ANTIFOULING MATERIALS
20180079912 · 2018-03-22
Inventors
Cpc classification
C09D187/00
CHEMISTRY; METALLURGY
C09D5/1681
CHEMISTRY; METALLURGY
International classification
C09D187/00
CHEMISTRY; METALLURGY
C07C229/36
CHEMISTRY; METALLURGY
Abstract
The invention provided herein presents a novel family of antifouling agents based on hydroxylated and fluorinated compounds.
Claims
1. A compound comprising at least one 3,4-dihydroxy-L-phenylalanin (DOPA) group and at least one fluorinated carbon group.
2. The compound according to claim 1, wherein said at least one fluorinated carbon group is a fluorinated alkyl group.
3. The compound according to claim 1, wherein said at least one fluorinated carbon group is perfluorinated.
4. The compound according to claim 1, having the general formula A-L-F, wherein A is DOPA, L is a covalent bond or a linker moiety linking A and F, and F is a fluorinated alkyl moiety.
5. The compound according to claim 4, wherein the linker is selected from substituted or unsubstituted carbon chain, optionally comprising two or more amino acids.
6. The compound according to claim 5, wherein the linker having the general structure ##STR00029## wherein each * denotes a point of connectivity; n is between 0 and 40; and m is between 1 and 40.
7. A method for preventing or arresting or minimizing or diminishing one or more of the following: (d) adsorption of organic and/or bio-organic materials to a surface; (b) adsorption of proteins and/or (poly)saccharides and (poly)lipids to a surface; (c) secretion from cells of multi-organism or of micro-organisms onto a surface; and (d) adsorption of cells of multi-organism or micro-organisms to a surface, the method comprising forming a coat of at least one compound according to claim 1 on at least a region of said surface.
8. A method for inhibiting settling, attachment, accumulation and dispersion of organisms, organism's secretion of an organic and/or bio-organic material on a surface, the method comprising contacting the surface with an effective amount of a formulation comprising a compound according to claim 1.
9. A formulation comprising at least one compound according to claim 1.
10. A film comprising at least one compound according to claim 1.
11. An article or a device comprising at least one surface region coated with a film according to claim 10.
12. A compound selected from ##STR00030##
13. A formulation comprising the compound: ##STR00031##
14. A biofouling film comprising the compound: ##STR00032##
Description
BRIEF DESCRIPTION OF THE DRAWINGS
[0158] In order to better understand the subject matter that is disclosed herein and to exemplify how it may be carried out in practice, embodiments will now be described, by way of non-limiting example only, with reference to the accompanying drawings, in which:
[0159]
[0160]
[0161]
[0162]
[0163]
[0164]
[0165]
[0166]
[0167]
[0168]
[0169]
[0170]
[0171]
[0172]
[0173]
[0174]
[0175]
[0176]
[0177]
DETAILED DESCRIPTION OF EMBODIMENTS
[0178] Biofouling is a process in which organisms and their by-products encrust a surface. It is one of the main concerns today in the health care system as the adsorption of pathogenic bacteria to medical devices causes hospital acquired infections. In addition, it is a major problem in the marine industry since the adsorption of marine organisms on ships hull leads to an increase in the consumption of fuel and delays in transportation. Many approaches to prevent biofouling have been suggested, however, they suffer from drawbacks such as release of toxic materials to the surroundings, low stability that limits their long-term application or complex and expensive synthesis.
[0179] The invention disclosed herein is based on the inventors development of antifouling coatings that are spontaneously formed by the self-assembly of a compounds such as peptides. The results presented clearly show that the coatings completely prevented the first stage of biofouling and abolished the adsorption of proteins to a substrate. In addition, the coating reduced significantly the amount of bacteria on the substrate.
[0180] The invention provides a peptide comprising at least two amino acids, at least one of said amino acids being 3,4-dihydroxy-L-phenylalanin (DOPA) and at least another of said amino acids being fluorinated.
[0181] In some embodiments, said peptide is antifouling.
[0182] In some embodiments, said fluorinated amino acid is bonded to said at least one DOPA.
[0183] In some embodiments, the peptide comprising between 3 and 8 amino acids. In some embodiments, the peptide comprising between 2 and 8 amino acids, between 3 and 6 amino acids or between 3 and 5 amino acids.
