanol and glycols,- vegetable and mineral oils,- ketones such as acetone; petroleum fractions such as mineral spirits and kerosene, chlorinated hydrocarbons, glycol esters glycol ester ethers, and the like. The liquid vehicle may contain at least one polar solvent, such as water, in admixture with an oily or oil-like low-volatility organic solvent, such as the mixture of aromatic and aliphatic solvents found in white spirits, also commonly called mineral spirits.

The liquid vehicle also may commonly include an emulsifier, a wetting agent, a dispersing agent or other surface active agent. Examples of suitable emulsifiers are the nonylphenol-ethylene oxide ethers, and polyoxyethylene sorbitol esters or polyoxyethylene sorbitan esters of fatty acids. For example, a useful formulation may contain the mixture of the active antifouling compounds dissolved in an organic solvent such as mineral spirits which in turn is emulsified with the aid of a suitable emulsifier in water as the primary liquid vehicle.

An aerosol preparation according to the invention is obtained in the usual manner by incorporating the active ingredients dissolved or suspended in a suitable solvent, in a volatile liquid suitable for use as a propellant, for example the mixture of chlorine and fluorine derivatives of methane and ethane commercially available under the trademark "Freon", or compressed air.

The balance of the compositions may include additional ingredients known to be useful in preservatives and coatings for aquatic application and related products. Such ingredients include fixatives such as carboxymethylcellulose, polyvinyl alcohol, paraffin and the like, co-solvents, such as ethylglycol acetate and methoxypropyl acetate and plasticizers such as benzoic acid esters and phthalates, e.g. dibutyl phthalate, dioctyl phthalate and didodecyl phthalate. Optionally, dyes, color pigments, corrosion inhibitors, chemical stabilizers or siccatives (dryers) such as cobalt octate and cobalt naphthenate also may be included depending on specific applications determined by those of skill in the art.

Such additional ingredients are not essential to the practice of the present invention but are included in particular formulations to optimize overall effectiveness and ease of application. The specific examples of suitable constituents for specific preparations as enumerated above are not intended to be limiting, and a wide variety of other possible additional ingredients will be recognized by those skilled in the art. Similarly, the quantity of such additional ingredients in any formulation is not critical. They generally can be used in an amount conventionally employed for products designed to be used in applications for protecting materials exposed to an aquatic environment. Normally, the totally formulated compositions may contain from about 0.1 % to 95 % by weight, and more usually from about 1 % to 50 % by weight of these additional ingredients on a total solids basis.

The antifouling compositions of the present invention can be applied by any of the techniques known in the art including for example, brushing, spraying, roll coating, and dipping. Generally, to obtain effective treatment, it should be sufficient to apply the composition to an amount to provide between 20 to 180 grams of the active ingredient or mixture of active ingredients per square meter of surface area to be treated (about 0.0040 to 0.037 pound per square foot) , with an amount of about 80 to 120 g/m² (about 0.016 to 0.025 lb/ft²) being more typical. Of course, higher rates of application can be used if determined by those of skill to be desirable. Compositions of the present invention can be prepared simply by mixing the various ingredients at a temperature at which they are not adversely affected, i.e., at a temperature of from about -5° C to 80° C, preferably at a temperature of from about -10° C to 45° C, and at a pressure of 450 mm Hg to 900 mm Hg, preferably at about 650 mm Hg. Preparation conditions are not critical. Equipment and methods conventionally employed in the manufacture of coating substances and similar compositions can be advantageously employed.

The antifouling compositions, when applied to any suitable aqueous surface, are effective in preventing the adherence of a wide variety of damaging aquatic surface- fouling organisms, by providing a slow release of nitric oxide at the interface between the structure and the aquatic environment. The antifouling compositions of the present invention are extremely effective as compared to conventional organic metal compositions and furthermore, pose less of a threat to the environment, and are less toxic to humans and aquatic life. A particular advantage of these compounds is that, in contrast to conventional antifouling biocides which act by killing the organisms, the nitric oxide-releasing compounds prevent adherence by temporarily and reverεibly immobilizing organisms which come in contact with the aqueous surface.

