WO2000016632A2

WO2000016632A2 - Environmentally benign crop protection agents

Info

Publication number: WO2000016632A2
Application number: PCT/US1999/022227
Authority: WO
Inventors: Randall S. Alberte; Richard C. Zimmerman
Original assignee: Phycogen, Inc.
Priority date: 1998-09-23
Filing date: 1999-09-23
Publication date: 2000-03-30
Also published as: WO2000016764A3; CA2345231C; US7108861B1; BR9913912A; AU6264099A; EP1115285A1; BR9914499A; WO2000016623A1; IL142131A0; JP2002526053A; EP1115289A2; CA2345233A1; AU6261499A; BR9914036A; AU6264399A; EP1115394A2; IL142130A0; IL142132A; IL142129A0; IL142132A0

Abstract

Disclosed are environmentally safe crop protection agents that interfere with the attachment of a broad range of plant pathogens to plant cells surfaces. The crop protection agents contain a compound represented by general structure (1), wherein X represents -0H, -0(aryl), -0(acyl), -0(sulfonyl), -CN, F, C1, or Br; Y represents O, S, Se, or NR; Z represents optionally substituted alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or-(CH2)m-R80; R represents independently for each occurence hydrogen, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2)m-R80; R80 represents independently for each occurence aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl; and m is an integer in the range 0 to 8 inclusive.

Description

Environmentally Benign Crop Protection Agents

Background of the Invention

There are more than 50,000 species of fungi. Fewer than 100 are pathogenic to humans. However, more than 10,000 fungi cause disease in plants. Fungal pathogens are extremely costly pests, affecting a broad range of crop plants. It is estimated that anywhere between 20-30% of crop production is lost to fungal pathogens alone worldwide with the greatest losses outside the United States (FAO Report). Losses from fungal pathogens are realized in every stage of crop production ranging from those fungi that attack seeds or germinating seed to those that attack the stems of seedlings, the roots, stems, vascular system, fruits and leaves of mature crops.

There are presently a number of widely used fungicide products on the market, including triazoles, anilides, dithiocarbamates, and benzimoidazoles. Total worldwide sales of fungicides in 1995 approached S60 billion with greater than 80% of the market being non- U.S.

Essentially all of the non-biological fungicides are toxic chemicals to the fungi, as well as to the environment when they enter drinking water supplies and natural waters and can be toxic to animals and humans through accidental contact. In addition, because many of the chemicals generate resistance in the target organisms. Biological control is based on inhibition by some microorganisms on the growth and action of pathogenic fungi, which cause rotting (See for example, U.S. Patent No. 4,975,277 and EP 0 781 843 Al). However, there can be great variations in the antifungic activity of different isolates or strains of the same species. In addition, the effectiveness of various strains may be diminished during storage and harvesting.

Summary of the Invention

In one aspect, the instant invention features crop protection compounds having the general structure 1 :

1 wherein

X represents -OH, -O(aryl), -O(acyl), -O(sulfonyl), -CN, F, Cl, or Br;

Y represents O, S, Se, or NR; Z represents optionally substituted alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2)_m-R8o;

R represents independently for each occurrence hydrogen, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2)_m-R-8o;

R₈o represents independently for each occurrence aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl; and m is an integer in the range 0 to 8 inclusive. Other preferred compounds are salts of the compounds in structure 1.

The instant claimed compounds interfere with the attachment of organisms to surfaces, thereby having broad applicability in effectively inhibiting the attachment of a variety of organisms. In addition, the compounds are relatively safe for wide-spread environmental use, as they naturally degrade into carbon dioxide and water, or simple organic acids.

In addition, certain preferred compounds have a relatively short half-life after release, rendering them particularly well-suited for widespread environmental use. Yet other preferred compounds can be readily synthesized. Particularly preferred compounds include: p-iso-butylphenyl chlorosulfate, p-tert- butylphenyl chlorosulfate, p-tert-amylphenyl chlorosulfate, p-tert-cumylphenyl chlorosulfate, 4-pentylphenyl chlorosulfate, 4-( 1 -methylheptyl)phenyl chlorosulfate, methyl chlorosulfate, octyl chlorosulfate, bisphenyl diacid sulfate, p-iso-butylphenyl sulfate, p-tert-butylphenyl sulfate, p-tert-amylphenyl sulfate, p-tert-cumylphenyl sulfate, 4-pentylphenyl sulfate, 4-(l- methylheptyl)phenyl sulfate, methyl sulfate, and octyl sulfate, p-sulfoxy cinnamic acid, p-sulfoxy ferulic- acid, m,p-disulfoxy caffeic acid, benzoic acid sulfate, vanillic acid sulfate, gentissic acid sulfate, gallic acid sulfate. protochateuic acid and zosteric acid and salts thereof.

Other features and advantages of the invention will be apparent from the following detailed description and claims.

Brief Description of the Drawings

Figure 1 is a bar graph plotting the average number of anthracnose lesions on the leaf and petioles of detached Chandler strawberries when treated with a range of concentrations of the sodium salt of zosteric acid. Anthracnose disease control efficacy of 73.6% was achieved with a 2% (w/v) solution of zosteric acid in phosphate buffer containing 0/1% Tween™ 20. Colletotrichum fragariae isolate CF63conidia (1.5 X10³ spores/mL) were used to induce disease.

Figure 2 is a graph showing the average disease severity and phytotoxicity ratings of Chandler strawberry whole plants when treated with a range of concentrations of the sodium salt of zosteric acid and an analog. Colletotrichum fragariae isolate isolate CF63conidia (1.5 X10⁶ spores/mL) were used to induce disease.

Figure 3 is a graph showing the result of microscopic studies indication that the sodium salt of zosteric acid and it TPPC analog were the most effective compounds at inhibiting spore attachment (C acutatum) to a glass surface. Figure 4 is a graph showing the results of microscopic evaluation of C. acutatum spores. As can be seen in the graph. 1% zosteric acid prevented 99% of the spores from germinating during the 24 hour incubation.

Figure 5 is a diagrammatic representation of the results of marine algae attachment assays measuring the abundance of algal biofilm development on the inert coating RTV-1 1 compared to biofilm development on RTV-1 1 with octyl sulfate incoφorated into the coating. Relative algal abundance represents the attachment of the marine algae to the tested surface. Error bars indicate 1 standard error of the mean (n = 3) for each treatment. The ratio of the optical densities measured at wavelengths 680 nm and 750 nm (A₆₈o/A ₅₀) at time O was used as a baseline reference for all samples. Figure 6 is a diagrammatic representation of the results of bacterial attachment assays performed with the maπne bacteπum Oceanosprύlum cultured m the presence and absence of either zosteπc acid or methyl sulfate

Figure 7 is a diagrammatic representation of the results of bacteπal attachment assays performed with the maπne bacteπum Oceanospr ilium cultured m the presence and absence of either zosteric acid or octyl sulfate

Figure 8 is a diagrammatic representation of the results of bacterial attachment assays performed with the bacterium Ahβromonas atlantica, performed m the presence and absence of either, zosteπc acid, octyl sulfate, or methyl sulfate Figure 9 is a diagrammatic representation of the results of fungal attachment and growth assays using the fungus Am eobasidium pullulans (a shower fungus that stams grout) grown in the presence and absence of zosteric acid, w here fungal abundance represents the attachment of A pullulans to the exposed surface

Figure 10 is a diagrammatic representation of the results of agglutination of the bacteπum Shewanella putrefaciens induced by the presence of increased amounts of zosteπc acid, measured by the percent transmission (%T) of the liquid cultures at wavelength 600 nm Agglutination is indicated by the concentration-dependent increase in %T of cultures grown in the presence of zosteric acid In this case, relatively high levels of %T exhibited by the zosteπc acid-exposed cultures do not reflect differences m growth, as counts of viable colony forming units exhibited no difference in cell density at eight hours

Detailed Description of the Invention

General The instant invention is based, at least part, on the finding described m detail m the following Example 1, that compounds of the invention inhibit attachment of parasitic fungi spores to plants, as well as hyphal production from previously attached spores Even after prolonged exposure, the presence of the compounds of the invention on the plants did not result in any toxic or growth inhibitory effect In addition, greenhouse studies revealed that the compound effectively controlled the disease on plants exposure to abnormal high spore pressures Again, no detectable phytotoxicity was observed In evaluations assessing fungal spore attachment on man-made surfaces, it was determined that a compound of the invention provided nearly 100% inhibition of attachment of two species of fungal pathogens. If fungal spores were allowed to attach, the compound provided 100%) inhibition of spore germination.