[0184] In some embodiments, each amino acid is bonded to said another amino acid via a peptidic bond. In some embodiments, at least two of said amino acids are bonded to each other through a covalent linker. In some embodiments, the peptide of the invention having the general formula A-L-F, wherein A is DOPA, L is a covalent bond or a linker moiety linking A and F, and F is a fluorinated amino acid moiety.
[0185] In some embodiments, said bond or linker associating A to L, or L to F is a non-hydrolyzable bond or linker group. In some embodiments, the linker is selected from substituted or unsubstituted carbon chain. In some embodiments, the linker is composed of two or more amino acids. In some embodiments, the linker comprises between 1 to 40 carbon atoms. In some embodiments, the linker is of the general structure
##STR00011## [0186] wherein [0187] each * denotes a point of connectivity; [0188] n is between 0 and 40; and [0189] m is between 1 and 40.
[0190] In some embodiments, two or more moieties are DOPA moieties. In some embodiments, the peptide comprises two or more fluorinated amino acids. In some embodiments, the peptide comprises two or more DOPA and two or more fluorinated amino acids moieties.
[0191] In some embodiments, the peptide comprises one or more DOPA and two or more fluorinated amino acids moieties. In some embodiments, the peptide comprises two or more DOPA and one or more fluorinated amino acids moieties. In some embodiments, said amino acid being fluorinated is selected amongst natural or unnatural amino acid, an amino acid analog, - or -forms, and L- or D amino acids. In some embodiments, the amino acid is selected amongst alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine valine, pyrrolysine and selnocysteine; and amino acid analogs such as homo-amino acids, N-alkyl amino acids, dehydroamino acids, aromatic amino acids and ,-disubstituted amino acids, cystine, 5-hydroxylysine, 4-hydroxyproline, a-aminoadipic acid, a-amino-n-butyric acid, 3,4-dihydroxyphenylalanine, homoserine, -methylserine, ornithine, pipecolic acid, ortho, meta or para-aminobenzoic acid, citrulline, canavanine, norleucine, d-glutamic acid, aminobutyric acid, L-fluorenylalanine, L-3-benzothienylalanine and thyroxine.
[0192] In some embodiments, the amino acid is selected amongst aromatic amino acids. In some embodiments, the aromatic amino acids are selected from tryptophan, tyrosine, naphthylalanine, and phenylalanine. In some embodiments, the amino acids are selected from phenylalanine and/or derivatives thereof.
[0193] In some embodiments, the phenylalanine derivatives are selected from 4-methoxy-phenylalanine, 4-carbamimidoyl-1-phenylalanine, 4-chloro-phenylalanine, 3-cyano-phenylalanine, 4-bromo-phenylalanine, 4-cyano-phenylalanine, 4-hydroxymethyl-phenylalanine, 4-methyl-phenylalanine, 1-naphthyl-alanine, 3-(9-anthryl)-alanine, 3-methyl-phenylalanine, m-amidinophenyl-3-alanine, phenylserine, benzylcysteine, 4,4-biphenylalanine, 2-cyano-phenylalanine, 2,4-dichloro-phenylalanine, 3,4-dichloro-phenylalanine, 2-chloro-penylalanine, 3,4-dihydroxy-phenylalanine, 3,5-dibromotyrosine, 3,3-diphenylalanine, 3-ethyl-phenylalanine, 3,4-difluoro-phenylalanine, 3-chloro-phenylalanine, 3-chloro-phenylalanine, 2-fluoro-phenylalanine, 3-fluoro-phenylalanine, 4-amino-L-phenylalanine, homophenylalanine, 3-(8-hydroxyquinolin-3-yl)-1-alanine, 3-iodo-tyrosine, kynurenine, 3,4-dimethyl-phenylalanine, 2-methyl-phenylalanine, m-tyrosine, 2-naphthyl-alanine, 5-hydroxy-1-naphthalene, 6-hydroxy-2-naphthalene, meta-nitro-tyrosine, (beta)-beta-hydroxy-1-tyrosine, (beta)-3-chloro-beta-hydroxy-1-tyrosine, o-tyrosine, 4-benzoyl-phenylalanine, 3-(2-pyridyl)-alanine, 3-(3-pyridyl)-alanine, 3-(4-pyridyl)-alanine, 3-(2-quinolyl)-alanine, 3-(3-quinolyl)-alanine, 3-(4-quinolyl)-alanine, 3-(5-quinolyl)-alanine, 3-(6-quinolyl)-alanine, 3-(2-quinoxalyl)-alanine, styrylalanine, pentafluoro-phenylalanine, 4-fluoro-phenylalanine, phenylalanine, 4-iodo-phenylalanine, 4-nitro-phenylalanine, phosphotyrosine, 4-tert-butyl-phenylalanine, 2-(trifluoromethyl)-phenylalanine, 3-(trifluoromethyl)-phenylalanine, 4-(trifluoromethyl)-phenylalanine, 3-amino-L-tyrosine, 3,5-diiodotyrosine, 3-amino-6-hydroxy-tyrosine, tyrosine, 3,5-difluoro-phenylalanine and 3-fluorotyrosine.