Having now generally described the present invention, the same will be more readily understood through reference to the following examples which are provided by way of illustration, and are not intended to be limiting of the present invention. ^g-"™!?¹* 1 Nitrosation of arom ^ »ιτππ₀ acids Methods a. Preparation of Nitroso-tyrosine

50 mmol of L-tyrosine (Sigma Chemical company; St. Louis, MO) were dissolved into 0.5 ml of distilled water. 250 mmol of NA"NO, (sodium N- [15] nitrite: MSD Isotopes, Merck Scientific; Rahway, NJ) were dissolved into 0.5 mL of I N HC1 (Fisher Scientific; Fair Lawn, NJ) and transferred immediately to the aqueous tyrosine solution with agitation by Vortex stirrer. Solution was capped and allowed to sit at room temperature for 30 minutes.

NMR measurements were made as follows:

(a) ¹⁵N-NMR: D₂0 was added and measurements were taken immediately;

(b) Η-NMR: After ^l5"N-NMR was completed, solution was removed and placed into a small round-bottom flask and water was removed in vacuo. D₂0was added to the dry off- white solid (this time as a solvent) and measurements were run immediately,-

(c) Infrared Spectroscopy: Fourier Transform Infrared Spectroscopy (FTIR) samples were prepared through removal of water (as in (b) ) and subsequent creation of a Nujol Mull using mineral oil.

(d) Ultraviolet and Visible Spectroscopy (UV-Vis) : Samples for UWis examination were used as per above prep without further modification. Samples were referenced to distilled water.

b. Nitrosylation of Phenylalanine. Tyrosine & L-Boc- Tvr(Bt)-OH

50 mmol of L-phenylalanine, L-tyrosine (Sigma Chemical Company; St. Louis, MO), or L-boc-tyr(Et) -OH (Bachem Bioscientific Incorporated; Philadelphia, PA) were dissolved into 0.5 ml of distilled water. 250 mmol of NA^lsN0₂ (sodium N- [15] nitrite) were dissolved into 0.5 ml of 1 N HCl (aq.) and transferred immediately to the aqueous amino acid solution with agitation by Vortex stirrer. Solution was capped and allowed to sit at room temperature for 30 minutes. ¹⁵N-NMR and Η-NMR were performed as per nitrosotyrosine above. Standard reference of tyrosine for FTIR was prepared as a Nujol Mull of pure crystalline L- tyrosine.

c. Nitrosation of Tryptophan

1.7 mM of tryptophan were reacted with equimolar NaN0₂ in 0.5 N HCl for time periods of 5, 10, 15 and 60 minutes at 25° C.

Results a. ^1SN-NMR data

All NMR [¹⁵N and ^lK] were run on two Bruker AM-500 MgHz spectrometers. Nitrosation of tyrosine at pH 0.3 gives signals at approximately 730 ppm and 630 ppm relative to saturated sodium N- [15] nitrite aqueous solution referenced at 587 ppm¹² (^1SN0₂) . A signal at 353 ppm (aqueous NO⁺) was also observed. Nitrosation of phenylalanine under the same conditions gave the signal at approximately 630 ppm but not the 730 ppm signal despite repeated attempts. Nitrosation of phenylalanine also yielded signals at 587ppm (excess, unprotonated nitrite) and 353ppm. Nitrosation of O-blocked tyrosine model, boc-tyr(Et) -OH, also yielded a signal at approximately 630 ppm; and others at 587 ppm and 353 ppm. Small signals in the range 450-495 ppm were observed for the tyrosine models, phe and boc-tyr(Et) -OH.

b. ^lH-NMR data

To further characterize the nitrosation of the phenolic functionality of L-tyr to the exclusion of C- nitrosation, proton-NMR was performed on nitrosated tyrosine; modification of L-tyr at the phenolic-OH would not appear in proton-NMR because of proton exchange with the deuterated solvent (D₂0) . Examination of the spectra showed the classic doublet of doublets at low field, which is characteristic of para-disubstituted benzene, thus excluding aromatic proton substitution. This, and other values in the spectra were characteristic of unmodified L- tyr.