By blocking spore attachment, an initial step in the infection process, the compounds of the invention provide a highly effective antifungal agent. In addition, since essentially all fungal plant pathogens use spores to recognize the host plant, attach, germinate, penetrate the host plant tissue and proliferate hyphae that allows the fungus access to the plant's nutrients for growth and reproduction, the compounds are broad-based antifungal agents. In addition a series of investigations on several species of bacteria, microalgae, macroalgal spores and invertebrates has confirmed that the inhibitory mode-of- action is through a non-toxic means (Zimmerman et al., (1995) US Patent 5,384,176; Zimmerman et al., (1997) US Patent 5,607,741 ; Todd et al., Phytocheimsti 34: 401-404 (1993); Sundberg et al.. Naval Research Reviews (1997) 4: 51-59).

Although the exact mechanism of action is not known, studies indicate that the mechanism involves binding of the compounds to a sulfate binding moiety on cells. The compound or a functional fragment thereof, must then be released for the inhibitory effect. If permanently tethered to a surface, the compounds and sulfate groups tend to promote rather than inhibit the attachment and growth of organisms on a surface.

Definitions

For convenience, certain terms employed in the specification, examples, and appended claims are described below.

The term "acylamino" is aπ-recognized and refers to a moiety that can be represented by the general formula:

wherein Rq is as defined above, and R'ι i represents a hydrogen, an alkyl, an alkenyl or -(CH2)_m- -8' where m and Rg are as defined above. The terms "alkenyl" and "alkynyl" refer to unsaturated aliphatic groups analogous in length and possible substitution to the alkyls described above, but that comprise a double or triple bond, respectively.

The terms "alkoxyl" or "alkoxy" as used herein refers to an alkyl group, as defined above, having an oxygen radical attached thereto. Representative alkoxyl groups include methoxy, ethoxy, propyloxy, tert-butoxy and the like. An "ether" is two hydrocarbons covalently linked by an oxygen. Accordingly, the substituent of an alkyl that renders that alkyl an ether is or resembles an alkoxyl, such as can be represented by one of -O-alkyl, -O- alkenyl, -O-alkynyl, -0-(CH₂)_m-R₈, where m and Rg are described above.

The term "alkyl" refers to the radical of saturated aliphatic groups, including straight- chain alkyl groups, branched-chain alkyl groups, cycloalkyl (alicyclic) groups, alkyl substituted cycloalkyl groups, and cycloalkyl substituted alkyl groups. In preferred embodiments, a straight chain or branched chain alkyl has 30 or fewer carbon atoms in its backbone (e.g., C1 -C₃₀ for straight chain, C₃-C₃₀ for branched chain), and more preferably 20 or fewer. Likewise, preferred cycloalkyls have from 3-10 carbon atoms in their ring structure, and more preferably have 5, 6 or 7 carbons in the ring structure.

Moreover, the term "alkyl" (or "lower alkyl") as used throughout the specification and claims is intended to include both "unsubstituted alkyls" and "substituted alkyls", the latter of which refers to alkyl moieties having substituents replacing a hydrogen on one or more carbons of the hydrocarbon backbone. Such substituents can include, for example, a halogen, a hydroxyl, a carbonyl (such as a carboxyl, an ester, a formyl, or a ketone), a thiocarbonyl (such as a thioester, a thioacetate, or a thioformate), an alkoxyl, a phosphoryl, a phosphonate, a phosphinate, an amino, an amido, an amidine, an imine, a cyano, a nitro, an azido, a sulfhydryl, an alkylthio, a sulfate, a sulfonate, a sulfamoyl, a sulfonamido, a sulfonyl, a heterocyclyl, an aralkyl, or an aromatic or heteroaromatic moiety. It will be understood by those skilled in the art that the moieties substituted on the hydrocarbon chain can themselves be substituted, if appropriate. For instance, the substituents of a substituted alkyl may include substituted and unsubstituted forms of amino, azido, imino, amido, phosphoryl (including phosphonate and phosphinate), sulfonyl (including sulfate, sulfonamido, sulfamoyl and sulfonate), and silyl groups, as well as ethers, alkylthios, carbonyls (including ketones, aldehydes, carboxylates, and esters), -CF₃, -CN and the like. Exemplary substituted alkyls are described below. Cycloalkyls can be further substituted with alkyls, alkenyls, alkoxys, alkylthios, aminoalkyls, carbonyl-substituted alkyls, -CF3, -CN, and the like.

The term "alkylthio" refers to an alkyl group, as defined above, having a sulfur radical attached thereto. In preferred embodiments, the "alkylthio" moiety is represented by one of - S-alkyl, -S-alkenyl, -S-alkynyl, and -S-(CH2)_m-R8, wherein m and R₈ are defined above. Representative alkylthio groups include methylthio, ethylthio, and the like.

The term "amido" is art recognized as an amino-substituted carbonyl and includes a moiety that can be represented by the general formula:

/

*ιo wherein R9, RI Q are as defined above. Preferred embodiments of the amide will not include imides which may be unstable.

The terms "amine" and "amino" are art recognized and refer to both unsubstituted and substituted amines, e.g., a moiety that can be represented by the general formula:

^R'ιo

/ ,κ10 l +

— N — N— K. or 10 ^l 9

^9 wherein R₉, R₁₀ and R' ₁₀ each independently represent a hydrogen, an alkyl, an alkenyl, -(CH2)_m-Rg, or R₉ and R₁₀ taken together with the N atom to which they are attached complete a heterocycle having from 4 to 8 atoms in the ring structure; Rg represents an aryl, a cycloalkyl, a cycloalkenyl, a heterocycle or a polycycle; and m is zero or an integer in the range of 1 to 8. In preferred embodiments, only one of R₉ or Rι ₀ can be a carbonyl, e.g., R₉, RI _Q and the nitrogen together do not form an imide. In even more preferred embodiments, R₉ and Ri o (and optionally R' 10) each independently represent a hydrogen, an alkyl, an alkenyl, or -(CH2)_m-Rg. Thus, the term "alkylamine" as used herein means an amine group, as defined above, having a substituted or unsubstituted alkyl attached thereto, i.e., at least one of R₉ and Ri O is an alkyl group.