[0194] In some embodiments, said fluorinated amino acid are selected from o-fluorophenylalanine, m-fluorophenylalanine and p-fluorophenylalanine.
[0195] In some embodiments, the peptide comprises between 2 and 12 amino acids, each amino acid being selected from aromatic amino acids. In some embodiments, the peptide comprises DOPA at one termini and a fluorinated aromatic amino acid selected from o-fluorophenylalanine, m-fluorophenylalanine and p-fluorophenylalanine at the other termini. In some embodiments, the peptide comprises DOPA at a mid-point amino acid along the peptide and a fluorinated aromatic amino acid selected from o-fluorophenylalanine, m-fluorophenylalanine and p-fluorophenylalanine at each of the peptide termini.
[0196] In some embodiments, the peptide is for use as an antifouling agent; e.g., for preventing or arresting or minimizing or diminishing one or more of the following:
[0197] (a) adsorption of organic and/or bio-organic materials to a surface;
[0198] (b) adsorption of proteins and/or (poly)saccharides and (poly)lipids to a surface;
[0199] (c) secretion from cells of multi-organism or of micro-organisms onto a surface; and
[0200] (d) adsorption of cells of multi-organism or micro-organisms to a surface.
[0201] The specific compounds of the invention are selected from:
##STR00012##
[0202] Peptide 1: (1S, 2S, 3S) A=B=D=E=H, CF
[0203] Peptide 2: (1S, 2S, 3R) A=B=D=E=H, CF
[0204] Peptide 3: (1S, 2R, 3S) A=B=D=E=H, CF
[0205] Peptide 4: (1S, 2R, 3R) A=B=D=E=H, CF
[0206] Peptide 5: (1S, 2S, 3S) A=B=C=D=E=F
##STR00013##
Peptide 6
[0207] ##STR00014##
[0208] Peptide 7: (1S, 2S, 3S) A=B=C=D=H, E=F
[0209] Peptide 8: (1S, 2S, 3R) A=B=C=D=H, E=F
[0210] Peptide 9: (1S, 2R, 3S) A=B=C=D=H, E=F
[0211] Peptide 10: (1S, 2R, 3R) A=B=C=D=H, E=F
##STR00015##
[0212] NH.sub.2-L-DOPA-L-(4-F)-Phe-COOH Peptide 15
[0213] NH.sub.2-L-DOPA-D-(4-F)-Phe-COOH Peptide 16
[0214] NH.sub.2-L-DOPA-L-(4-F)-Phe-L-(4-F)-Phe-COOMe Peptide 17.
[0215] The invention also contemplates formulations comprising peptide compounds as described herein. The formulation may be a ready-for-use antifouling formulation.
[0216] The invention also provides a film or a coat comprising at least one peptide of the invention. The film is preferably antifouling and/or anti-biofilm.
[0217] The film may be part of an article or a device comprising at least one surface region coated with a film according to the invention. The article or device may be selected from a marine vessel, a hull of a marine vessel, a medical device, a contact lens, a food processing apparatus, a drinking water dispensing apparatus, a pipeline, a cable, a fishing net, a pillar of a bridge and a surface region of a water immersed article.
[0218] The film in such devices or articles are for preventing biofouling caused by an organism selected from bacteria, diatoms, hydroids, algae, bryozoans, protozoans, ascidians, tube worms, asiatic clams, zebra mussels and barnacles. In some embodiments, the organisms are bacteria. In some embodiments, the bacteria is selected from Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumonia, Chlamydia psittaci, Chlamydia trachomatis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheria, Enterococcus faecalis, Enterococcus faecium, Escherichia coli (E. coli), Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic E. coli, Francisella tularensis, Haemophilus influenza, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans Streptococcus pneumonia, Streptococcus pyogenes, Treponema pallidum, Vibrio cholera, Vibrio harveyi and Yersinia pestis.