c. FTIR data

All FTIR were run on a Nicolet 5ZDX FT-IR Spectrometer. FRIR of a Nujol Mull of L-tyrosine showed a very characteristic and well-documented alcoholic stretch in the spectra due to the phenolic-OH. This spectrum lacked any signal (s) at the 1680-1610 cm^'1 range that coincides with the 0-N=0 stretch (not shown) . FTIR of nitrosated L-tyrosine showed no evidence of alcoholic-OH stretches and contained two small bands in the range of 1680-1610 cm^"1 that could possibly account for the expected O-N=0 stretch (Wade, L.G. , Organic Chemistry (1st Ed.) Prentice-Hall Inc., Englewood Cliffs, N.J. : 1987. p. 1334) .

d. UV-Vis data

All UV-Vis spectroscopy was performed using a Gilford Response UV-Vis Spectrophotometer (CIBA-Corning, Oberlin, OH) . Treatment of L-tyrosine with aqueous sodium nitrite at pH 0.3 (0.5N HCl) resulted in a yellow solution with an absorption maximum at 361 nm. This result is similar to, but differs from, previously reported results with nitrosated L-tyrosine. Ortho-ring substituted L-nitro- tyrosine (Sigma) absorbs at 356 nm at pH 0.3.

Treatment of phenylalanine with sodium nitrite at pH 0.3 gives a rapidly changing UV spectrum with a peak increasing in wavelength from 318 nm at 5 min. to a maximum unchanging peak at 527 nm by 30 min.

W-rΛmrfΛ 2

Tttimpbilization of barnacles bv exposure to nitric oxide

Materials And Methods

Cyprid stage barnacle larvae, Balanus amphi tri te, were obtained upon demand from cultures sustained at the Duke University Marine Lab. The cyprids were tested within an hour of the time they were removed from cold storage (6°C) .

Bichamber (Granger Cell) Experiments

Testing cells were made of two chambers separated by a hydrophobic, gas permeable membrane. In the lower chamber 250 μl of 0.25 M aqueous succinic acid (C₄H₆0₄)was mixed with 250 μl, 500 μl, or 1 ml of 1 M aqueous sodium nitrite NO*. The upper chamber was filled with 100 kD filtered seawater (100 kD CH₂0) , and contained the cyprids.

The amount of NO* produced varied with the amount of NaN0₂, solution used. After a period of exposure to NO*, the chamber with the cyprids was either flushed with fresh seawater, or removed from contact with the NO* producing solution.

Tissue Culture Cell Experiments

Tests were carried out in the cells of a twenty-four well tissue culture plate. Each cell had a fluid capacity of approximately 3 ml. One method of NO* delivery used in these tests was the release of NO* from nucleophile/nitric oxide Keefer salts. These NO* impregnated salts are stable as solids and in high pH solutions, such as sodium hydroxide (NaOH, pH 12.2). When put into solutions of lower pH, such as seawater (pH 8.3) , they release NO*. The total yield of NO* and rate of release have been determined for each of these compounds (Maragos et al., J. Med. Chem. 34:3242-3247 (1991) ) .

The compound used in these experiments was called DEA/NO, or diethylamine/nitric oxide (molecular formula Et^-N- (ONa) -N=0) . The NO* yield, or E_NO for this compound is 1.5 +/- 0.11 moles of NO* per mole of reagent at pH 7.4 and 37° C. The exact yield at room temperature and pH of approximately 8 is not known, but is assumed to be in the same range as that for physiological conditions. Stock solutions of 10^"2 M DEA/NO in 10 mM NaOH were made and kept cold. From these relatively stable solutions, small doses (10 μl to 100 μl) were taken for each test.

Sodium nitroprusside (SNP) was also used as a NO* releasing compound. SNP releases one mole of NO* per mole of starting material. SNP dissolved in 100 kD CH₂0 was added directly to the cells. Similar experiments were performed with S-nitroso-cysteine, S-nitroso-glutathione, S-nitroso-N-acetylcysteine, S-nitroso-albumin, S-nitroso- tryprophan and S-nitroso-phenylalanine.