SUBSTTTUTE SHEET (RULE 26) An "aprotic solvent" means a non-nucleophilic solvent having a boiling point range above ambient temperature, preferably from about 25°C to about 190°C, more preferably from about 80°C to about 160°C, most preferably from about 80°C to 150°C, at atmospheric pressure. Examples of such solvents are acetonitrile, toluene, DMF, diglyme, THF or DMSO. The term "aryl" as used herein includes 5-, 6- and 7-membered single-ring aromatic groups that may include from zero to four heteroatoms, for example, benzene, pyrrole, furan, thiophene, imidazole, oxazole, thiazole, triazole, pyrazole, pyridine, pyrazine, pyridazine and pyrimidine, and the like. Those aryl groups having heteroatoms in the ring structure may also be referred to as "aryl heterocycles" or "heteroaromatics". The aromatic ring can be substituted at one or more ring positions with such substituents as described above, for example, halogen, azide, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, sulfonamido. ketone, aldehyde, ester, heterocyclyl, aromatic or heteroaromatic moieties, -CF3, -CN. or the like. The term "aryl" also includes polycyclic ring systems having two or more rings in which two or more carbons are common to two adjoining rings (the rings are "fused") wherein at least one of the rings is aromatic, e.g., the other rings can be cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls.

The term "carbocycle", as used herein, refers to an aromatic or non-aromatic ring in which each atom of the ring is carbon. The term "carbonyl" is art recognized and includes such moieties as can be represented by the general formula:

wherein X is a bond or represents an oxygen or a sulfur, and R_j 1 represents a hydrogen, an alkyl, an alkenyl, -(CH2)_m-Rg or a pharmaceutically acceptable salt, R'i _j represents a hydrogen, an alkyl, an alkenyl or -CCH2)_m-R8₅ where m and R₈ are as defined above. Where X is an oxygen and R_j 1 or R'ι 1 is not hydrogen, the formula represents an "ester". Where X is an oxygen, and R1 _j is as defined above, the moiety is referred to herein as a carboxyl group, and particularly when Ri _j is a hydrogen, the formula represents a "carboxylic acid". Where X is an oxygen, and R'ι 1 is hydrogen, the formula represents a "formate". In general, where the oxygen atom of the above formula is replaced by sulfur, the formula represents a "thiolcarbonyl" group. Where X is a sulfur and R1 1 or R'1 1 is not hydrogen, the formula represents a "thiolester." Where X is a sulfur and Ri i is hydrogen, the formula represents a "thiolcarboxylic acid." Where X is a sulfur and R1 1 ' is hydrogen, the formula represents a "thiolformate." On the other hand, where X is a bond, and R_] i is not hydrogen, the above formula represents a "ketone" group. Where X is a bond, and R1 1 is hydrogen, the above formula represents an "aldehyde" group.

A "coating" refers to any temporary, semipermanent or permanent layer or covering. A coating can be a gas, vapor, liquid, paste, semi-solid or solid. In addition a coating can be applied as a liquid and solidify into a hard coating. Examples of coatings include sprays, liquids, gases, vapors, gels, powders, waters, wetters, detergents, oils.

"Contacting" as used herein refers to any means for providing the compounds of the invention to a plant of plant component. Contacting can include spraying, wetting, immersing, dipping, painting, bonding, adhering or otherwise providing a surface with a compound of the invention. The phrase "effective amount" refers to an amount of the disclosed antifouling compounds that reduces the number of organisms that attach to a defined surface (cells/mm²) of a plant or plant component relative to the number that attach to an untreated surface. Particularly preferred are amounts that reduce the number of organisms that attach to the surface by a factor of at least 2. Even more preferred are amounts that reduce the surface attachment of organisms by a factor of 4, more preferably by a factor of 6, 8, 10 or more. Especially preferred is that amount, which will completely inhibit fungal growth (i.e. inhibit the spread of fungal mycelia) without causing necrotic tissue to the plant.

The phrase "electron-withdrawing group" is recognized in the art, and denotes the tendency of a substituent to attract valence electrons from neighboring atoms, i.e., the substituent is electronegative with respect to neighboring atoms. A quantification of the level of electron-withdrawing capability is given by the Hammett sigma (σ) constant. This well known constant is described in many references, for instance, J. March, Advanced Organic Chemistry. McGraw Hill Book Company, New York. (1977 edition) pp. 251-259. The Hammett constant values are generally negative for electron donating groups (σ[P] = - 0.66 for b) and positive for electron withdrawing groups (σ[P] = 0.78 for a nitro group), σ[P] indicating para substitution. Exemplary electron-withdrawing groups include nitro, ketone,

SUBSTTTUTE SHEET (RULE 26) aldehyde, sulfonyl, trifluoromethyl, -CN, chloride, and the like. Exemplary electron-donating groups include amino, methoxy, and the like.

The term "half-life" refers to the amount of time required for half of a compound to be eliminated or degraded by natural processes. Preferred compounds have a half-life of less than one year. Particularly preferred are half-lives in the range of 1 to 60 days in the environment.

The term "heteroatom" as used herein means an atom of any element other than carbon or hydrogen. Preferred heteroatoms are nitrogen, oxygen, sulfur and phosphorous.

The terms "heterocyclyl" or "heterocyclic group" refer to 3- to 10-membered ring structures, more preferably 3- to 7-membered rings, whose ring structures include one to four heteroatoms. Heterocycles can also be polycycles. Heterocyclyl groups include, for example, thiophene, thianthrene, furan, pyran, isobenzofuran, chromene, xanthene, phenoxathiin, pyrrole, imidazole, pyrazole, isothiazole, isoxazole, pyridine, pyrazine, pyrimidine, pyridazine, indolizine, isoindole, indole, indazole, purine, quinolizine, isoquinoline, quinoline, phthalazine, naphthyridine, quinoxaline, quinazoline, cinnoline, pteridine, carbazole, carboline, phenanthridine, acridine, perimidine, phenanthroline, phenazine, phenarsazine, phenothiazine, furazan, phenoxazine, pyrrolidine, oxolane, thiolane, oxazole, piperidine, piperazine, morpholine, lactones, lactams such as azetidinones and pyrrolidinones, sultams, sultones, and the like. The heterocyclic ring can be substituted at one or more positions with such substituents as described above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, amino, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like.

Unless the number of carbons is otherwise specified, "lower alkyl" as used herein means an alkyl group, as defined above, but having from one to ten carbons, more preferably from one to six carbon atoms in its backbone structure. Likewise, "lower alkenyl" and "lower alkynyl" have similar chain lengths. Preferred alkyl groups are lower alkyls. In preferred embodiments, a substituent designated herein as alkyl is a lower alkyl.

As used herein, the term "nitro" means -NO2; the term "halogen" designates -F, -Cl, - Br or -I; the term "sulfhydryl" means -SH; the term "hydroxyl" means -OH; and the term "sulfonyl" means -SO_?-. The terms ortho, meta and para apply to 1,2-, 1,3- and 1,4-disubstituted benzenes, respectively. For example, the names 1,2-dimethylbenzene and ort/zo-dimethylbenzene are synonymous.

A "phosphoryl" can in general be represented by the formula:

wherein Q\ represented S or O, and R46 represents hydrogen, a lower alkyl or an aryl. When used to substitute, e.g., an alkyl, the phosphoryl group of the phosphorylalkyl can be represented by the general formula:

wherein Q\ represented S or O, and each R46 independently represents hydrogen, a lower alkyl or an aryl, Q2 represents O, S or N. When Q1 is an S, the phosphoryl moiety is a "phosphorothioate".

"Plant" as used herein refers to any member of the plant kingdom, at any stage of its life cycle , including seeds, germinated seeds, seedlings, or mature plants. "Plant cell" refers to a cell from a plant or plant component.

"Plant component" refers to a portion or part of a plant. Examples include: seeds, roots, stems, vascular systems, fruits (further including pip fruits (e.g. apples, pears, quinces)), citrus fruits (oranges, lemons, limes, grapefruits, mandarins, nectarines), stone fruits (peaches apricots, plums, cherrries, avocados, grapes), berries (strawberries, blueberries, raspberries, blackberries)), leaves, grains and vegetables.