[0219] In some embodiments, bacteria are Escherichia coli (E. Coli). In some embodiments, the bacteria are P. aeruginosa.
[0220] The invention also provide use of a peptide according the invention for preventing or arresting adsorption of secretion products of cells of muli-cellular organism or of microorganisms to a surface of a dialysis unit to prevent adherence of blood cells or of proteins secreted from blood cells from a patient being treated by the unit.
[0221] The invention further provides a method for inhibiting settling, attachment, accumulation and dispersion of organisms, organism's secretion of an organic and/or bio-organic material on a surface, the method comprising contacting the surface with an effective amount of a formulation comprising a peptide according to the invention.
[0222] In another aspect, the invention provides a film or a coat comprising a compound having at least one antifouling moiety and at least one surface-adsorbing moiety, wherein the at least one antifouling moiety is selected amongst fluorine (F) and a group comprising at least one fluorine atom and said at least one surface-adsorbing moiety being selected amongst 3,4-dihydroxy-L-phenylalanin (DOPA) and DOPA containing groups. In some embodiments, the film or coat is formed on a surface region of a device or an article.
[0223] The invention also provides a film or a coat comprising a bifunctional compound comprising at least one antifouling moiety and at least one surface-adsorbing moiety (or group), wherein the at least one antifouling moiety is selected amongst fluorine (F) and at least one group comprising a fluorine atom and said at least one surface-adsorbing moiety being selected amongst dihydroxy-amino acids and dihydroxy-amino acid containing groups, said at least one antifouling moiety and said at least one surface-adsorbing moiety being associated to each other via a covalent bond or via a linker moiety. The film or coat may comprise at least one antifouling moiety and at least one surface-adsorbing moiety, wherein the at least one antifouling moiety being selected amongst fluorine (F) and at least one group comprising a fluorine atom and said at least one surface-adsorbing moiety is selected amongst 3,4-dihydroxy-L-phenylalanin (DOPA) and DOPA containing groups, and wherein said at least one antifouling moiety and said at least one surface-adsorbing moiety being associated to each other via a covalent bond or via a linker moiety.
[0224] In some embodiments, said compound being of the general formula A-L-F, wherein A is a surface-adsorbing moiety, L is a covalent bond or a linker moiety linking A and F, and F is an antifouling moiety, and wherein each of A, L and F are associated to each other via a non-hydrolyzable bond.
[0225] The film or coat is antifouling for preventing or arresting adsorption of organic and/or bio-organic materials to said surface, or for preventing or arresting adsorption of secretion products of cells of multi-cellular organisms or of microorganisms to a surface.
[0226] In some embodiments, the surface-adsorbing moiety is DOPA being linked, associated or bonded to an atom on said linker moiety. In some embodiments, said linker moiety is a one-carbon chain. In some embodiments, the linker moiety is selected from substituted or unsubstituted carbon chain. In some embodiments, the linker moiety is selected from amino acids and peptides. In some embodiments, the linker moiety comprises between 1 to 40 carbon atoms. In some embodiments, the linker moiety is substituted by one or more functional groups selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted cycloalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted NR.sub.1R.sub.2, substituted or unsubstituted OR.sub.3, substituted or unsubstituted SR.sub.4, substituted or unsubstituted S(O)R.sub.5, substituted or unsubstituted alkylene-COOH, and substituted or unsubstituted ester.
[0227] In some embodiments, the linker moiety is of the general structure
##STR00016## [0228] wherein [0229] each * denotes a point of connectivity; [0230] n is between 0 and 40; and [0231] m is between 1 and 40.
[0232] In some embodiments, n is between 1 and 12. In some embodiments, n is between 1 and 8. In some embodiments, n is between 1 and 6. In some embodiments, m is between 1 and 20. In some embodiments, m is between 1 and 12. In some embodiments, m is between 1 and 8. In some embodiments, m is between 1 and 6.
[0233] In some embodiments, one or more of the (CH.sub.2).sub.n groups are substituted.
[0234] In some embodiments, the linker moiety is an amino acid comprising 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 or 31 or 32 or 33 or 34 or 35 or 36 or 37 or 38 or 39 or 40 amino acids.
[0235] In some embodiments, the compound is constructed of two amino acids bonded to each other via an amide bond, wherein one amino acid is DOPA and the other being a fluorinated amino acid. In some embodiments, the antifouling moieties are bonded to the linker at one end and the surface-adsorbing moieties at the other end of the linker moiety. In some embodiments, the antifouling moieties and the surface-adsorbing moieties are at alternating positions along the linker moiety.