For all experiments done in the tissue culture cells using either Keefer salts or SNP, cyprids in 1 ml of 100 kD CH₂0 were exposed to NO* concentrations from 10^"3 M to 10^"12 M. In the case of S- and N-nitroso amino acids, concentrations ranged from 10^'3 - 10^"9 M.

In these tests, 60 μM hemoglobin (HbA) in filtered seawater was used to scavenge and sequester NO* in the test chambers. This was done after the effects of NO* were observed to determine the rate at which the cyprids recovered from the exposure to NO*. Observation of these tests was done using a microscope. Video documentation of the tests was made with a VHS camera, without the use of a microscopic lens. These tapes were later reviewed to more accurately estimate the time of onset of NO* action, and the time of recovery from NO* exposure.

Ultraviolet Spectrophotometry

UV spectra of samples of DEA/NO in IOOkD CH₂0 were obtained, using a diode array spectrophotometer. Spectra of the changes in the structure of hemoglobin upon introduction to a cuvette in which DEA/NO had been reacting in seawater were also collected.

Oxygen Probes

Experiments were conducted using two different concentrations of SNP. SNP was added to bottles filled with aerated 24° C 100 kD CH₂0, salinity 35 ppt. The bottles were sealed by the insertion of the 0₂ probes. P0₂ was monitored for the test bottles and for simultaneous controls for approximately one half hour during both trials.

Results

Bichamber (Granger Cell) Experiments

In the experiments using the bichamber testing cells and a mixture of succinic acid and NaN0₂ to release NO*, a behavior-altering effect was observed. All three concentrations of NO* caused the cyprids to become immobile and sink to the bottom of the chamber. The onset time of this effect varied from 5 to 40 minutes until full effect was observed.

Tissue Culture Cell Experiments In these experiments, using both DEA/NO and SNP, similar results to those of the earlier experiments were observed. The onset time for cyprid immobility to occur was 5 min to 15 min. Effective concentrations for both compounds ranged from 10³ M to 10^'5 M, with time required for immobilization decreasing as compound concentration decreased. At these concentrations, greater than 90% of the cyprids were affected, and sank to the bottom of the test chambers. Some individuals retracted their swimming appendages and antennuies, and remained completely motionless. Others would occasionally "kick" or move their antennuies, but these individuals did not swim normally through the water column. At concentrations between 10^s M and 10^'10 M the effect of NO* became difficult to measure. There was some apparent slowing of movement, but no consistent cessation of swimming activity was observed. The time from addition of NO* releasing compounds until the onset of behavioral effect was longer than that of the higher concentration (20 to 30 min) .

On the video tapes, it was easy to see when the cyprids were active, but their appendages and antennuies were not discernable. The use of a microscope was necessary to see these details.

Results with S-nitrosothiols (RSNOs) revealed similar results with some variation, but all caused effects within the range of 10^~3 - 10^"6 M. The effects of nitroso- tryptophan were distinct from other compounds. The effects were more potent and more temporary. Organisms slowed with very jerky movements and then came to a standstill. After a few minutes the organisms began to move again.

After the addition of HbA to the test cells, the cyprids would quickly become active again. Active cyprids dart about the water column in bursts of movement interspersed with brief periods of immobility. In these tests recovery to fully active condition took from approximately one to one and one half (1.5) minutes for the earliest individuals, to approximately five (5) minutes for the slowest individuals. In the trials with NO* concentrations lower than 10^s M, the addition of HbA increased activity, suggesting that the NO* did have some behavior-altering effect when administered at this concentration.

Ultraviolet Soectrophotometry

UV spectra of samples of DEA/NO in 100 kD CH₂0 were obtained. In separate trials, 15 μl and 30 μl of 10'² M DEA/NO in 10 mM NaOH were added to cuvettes containing I ml of 100 kD CH₂0 and mixed. Maximum absorbance was in the area of lambda 244 to 246 as was expected (Maragos et al . , J. Med. Chem. 34:3242-3247 (1991)) . Absorbance increased with increased concentration of DEA/NO (Figure 1) .