A "plant pathogen" refers to an organism (bacteria, virus, protist, algae or fungi) that infects plants of plant components. Examples include molds, fungi and rot that typical use spores to infect plants or plant components (e.g fruits, vegetables, grains, stems, roots). Spores must recognize the host, attach, germinate, penetrate host tissues, and proliferate hyphae that will allow the fungus access to nutrients for growth and reproduction. Examples

1 1

SUBSTTTUTE SHEET (RULE 26) include Botr\>tιs sp (B cinei ea), Pemcillium sp ( P expansion P italicum, P digitalum), Rluzopus sp (R sulonifet . R mgricans), Alternaria sp (A alternata A solani), Diploidia sp (Dtploidia natalensesi Monilinta sp (M fructicola), Pseudomonas sp (P cepacia ) Xanthomonas sp Erwima sp and Corvnebacterium Cladosportum sp (C fulva), Phytophtora sp (P infestans) Colletotricum spp (C coccoides C fragariae C gloesponoides), Fusarium spp (F h copersici), Verticillium spp (V alboatrum V dahhae), Unicula spp (U necator), Plasmopai a spp (P viticola). Guignardia spp (G bidwellu), Cercospora spp (C ai achidtcola) Scelrotinia spp (S scerotwrum), Puccinia spp (P arachidis), Aspergillus spp (A flavus) Ventuna spp (V inaequahs,) Podosphaera spp (P leucotncha) , Pγthiun spp , Sphaerotheca (S macularis) and Bacillus spp (B subitlts)

A "polar, aprotic solvent" means a polar solvent as defined above which has no available hydrogens to exchange w ith the compounds of this invention during reaction, for example DMF, acetomtπle, diglyme DMSO, or THF

A "polar solvent" means a solvent which has a dielectric constant (ε) of 2 9 or greater, such as DMF, THF, ethylene glycol dimethyl ether (DME), DMSO, acetone, acetomtπle, methanol, ethanol, isopropanol, n-propanol. t-butanol or 2-methoxyethyl ether Preferred solvents are DMF, DME, NMP, and acetonitπle

The terms "polycyclyl" or "polycychc group" refer to two or more πngs (e g , cycloalkyls, cycloalkenyls, cycloalkynyls, aryls and/or heterocyclyls) in which two or more carbons are common to two adjoining πngs, e g , the πngs are "fused rings" Rings that are joined through non-adjacent atoms are termed "bπdged" πngs Each of the rings of the polycycle can be substituted with such substituents as descπbed above, as for example, halogen, alkyl, aralkyl, alkenyl, alkynyl, cycloalkyl, hydroxyl, ammo, nitro, sulfhydryl, imino, amido, phosphonate, phosphinate, carbonyl, carboxyl, silyl, ether, alkylthio, sulfonyl, ketone, aldehyde, ester, a heterocyclyl, an aromatic or heteroaromatic moiety, -CF3, -CN, or the like

The phrase "protecting group" as used herein means temporary modifications of a potentially reactive functional group which protect it from undesired chemical transformations Examples of such protecting groups include esters of carboxy c acids, silyl ethers of alcohols, and acetals and ketals of aldehydes and ketones, respectively The field of protecting group chemistry has been reviewed (Greene, T W , Wuts, P G M Protective Groups in Organic Synthesis, 2^nd ed Wiley New York, 1991) "Release rate" or "flux" refers to the rate of delivery or diffusion of a compound to and ultimately from a surface. The release rate may be constant or sustained over a period of time or may be variable. However, constant, controlled or sustained release rates are generally preferred. Steady state or sustained release may be effected by use of a reservoir membrane (i.e. a two layer coating in which one layer contains the active agent and the other creates a membrane through which the active agent can be released). The active agent could alternatively be microencapsulated within any of a variety of matrices for sustained release. Preferred release rates are in the range of about 100 to about 200 μgcm^"2d^"' is useful for temporary uses of uses that require reapplication. For more sustained applications, preferred release rates are in the range of about 1 to about 100 μgcirf 7 d^" 1 ., more preferably in the range of about 1-50 and still more preferably in the range of about 1-25 or better yet, 1-15.

The term "soluble" refers to the ability to be loosened or dissolved. A "solubilized" compound has been loosened or dissolved (e.g. into a fluid).

As used herein, the term "substituted" is contemplated to include all permissible substituents of organic compounds. In a broad aspect, the permissible substituents include acyclic and cyclic, branched and unbranched, carbocyclic and heterocyclic, aromatic and nonaromatic substituents of organic compounds. Illustrative substituents include, for example, those described hereinabove. The permissible substituents can be one or more and the same or different for appropriate organic compounds. For purposes of this invention, the heteroatoms such as nitrogen may have hydrogen substituents and/or any permissible substituents of organic compounds described herein which satisfy the valencies of the heteroatoms. This invention is not intended to be limited in any manner by the permissible substituents of organic compounds.

It will be understood that "substitution" or "substituted with" includes the implicit proviso that such substitution is in accordance with permitted valence of the substituted atom and the substituent, and that the substitution results in a stable compound, e.g., which does not spontaneously undergo transformation such as by rearrangement, cyclization, elimination, etc.

The term "sulfate" is art recognized and includes a moiety that can be represented by the general formula:

0

I I

— 0—5-OK

I I

0 in which R41 is as defined above.

A "sulfate binding moiety" refers to a moiety that is capable of binding or otherwise associating with a sulfate or sulfonate group.

The term "sulfonate" is art recognized and includes a moiety that can be represented by the general formula:

0 I I — S-OR,,, I I

0

in which R41 is an electron pair, hydrogen, alkyl, cycloalkyl, or aryl.

The terms "sulfoxido" or "sulfinyl", as used herein, refers to a moiety that can be represented by the general formula:

0 — 5- ^ in which R is selected from the group consisting of hydrogen, alkyl, alkenyl, alkynyl, cycloalkyl, heterocyclyl, aralkyl, or aryl.

Analogous substitutions can be made to alkenyl and alkynyl groups to produce, for example, aminoalkenyls, aminoalkynyls, amidoalkenyls, amidoalkynyls, iminoalkenyls, iminoalkynyls, thioalkenyls, thioalkynyls, carbonyl-substituted alkenyls or alkynyls.

The term "surface" as used herein, refers to any interface between an object and a fluid environment, which permits at least intermittent contact between the object and the fluid environment. Fluids contacting the surfaces can be stagnant or flowing, and can flow intermittently or continuously, with laminar or turbulent or mixed rheologies. A surface upon which a biofilm can form can be dry at times with sporadic fluid contact, or can have any degree of fluid exposure including total immersion. Fluid contact with the surface can take place via aerosols or other means for air-borne fluid transmission.