Spectra of the changes in the structure of hemoglobin upon introduction to a cuvette in which DEA/NO had been reacting in seawater were also collected. In a cuvette, 15 μl of 10^-2 M DEA/NO in 10 mM NaOH was added to 1.5 ml 100 kD CH₂0. Approximately 30 min later, 0.5 ml of that solution was removed and 0.5 ml 60 μM HbA was added. A progression of spectra showing the conversion of HbA to Met-Hemoglobin over a 55 min period were collected (Figure 2) . oxyHbA converts to Met-Hb in the presence of NO*. The assumption that the normal reaction progression of NO* (Pogrebnaya et al . , Chem. Abstr. 78:285, α76393k (1972)) occurs in seawater explains the appearance of spectra that belong to Met-Hb and not HB-NO. Oxygen Probes

SNP was added to bottles filled with aerated 24° C IOOkD CH₂0, salinity 35 ppt. The concentrations of SNP, and presumably NO*, in these bottles were 1 mM and 3.5 mM respectively in two separate trials. P0₂ was monitored for the test bottles and for simultaneous controls for approximately one half hour during both trials. In none of these trials did the P0₂ in the test bottles change enough to be discerned from background fluctuation of the meters in the control bottles.

Discussion

Nitric oxide (NO*) or alternative nitrogen monoxides (i.e. NO⁺ or NO^") have a behavior-altering effect on cyprid stage barnacle larvae. This effect can be described as anesthetic or narcotic. The effect is non-toxic, and can be reversed by the removal of NO* from the environment that the larvae are in, or by removal of the larvae from the source of the NO*.

By using new methods to produce NO* in the presence of barnacle larvae this study has demonstrated that NO* has a physiological effect upon marine invertebrate larvae. The data from the oxygen probe trials, and the assumption of a third order reaction required to convert NO* to N0₂ ^", - (Pogrebnaya et al., Chem. Abstr. 78:285, α76393k (1972)) suggest that NO* in seawater is not deoxygenating the chambers that the cyprids are in. Thus, the observed behavioral effect is not attributable to external suffocation or exhaustion Of 0₂ in the testing environment.

The exact mechanism of action by which NO* affects the cyprids is not clear. While the invention is not predicated upon or limited to a particular theory, it is possible that NO* is binding to a receptor site, and that it may act as a blocker of transmission between neurons, either in the nerve center or between sensory and motor neutrons. Being readily diffusible, NO* may be affecting respiration by competing with oxygen in areas of 0₂ diffusion from the water. For both of these mechanisms, increasing in the concentration of NO* in the environment would result in a more rapid onset of NO*-mediated symptoms.

As the above experimental results demonstrate, NO*- releasing compounds are effective in immobilizing barnacles. Therefore, these compounds have significant potential for use as non-toxic antifouling compounds, by providing a clean alternative to the highly toxic chemicals that are commonly used today.

Having now fully described this invention, it will be appreciated by those skilled in the art that the same can be performed within a wide range of equivalent parameters, concentrations and conditions without departing from the spirit and scope of the invention and without undue experimentation.

While this invention has been described in connection with specific embodiments thereof, it will be understood that it is capable of further modifications. This application is intended to cover any variations, uses, or adoptions of the inventions following, in general, the principles of the invention and including such departures from the present disclosure as come within known or customary practice in the art to which the invention pertains and as may be applied to the essential features hereinbefore set forth as follows in the scope of the appended claims.

Claims

What Iff πaimed Is;

1. A method for preventing aquatic surface-fouling organisms from attaching to an aqueous surface comprising:

treating said aquatic surface with a composition comprising at least one nitric oxide-releasing compound in an amount sufficent to prevent said aquatic surface-fouling organisms from attaching to said aquatic surface.

2. The method of claim 1 wherein the treating is effected by exposing said aquatic surface to gaseous nitric oxide or a compound which delivers nitric oxide.

3. The method of claim 2 wherein the compound is selected from the group consisting of S-nitrosothiols, compounds that include at least one -O-NO group, N- nitrosoamines, C-nitroso compounds including at least one - C-NO group, nitrates having at least one -0-N0₂ group, nitroso-metal compounds, N-oxo-N-nitrosoamines, and thionitrates.

4. The method of claim 3 wherein the s-nitrosothiol is selected from the group consisting of those having the structures:

(i) CH₃(CH₂)_xSNO

wherein x equals 2 to 20;

(ii) HS(CH₂)_xSNO

wherein x equals 2 to 20; and

(iii) ONX(CH₂)-Y wherein x equals 2 to 20 and Y is selected from the group consisting of fluoro, C,-C₆ alkoxy, cyano, carboxamido, C₃-C_j cycloalkyl, aralkoxy, C₂-C₆ alkysulfinyl, arylthio, C_j- alkylamino, C₂-C₁₃ N,N-dialkylcarbamoyl, amino, hydroxyl, carboxyl, hydrogen, nitro and aryl; wherein aryl includes benzyl, naphthyl and anthracenyl groups.

5. The method of claim l wherein the nitric oxide releasing compound comprises a compound which generates nitric oxide by its own decomposition.

6. The method of claim l wherein the nitric oxide- releasing compound is selected from the group consisting of lipophilic thionitrates, lipophilic organic nitrates and lipophilic nitro(so) aromatic compounds.

7. The method of claim 6 wherein the lipophilic thionitrite is selected from the group consisting of propylthionitrite, butylthionitrite, pentylthionitrite, dodecathionitrite, and benzenylthionitrite.

8. The method of claim 6 wherein the lipophilic organic nitrate and nitro(so) aromatic compounds include compounds which contain nitric oxide in combination with carbon, sulfur, nitrogen, oxygen, a redox metal or an amino acid.

9. The method of claim 8 wherein the nitro(so) aromatic compound is a nitrosated indole.

10. The method of claim 9 wherein the nitrosated indole is a nitrosated tryptophan or derivative thereof.

11. The method of claim 1 wherein said treating comprises coating onto such a surface a composition containing an antifouling effective amount of one or more lipophilic compounds selected from the group consisting of: (i) a compound having the fomula

(X_l. ( O) _D. wherein X is selected from the group consisting of 0, C, N, S, N0₂, N0₃, Al, Fe, Cr, Cu, an amine, an amino acid and a peptide comprising at least two amino acids linked by peptide bonds,- m is 1 or 2; and n is any integer; (ii) a compound having the formula X₂R,S-NO wherein X₂ is selected from the group consisting of H, HS and NO; and R, is selected from the group consisting of H, C₂-C₂₀ alkyl, C₃-Cβ aryl, Cj-Cβ cycloalkyl and benzyl; and (Hi) a compound having the formula

ONSRjY wherein Rj is a C₂-C₂₀ alkyl and Y is selected from the group consisting of fluoro, - alkoxy, cyano, carboximido, C₃-Cβ cycloalkyl, arylalkoxy, C₂-C₆ alkylsulfinyl, arylthio, C,-Cβ alkylamino, j-C_u dialkylamino, hydroxy, carbamoyl, C_t- N-alkylcarbamoyl, C₂-C,₅ N,N-dialkylcarbamoyl, amino, hydroxyl, carboxyl, hydrogen, nitro, and aryl.

12. A composition for preventing aquatic surface- fouling organisms from attaching to an aqueous surface, comprising an antifouling effective amount of at least one nitric oxide-releasing compound in combination with a coating substance.

13. The composition of claim 12 wherein said coating substance contains a solvent and a binder.

14. The composition of claim 12 wherein the nitric oxide-releasing compound comprises a compound which generates nitric oxide through its chemical interaction with another compound.

15. The composition of claim 14 wherein said nitric oxide-releasing compound is a nitrite-reducing compound.

16. The composition of claim 14 wherein said nitrite- reducing compound is a redox dye selected from the group consisting of methylene blue and indium red.

17. The composition of claim 14 wherein said nitrite- reducing compound is nitrite reductase.

18. The method of claim 17 wherein said indol is tryptophan.

19. The composition of claim 17 which further comprises a reducing agent.

20. The method of claim 1 wherein nitric oxide is used as a gas.

21. The method of claim 1 which comprises cleaving NO* from nitrites by photolysis.