"Sustained release" or "controlled release refers to a relatively constant or prolonged release of a compound of the invention from a surface. This can be accomplished through the use of diffusional systems, including reservoir devices in which a core of a compound of the invention is surrounded by a porous membrane or layer, and also matrix devices in which the compound is distributed throughout an inert matrix. Microencapsulation techniques can also be used to maintain a sustained focal release of a compound of the invention Microencapsulation may also be used for providing improved stability The encapsulated product can take the form of for example, spheres, aggregates of core material embedded in a continuum of wall mateπal, or capillary designs The core material of a microcapsule containing a compound of the m\ ention may be in the form of a liquid droplet, an emulsion, a suspension of solids, a solid particle, or a crystal The skilled artisan will be aware of numerous mateπals suitable for use as microcapsule coating materials, including, but not limited to, organic polymers, hydrocolloids, hpids, fats, carbohydrates, waxes, metals, and inorganic oxides Silicone polymers are the most preferred microcapsule coating mateπal for treatment of surfaces Microencapsulation techniques are well known in the art and are descπbed in the Encyclopedia of Polymer Science and Engineering, Vol 9, pp 724 et seq (1989) hereby incorporated by reference

The abbreviations Me, Et, Ph, Tf, Nf, Ts, Ms, and dba represent methyl, ethyl, phenyl, tπfluoromethanesulfonyl, nonafluorobutanesulfonyl, /j-toluenesulfonyl, methanesulfonyl, and dibenzylideneacetone, respectively A more comprehensive list of the abbreviations utilized by organic chemists of ordinary skill in the art appears in the first issue of each volume of the Journal of Organic Chemistry, this list is typically presented in a table entitled Standard List of Abbreviations The abbreviations contained in said list, and all abbreviations utilized by organic chemists of ordinary skill in the art are hereby incorporated by reference For purposes of this invention, the chemical elements are identified in accordance with the Peπodic Table of the Elements, CAS version, Handbook of Chemistry and Physics, 67th Ed , 1986-87, inside cover Also for purposes of this invention, the term "hydrocarbon" is contemplated to include all permissible compounds having at least one hydrogen and one carbon atom In a broad aspect, the permissible hydrocarbons include acyclic and cyclic, branched and unbranched, carbocychc and heterocyclic, aromatic and nonaromatic organic compounds which can be substituted or unsubstituted

Compositions of the Invention

In certain embodiments, the compositions of the present invention compπse an anti- fouling compound represented by general structure 1

15

SUBSTTTUTE SHEET (RULE 26)

1 wherein

X represents -OH, -O(aryl), -O(acyl), -O(sulfonyl), -CN, F, Cl, or Br;

Y represents O, S, Se, or NR; Z represents optionally substituted alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2)_m-R₈o;

R represents independently for each occurrence hydrogen, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2)_m-R o;

R o represents independently for each occurrence aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl; and m is an integer in the range 0 to 8 inclusive.

Particularly stable compounds are represented by general structure 1 and the attendant definitions, wherein X represents -OH, F, Cl, or Br. In other preferred embodiments, the compositions of the present invention comprise an anti-fouling compound represented by general structure 1 and the attendant definitions, wherein Y represents O.

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein Z represents optionally substituted alkyl, aryl, or -(CH2)_m-R₈o-

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or hetero arylphenyl . In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein Z represents methyl, octyl, 4-(2-methylpropyl)phenyl, 4-(l,l-dimethylethyl)phenyl, 4-(l,l- dimethylpropyl)phenyl, 4-pentylphenyl, 4-(l -methyl- l-phenylethyl)phenyl, or 4-(l- methylheptyl)phenyl.

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein R represents H or alkyl.

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein X represents -OH, F, Cl, or Br; and Y represents O.

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein X represents -OH or Cl; and Y represents O. In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein X represents -OH, F, Cl, or Br; and Z represents optionally substituted alkyl, aryl, or -(CH2)_m-

R₈o-

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein X represents -OH or Cl; and Z represents optionally substituted alkyl, aryl, or -(CH2)_m-R o-

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein X represents -OH, F, Cl, or Br; and Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein X represents -OH or Cl; and Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl. In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein X

17

SUBSTTTUTE SHEET (RULE 26) represents -OH, F, Cl, or Br; and Z represents methyl, octyl, 4-(2-methylpropyl)phenyl, 4- (l,l-dimethylethyl)phenyl, 4-(l,l -dimethylpropyl)phenyl, 4-pentylphenyl, 4-( 1 -methyl- 1- phenylethyl)phenyl, or 4-(l-methylheptyl)phenyl.

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein X represents -OH or Cl; and Z represents methyl, octyl, 4-(2-methylpropyl)phenyl, 4-(l,l- dimethylethyl)phenyl, 4-(l,l-dimethylpropyl)phenyl, 4-pentylphenyl, 4-( 1 -methyl- 1- phenylethyl)phenyl, or 4-(l-methylheptyl)phenyl.

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein Y represents O; and Z represents optionally substituted alkyl, aryl, or -(CH2)_m-R8o-

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein Y represents O; and Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein Y represents O; and Z represents methyl, octyl, 4-(2-methylpropyl)phenyl, 4-(l ,l- dimethylethyl)phenyl, 4-(l,l-dimethylpropyl)phenyl, 4-pentylphenyl, 4-( 1 -methyl- 1- phenylethyl)phenyl, or 4-(l -methylheptyl)phenyl.

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein X represents -OH, F, Cl, or Br; Y represents O; and Z represents optionally substituted alkyl, aryl, or -(CH2)_m-R₈o-

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein X represents -OH or Cl; Y represents O; and Z represents optionally substituted alkyl, aryl, or - (CH₂)_m-Rso.

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein X represents -OH, F, Cl, or Br; Y represents O; and Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein X represents -OH or Cl; Y represents O; and Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein X represents -OH, F, Cl, or Br; Y represents O; and Z represents methyl, octyl, 4-(2- methylpropyl)phenyl, 4-(l,l-dimethylethyl)phenyl, 4-(l,l-dimethylpropyl)phenyl, 4- pentylphenyl, 4-(l -methyl- l-phenylethyl)phenyl, or 4-(l-methylheptyl)phenyl.

In certain embodiments, the compositions of the present invention comprise an anti- fouling compound represented by general structure 1 and the attendant definitions, wherein X represents -OH or Cl; Y represents O; and Z represents methyl, octyl, 4-(2- methylpropyl)phenyl, 4-(l,l-dimethylethyl)phenyl, 4-(l,l-dimethylpropyl)phenyl, 4- pentylphenyl, 4-(l -methyl- l-phenylethyl)phenyl, or 4-(l-methylheptyl)phenyl.

One of skill in the art will recognize that the composition of the invention can be varied as required to optimize the overall chemical properties of the particular compound for specific uses, while retaining the activity. For example, the length of an alkyl chain can be extended or shortened to control the rate of dissolution of the compound from a structure or a coating. Alternatively, additional functional groups can be added to the alkyl chain to further vary the chemical nature of the molecule.

The present invention is further illustrated by the following examples, which should not be construed as limiting in any way. The contents of all cited references including literature references, issued patents and published patent applications as cited throughout this patent application are hereby expressly incoφorated by reference.

19

SUBSTTTUTE SHEET (RULE 26) Examples

Example 1 : Inhibition of Surface Attachment of C. fragariae bv Zosteric Acid

In vitro evaluation of zosteric acid for disease control efficacy indicated that the sodium salt of zosteric acid was an effective non-toxic fungal control agent for Strawberry (cv. Chandler-susceptible variety) anthracnose caused by Colletotrichum fragariae. Detached leaf and petiole assays were employed to determine an effective dose. An optimum effective concentration of about 2% (which is within a range that is commercially viable) was obtained when the agent was applied in phosphate buffer (pH 7.0) containing 0.1% Tween 20, and when plant tissues were sprayed to wetness with a spore density of 1.5 x 10⁶ spores/mL (Fig. 1).

Greenhouse evaluations of zosteric acid for efficacy in control of anthracnose crown rot of strawberry confirmed that the optimal concentration of zosteric acid was 2% for C. fragariae. It was found that the disodium salt of zosteric acid showed very low or no detectable phytotoxicity. Spore attachment assays ascertained that the most effective analogs, where the sodium salt of zosteric acid and the TPPC analog (Figure 3). The 1 % zosteric acid prevented spores from germinating during the 24 hour incubation while the lower concentrations (0.1 and 0.01%) reduced the number of spores germinating (Figure 4). Post-harvest treatment of ripe blueberries with zosteric acid sodium salt and a zosteric acid analog followed by innoculation with C. acutatum resulted in a decrease of about 50%) in the number of infected berries in the 0.67 and 2% zosteric acid analog treatment.

Example 2: Inhibition of Surface Attachment of Marine Bacteria bv Alkyl Sulfates Octyl sulfate is an alkyl sulfate surfactant with extensive industrial applications, and is manufactured by several large chemical companies. To investigate potential AF properties of the sulfate ester octyl sulfate, it w as incoφorated into an inert coating material that was then coated onto a surface to be exposed to conditions that support the formation of marine algal biofilms. Materials and Methods

A 30%(w/v) solution of octyl sulfate in water (Stepan Chemical Co.) was evaporated to dryness under a stream of room temperature air, to recover pure octyl sulfate (Fig. 5). The dry octyl sulfate was incoφorated into RTV-11 silicone polymer at a loading of 25% (wt/wt) (RTV-1 1 silicone, catalyst and primer obtained from General Electric). The mixture was applied to three glass slides previously primed with silicone primer, and allowed to cure to dryness. Three primed glass slides coated with pure RTV-1 1 served as agent-free controls. After complete drying, the absoφtion properties of each slide were measured using a Shimadzu UN-2101 spectrophotometer fitted with an integrating sphere. Slides were then placed in a tank of running raw seawater and allowed to incubate outdoors in natural sunlight for 26 days. Water temperature was nominally 15 C. Spectrophotometric determination of biofilm accumulation was measured on each slide periodically. Relative algal biomass was calculated as the ratio of absoφtion at 680 nm, contributed by chlorophyll a, to that at 750 nm, a wavelength not absorbed by chlorophyll, to correct for differences in turbidity and scattering properties of the different slides.

Results

As shown in Figure 5, octyl sulfate incoφorated into RTV-1 1 silicone, and then coated onto glass slides, significantly inhibited the formation of natural marine algal biofilms in natural seawater. After 26 days of incubation in running seawater, algal biofilm development on the octyl sulfate containing coatings was five fold less than that of controls lacking octyl sulfate, indicating that octyl sulfate possesses strong AF activity.

Studies were performed to evaluate the ability of the sulfate ester molecules octyl sulfate and methyl sulfate, to inhibit adhesion of the marine bacteriums Oceanospr ilium and Alteromonas atlantica to glass surfaces.

Materials and Methods

Oceanosprillum adhesion test Each test consisted of a control set (with no sulfate esters) and sample sets containing the test molecules. The first test group consisted of a control sample set, a zosteric acid (5 mM) sample set, and an octyl sulfate (5 mM) sample set.

The second test group consisted of a control sample set, a zosteric acid (5 mM) sample set, and a methyl sulfate (5 mM) sample set. Sample sets consisted of five 50 mL sterile centrifuge tubes, with each tube containing a glass microscope slide, 50 ml of artificial seawater (ASTM - American Society for testing and materials) with the dissolved sulfate ester, inoculated with an Oceanosprillum culture at 1 x 10⁶ cells/mL. Sample sets were incubated at 23 C, with shaking so that the surface of the slides were horizontal. Over a 6- hour period, individual tubes were taken from the sample sets and tested for microbial adhesion.

Alteromonas atlantica adhesion tests. The tests consisted of a control sample set, a zosteric acid (5 mM) sample set, an octyl sulfate (5 mM) sample set, and a methyl sulfate (5 mM) set. A sample set consisted of six 60 mL sterile centrifuge tubes. Each tube contained a glass microscope slide and 50 mL of modified ASTM seawater (American Society for Testing and Materials (1986) Dl 141 -86, ASTM, Philadelphia, PA) with dissolved agent, inoculated with Alteromonas atlantica culture to an initial cell density of 1 x 10⁶ cells/mL. The modified seawater consisted of normal ASTM seawater ingredients, however the carbon source glycerol, was only 1000th the normal strength, 0.1 L/L instead of 100 L/L, and was void of an amino acid source (casamino acids), in order to allow enough carbon for attachment, but not for significant cell growth.

Determination of bacterial adhesion. Samples were removed from the shaker and 1 mL of 50X acridine orange stain (0.5 g/L acridine powder in water) was added to the tube. The stain was allowed to react for 4 minutes. The slides were then removed and fitted with a long cover slip and immediately counted with an epifluorescent microscope fitted with a 100X (oil) objective lens on the under side of the slide. The size of the counting field was 10 X 10 μm. A total of 20 counts per slide were performed and averaged to yield the number of cells per μm², which was in turn converted to cells per mm². Error was assigned at 10% which is the standard accepted error for direct counting of bacterial cells.

Results As shown in Figure 6, the presence of octyl sulfate or methyl sulfate in the medium significantly reduced bacterial adhesion to the glass slides when compared to controls in which no sulfate ester molecule was present. Methyl sulfate inhibited Oceanosprillum adhesion to an extent similar to the proven AF agent zosteric acid, with each compound promoting roughly a two fold reduction in bacterial attachment, relative to control. As shown in Figure 4, octyl sulfate inhibited Oceanosprillum adhesion to an even greater extent than zosteric acid.

As shown in Figure 8, the presence of dissolved zosteric acid, octyl sulfate, or methyl sulfate produced a significant reduction in the marine bacterium, Alteromonas atlantica adhesion relative the controls. The presence of methyl sulfate had the most dramatic effect upon adhesion, with adhesion remaining constant after 120 minutes at 150,000 cells/mm , while controls had greater than 700,000 cells/mm². Octyl sulfate also inhibited adhesion, demonstrating a slightly higher inhibitory activity than zosteric acid..

Example 3 : Inhibition of Fungal Surface Attachment and Mvcelial Development

To determine the effectiveness of sulfate esters at inhibiting fungal biofouling, the ability of zosteric acid to inhibit attachment of the fungus Aureobasidium pullulans to surfaces was examined.

Materials and Methods

Aureobasidium pullulans (ATCC 34261) was grown on potato-dextrose agar and harvested according to ASTM G-21-90 protocols (.American Society for Testing and Materials (1986) Dl 141 -86, ASTM, Philadelphia, PA). The resulting spore suspension was used to inoculate liquid culture tubes containing 35 mL of growth medium (nutrient salts with 5 mM sucrose) and 15 mM zosteric acid. Zosteric acid- free medium was prepared as a control. A sterile microscope slide was added to each tube, the tubes were sealed and placed on a rotary shaker table at room temperature. One tube w as harvested each day by removing the slide and counting the number of attached spores by direct microscopic counts, as described above.

23

SUBSTTTUTE SHEET (RULE 26) Results

Fungal spores were obsen ed to grow in both the presence and absence of zosteπc acid, as indicated by the clouding of all tubes after Day 1 However, as shown in Fig 9, the presence of zosteπc acid prevented the attachment of the fungus to the glass slides After 5 days incubation with A pullalans less than 20 germinated fungal colonies/mnr were observed on slides incubated in the additional presence of zosteπc acid, compared to more than 600 germinated fungal colonies/mirr on control slides Furthermore, fungal colonies in the media of zosteπc acid free cultures were composed of multi-cellular (>20 cells) filaments, indicative of mycehal growth In contrast, colonies m the zosteπc acid treated cultures were generally small and round, exhibiting no evidence of filamentous growth or mycehal development

Example 4 Sulfate Esters Bind Cell Surfaces of Shewanella putrefaciens

To investigate the mechanism behind the AF activity of sulfate esters, polyclonal antibodies specific for the sulfate ester zosteπc acid were generated (BAbCo, Berkeley, CA) Preliminary testing of these antibodies for cross reactivity towards related compounds lacking the sulfate ester group (cinnamic acid, feruhc acid, coumaπc acid) showed no cross reactivity, suggesting that the specific domain recognized by the antibodies probably includes the sulfate ester group These antibodies were then used to investigate whether the sulfate ester AF agent zosteπc acid directly binds fouling organisms

The maπne bacteπum Shewanella putrefaciens were grown in cultures containing zosteric acid and were subsequently examined for bound zosteπc acid using immuno-gold staining with the antibody descπbed above Electron microscopic examination of immunoprobed S putrefaciens detected zosteπc acid molecules bound to the surface of the bacteπa Furthermore, zosteric acid was observed to be present at high incidence at the sites of cell adhesion In contrast to these agglutination sites, the majoπty of the cell surfaces as well as the continuous boundaπes between daughter cells in dividing chains, showed no evidence of bound zosteric acid, as indicated by a lack of immuno-gold staining These results indicate that sulfate esters bind to the surfaces of bacteπal cells and suggest a possible relationship between sulfate ester binding sites and the sites of bacteπal agglutination Example 5 Zosteπc Acid Promotes Bacteπal Agglutination

To further investigate the role of sulfate esters in agglutination, the ability of sulfate esters to facilitate the agglutination of bacteπal cells was investigated Log-phase cultures grown in the presence of zosteπc acid were monitored spectrophotometπcally (OD₆oo) for growth, and for agglutination in the presence of increasing amounts of zosteric acid

Materials and Methods

Cell Surface Binding Assays The maπne bacterium Shewanella putrefaciens was grown in maπne broth m the presence of 16mM zosteric acid Dense log phase cells were harvest after 5 hours growth, and preserved in 0 5 X Kamofsky's fixative (2% formaldehyde, 2 5% gluturaldehyde, 0 05 M sodium cacodylate. 0 25 M sucrose, pH 7 4) for 2 hours, and then transferred to a cacodylate buffer (0 05 M sodium cacodylate, pH 7 4) for storage Cells were prepared foi electron microscopic examination using immuno-gold staining techniques (Harlow, E and Lame, D , Antibodies, A Laboratory Manual, Cold Spπng Harbor Laboratory, 359-421 , Roth et al , J Histochem Cytochem 26 1074-1081 (1978)) The pπmary antibody used m this study was an anti-zosteπc acid polyclonal antibody (BAbCo, Richmond, CA)

Bactenal agglutination assays Log-phase cultures of Shewanella putrefaciens were grown in complete seawater medium containing zosteric acid at a range of concentrations up to 20 mM Cultures were counted for viable colony forming units at eight hours

Results

Although zosteπc acid concentrations up to 16 mM did not inhibit the growth of S putrefaciens m liquid culture, the presence of zosteπc acid caused significant agglutination of S putrefaciens m a concentration dependent manner The agglutination observed was visible to the naked eye, and was more quantitatively detected as a decrease in optical density absorbance in cultures containing zosteπc acid (Fig 10) Counts of viable colony forming units at eight hours revealed no difference in cell density among the different cultures, thus the observed differences in absoφtion resulted from differences in bacteπal agglutination, not differences in growth (cell division) rates among the cultures. Thus, zosteric acid promoted cell agglutination, but did not

Equivalents Those skilled in the art will recognize, or be able to ascertain using no more than routine experimentation, many equivalents of the specific embodiments of the invention described herein. Such equivalents are encompassed by the following claims.

Claims

1. A plant cell surface comprising an effective amount of bioavailable anti-fouling compound represented by general structure 1 :

O

z Y s X o

1 wherein

X represents -OH, -O(aryl). -O(acyl), -O(sulfonyl), -CN, F, Cl, or Br;

Y represents O, S, Se, or NR;

Z represents optionally substituted alkyl, heteroalkyl, cycloalkyl, heterocycloalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2)_m-R₈o;

R represents independently for each occurrence hydrogen, alkyl, heteroalkyl, aryl, heteroaryl, aralkyl, heteroaralkyl, or -(CH2)_m-R₈o;

R₈o represents independently for each occurrence aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl; and m is an integer in the range 0 to 8 inclusive, or a salt thereof, wherein the compound or a biologically active fragment thereof can be released from the surface in the presence of a liquid or vapor.

2. A plant cell surface of claim 1, wherein X represents -OH, F, Cl, or Br.

3. A plant cell surface of claim 1, wherein Y represents O.

4. A plant cell surface of claim 1. wherein Z represents optionally substituted alkyl, aryl, or -

5. A plant cell surface of claim 1, wherein Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

6. A plant cell surface of claim 1, wherein Z represents methyl, octyl, 4-(2- methylpropyl)phenyl, 4-(l,l-dimethylethyl)phenyl, 4-(l,l-dimethylpropyl)phenyl, 4- pentylphenyl, 4-(l -methyl- l-phenylethyl)phenyl, or 4-(l-methylheptyl)phenyl.

7. A plant cell surface of claim 1, wherein R represents H or alkyl.

8. A plant cell surface of claim 1, wherein X represents -OH, F, Cl, or Br; and Y represents O.

9. A plant cell surface of claim 1, wherein X represents -OH or Cl; and Y represents O.

10. A plant cell surface of claim 1, wherein X represents -OH, F, Cl, or Br; and Z represents optionally substituted alkyl, aryl, or -(CH2)_m-R₈o-

11. A plant cell surface of claim 1, wherein X represents -OH or Cl; and Z represents optionally substituted alkyl, aryl, or -(CH2)_m-R₈o-

12. A plant cell surface of claim 1, wherein X represents -OH, F, Cl, or Br; and Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

13. A plant cell surface of claim 1, wherein X represents -OH or Cl; and Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

14. A planr cell surface of claim 1, wherein X represents -OH, F, Cl, or Br; and Z represents methyl, octyl, - 4-(2-methylpropyl)phenyl, 4-(l,l-dimethylethyl)phenyl, 4-(l,l- dimethylpropyOphenyl, 4-pentylphenyl, 4-(l -methyl- l-phenylethyl)phenyl, or 4-(l- methylheptyl)phenyl.

15. A plant cell surface of claim 1. wherein X represents -OH or Cl; and Z represents methyl, octyl, 4-(2-methylpropyl)phenyl, 4-(l ,l-dimethylethyl)phenyl, 4-(l ,l-dimethylpropyl)phenyl,

4-pentylphenyl, 4-(l -methyl- l-phenylethyl)phenyl, or 4-(l -methylheptyl)phenyl.

16. A plant cell surface of claim 1, wherein Y represents O; and Z represents optionally substituted alkyl. aryl, or -(CH2)_m-R8o-

17. A plant cell surface of claim 1 , wherein Y represents O; and Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

18. A planr cell surface of claim 1 , wherein Y represents O; and Z represents methyl, octyl, 4- (2-methylpropyl)phenyl, 4-(l,l-dimethylethyl)phenyl, 4-(l,l-dimethylpropyl)phenyl, 4- pentylphenyl, 4-(l -methyl- l-phenylethyl)phenyl, or 4-(l-methylheptyl)phenyl.

19. A plant cell surface of claim 1 , wherein X represents -OH, F, Cl, or Br; Y represents O; and Z represents optionally substituted alkyl, aryl. or -(CH2)_m-R₈o-

20. A plant cell surface of claim 1 , wherein X represents -OH or Cl; Y represents O; and Z represents optionally substituted alkyl, aryl, or -(CH2)_m-R₈o-

21. A plant cell surface of claim 1 , wherein X represents -OH, F, Cl, or Br; Y represents O; and Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

22. A plant cell surface of claim 1 , wherein X represents -OH or Cl; Y represents O; and Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

23. A plant cell surface of claim 1, wherein X represents -OH, F, Cl, or Br; Y represents O; and Z represents methyl, octyl, 4-(2-methylpropyl)phenyl, 4-(l,l-dimethylethyl)phenyl, 4- (l,l-dimethylpropyl)phenyl, 4-pentylphenyl, 4-(l -methyl- l-phenylethyl)phenyl, or 4-(l- methylheptyl)phenyl.

24. A plant cell surface of claim 1 , wherein X represents -OH or Cl; Y represents O; and Z represents methyl, octyl, 4-(2-methylpropyl)phenyl, 4-(l,l-dimethylethyl)phenyl, 4-(l,l- dimethylpropyl)phenyl, 4-pentylphenyl, 4-(l -methyl- l-phenylethyl)phenyl, or 4-(l- methy lhepty l)phenyl .

25. A plant cell surface of claim 1, wherein the surface is a coating.

26. A plant cell surface of claim 25, wherein the coating is temporary

27. A plant cell surface of claim 25, wherein the coating is semi-permanent.

28. A plant cell surface of claim 25, wherein the coating is permanent.

29. A plant cell surface of claim 1 , wherein the effective amount reduces the number of plant pathogens on a plant cell surface over a defined period of time by a factor of 4 relative to a control plant cell, which does not comprise the compound.

30. A plant cell surface of claim 1, wherein the effective amount reduces the number of pathogens on a plant cell surface over a defined period of time by a factor of 8 relative to a control plant cell, which does not comprise the compound.

31. A plant cell surface of claim 1, wherein the effective amount reduces the number of pathogens on a plant cell surface over a defined period of time by a factor of 10.

32. A plant cell surface of claim 1, wherein the effective amount reduces the number of pathogens on a plant cell surface over a defined period of time by a factor of 15.

33. A plant cell surface of claim 1, wherein the release of the compound is at a constant rate.

34. A coating for contacting a plant cell surface comprising an effective amount of an anti- fouling compound represented by general structure 1 :

O

Z Y S X

1 wherein

X represents -OH, -O(aryl), -O(acyl), -O(sulfonyl), -CN, F, Cl, or Br;

Rso represents independently for each occurrence aryl, cycloalkyl, cycloalkenyl, heterocyclyl, or polycyclyl; and m is an integer in the range 0 to 8 inclusive or a salt thereof , wherein the coating releases the compound or a biologically active fragment thereof when in contact with a liquid or vapor.

35. A coating of claim 34, wherein X represents -OH, F, Cl, or Br.

36. A coating of claim 34, wherein Y represents O.

37. A coating of claim 34, wherein Z represents optionally substituted alkyl, aryl, or - (CH₂)_m-R₈o.

38. A coating of claim 34, wherein Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

39. A coating of claim 34, wherein Z represents methyl, octyl, 4-(2-methylpropyl)phenyl, 4- (l,l-dimethylethyl)phenyl, 4-(l,l-dimethylpropyl)phenyl, 4-pentylphenyl, 4-( 1 -methyl- 1- phenylethyl)phenyl, or 4-(l-methylheptyl)phenyl.

40. A coating of claim 34, wherein R represents H or alkyl.

41. A coating of claim 34, wherein X represents -OH, F, Cl, or Br; and Y represents O.

42. A coating of claim 34, wherein X represents -OH or Cl; and Y represents O.

43. A coating of claim 34, wherein X represents -OH, F, Cl, or Br; and Z represents optionally substituted alkyl, aryl, or -(CH2)_m-R₈o-

44. A coating of claim 34, wherein X represents -OH or Cl; and Z represents optionally substituted alkyl, aryl, or -(CH2)_m-R₈o.

45. A coating of claim 34, wherein X represents -OH, F, Cl, or Br; and Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

46. A coating of claim 34, wherein X represents -OH or Cl; and Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

47. A coating of claim 34, wherein X represents -OH, F, Cl, or Br; and Z represents methyl, octyl, 4-(2-methylpropyl)phenyl, 4-(l,l-dimethylethyl)phenyl, 4-(l,l-dimethylpropyl)phenyl, 4-pentylphenyl, 4-(l -methyl- l-phenylethyl)phenyl, or 4-(l-methylheptyl)phenyl.

48. A coating of claim 34, wherein X represents -OH or Cl; and Z represents methyl, octyl, 4- (2-methylpropyl)phenyl, 4-(l,l-dimethylethyl)phenyl, 4-(l,l-dimethylpropyl)phenyl, 4- pentylphenyl, 4-(l -methyl- l-phenylethyl)phenyl, or 4-(l-methylheptyl)phenyl.

49. A coating of claim 34, wherein Y represents O; and Z represents optionally substituted alkyl, aryl, or -(CH2)_m-R₈o-

50. A coating of claim 34, wherein Y represents O; and Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

51. A coating of claim 34, wherein Y represents O; and Z represents methyl, octyl, 4-(2- methylpropyl)phenyl, 4-( 1,1 -dimethyl ethyl)phenyl, 4-(l,l-dimethylpropyl)phenyl, 4- pentylphenyl, 4-(l -methyl- l-phenylethyl)phenyl, or 4-(l-methylheptyl)phenyl.

52. A coating of claim 34, wherein X represents -OH, F, Cl, or Br; Y represents O; and Z represents optionally substituted alkyl, aryl, or -(CH2)_m-R8o-

53. A coating of claim 34, wherein X represents -OH or Cl; Y represents O; and Z represents optionally substituted-alkyl, aryl, or -(CH2)m^_R₈₀-

^•u

54. A coating of claim 34, wherein X represents -OH, F, Cl, or Br; Y represents O; and Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

55. A coating of claim 34, wherein X represents -OH or Cl; Y represents O; and Z represents optionally substituted alkylphenyl, heteroalkylphenyl, arylphenyl, or heteroarylphenyl.

56. A coating of claim 34, wherein X represents -OH, F, Cl, or Br; Y represents O; and Z represents methyl, octyl, 4-(2-methylpropyl)phenyl, 4-(l,l-dimethylethyl)phenyl, 4-(l,l- dimethylpropyl)phenyl, 4-pentylphenyl, 4-(l -methyl- 1 -phenylethyl)phenyl, or 4-(l- methy lheptyl)pheny 1.

57. A coating of claim 34, wherein X represents -OH or Cl; Y represents O; and Z represents methyl, octyl, 4-(2-methylpropyl)phenyl, 4-(l,l-dimethylethyl)phenyl, 4-(l,l- dimethylpropyl)phenyl, 4-pentylphenyl, 4-(l -methyl- l-phenylethyl)phenyl, or 4-(l- methylhepty l)pheny 1.

58. A coating of claim 57, wherein the coating is temporary.

59. A coating of claim 57, wherein the coating is semi-permanent.

60. A coating of claim 57, wherein the coating is permanent.

61. A coating of claim 34, wherein the effective amount reduces the number of plant pathogens on a plant cell surface over a defined period of time by a factor of 4 relative to a control plant cell, which does not comprise the compound.

62. A coating of claim 34, wherein the effective amount reduces the number of pathogens on a plant cell surface over a defined period of time by a factor of 8 relative to a control plant cell, which does not comprise the compound.

63. A coating of claim 34, wherein the effective amount reduces the number of pathogens on a plant cell surface over a defined period of time by a factor of 10.

64. A coating of claim 34, wherein the effective amount reduces the number of pathogens on a plant cell surface over a defined period of time by a factor of 15.

65. A coating of claim 34, wherein the release of the compound is at a constant rate

66. A coating of claim 34, which is a liquid.

67. A coating of claim 34, which is a gas or vapor.

68. A coating of claim 34, which is a paste or other semi-solid state.

69. A coating of claim 34, which is a solid.

70. A coating of claim 34, which is a liquid and solidifies into a hard coating on a surface.