[0236] In some embodiments, the linker moiety comprises or consists a peptide of two or more amino acids.
[0237] In some embodiments, the compound is a peptide having at least two amino acids, at least one DOPA and at least fluorinated group, which may or may not be a fluorinated amino acid. In some embodiments, the peptide comprises between 2 and 40 amino acids. In some embodiments, the peptide comprises 2, or 3, or 4, or 5, or 6, or 7, or 8 or 9 or 10 or 11 or 12 amino acids.
[0238] In some embodiments, said antifouling moiety is a fluorinated amino acid selected amongst natural or unnatural amino acid, an amino acid analog, - or -forms, and L- or D amino acids. In some embodiments, the amino acid is selected amongst alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine valine, pyrrolysine and selnocysteine; and amino acid analogs such as homo-amino acids, N-alkyl amino acids, dehydroamino acids, aromatic amino acids and ,-disubstituted amino acids, cystine, 5-hydroxylysine, 4-hydroxyproline, a-aminoadipic acid, a-amino-n-butyric acid, 3,4-dihydroxyphenylalanine, homoserine, -methylserine, ornithine, pipecolic acid, ortho, meta or para-aminobenzoic acid, citrulline, canavanine, norleucine, d-glutamic acid, aminobutyric acid, L-fluorenylalanine, L-3-benzothienylalanine and thyroxine.
[0239] In some embodiments, the amino acid is selected amongst aromatic amino acids. In some embodiments, said aromatic amino acids are selected from tryptophan, tyrosine, naphthylalanine, and phenylalanine.
[0240] In some embodiments, the amino acids are selected from phenylalanine and derivatives thereof. In some embodiments, the phenylalanine derivatives are selected from 4-methoxy-phenylalanine, 4-carbamimidoyl-1-phenylalanine, 4-chloro-phenylalanine, 3-cyano-phenylalanine, 4-bromo-phenylalanine, 4-cyano-phenylalanine, 4-hydroxymethyl-phenylalanine, 4-methyl-phenylalanine, 1-naphthyl-alanine, 3-(9-anthryl)-alanine, 3-methyl-phenylalanine, m-amidinophenyl-3-alanine, phenylserine, benzylcysteine, 4,4-biphenylalanine, 2-cyano-phenylalanine, 2,4-dichloro-phenylalanine, 3,4-dichloro-phenylalanine, 2-chloro-penylalanine, 3,4-dihydroxy-phenylalanine, 3,5-dibromotyrosine, 3,3-diphenylalanine, 3-ethyl-phenylalanine, 3,4-difluoro-phenylalanine, 3-chloro-phenylalanine, 3-chloro-phenylalanine, 2-fluoro-phenylalanine, 3-fluoro-phenylalanine, 4-amino-L-phenylalanine, homophenylalanine, 3-(8-hydroxyquinolin-3-yl)-1-alanine, 3-iodo-tyrosine, kynurenine, 3,4-dimethyl-phenylalanine, 2-methyl-phenylalanine, m-tyrosine, 2-naphthyl-alanine, 5-hydroxy-1-naphthalene, 6-hydroxy-2-naphthalene, meta-nitro-tyrosine, (beta)-beta-hydroxy-1-tyrosine, (beta)-3-chloro-beta-hydroxy-1-tyrosine, o-tyrosine, 4-benzoyl-phenylalanine, 3-(2-pyridyl)-alanine, 3-(3-pyridyl)-alanine, 3-(4-pyridyl)-alanine, 3-(2-quinolyl)-alanine, 3-(3-quinolyl)-alanine, 3-(4-quinolyl)-alanine, 3-(5-quinolyl)-alanine, 3-(6-quinolyl)-alanine, 3-(2-quinoxalyl)-alanine, styrylalanine, pentafluoro-phenylalanine, 4-fluoro-phenylalanine, phenylalanine, 4-iodo-phenylalanine, 4-nitro-phenylalanine, phosphotyrosine, 4-tert-butyl-phenylalanine, 2-(trifluoromethyl)-phenylalanine, 3-(trifluoromethyl)-phenylalanine, 4-(trifluoromethyl)-phenylalanine, 3-amino-L-tyrosine, 3,5-diiodotyrosine, 3-amino-6-hydroxy-tyrosine, tyrosine, 3,5-difluoro-phenylalanine and 3-fluorotyrosine.
[0241] In some embodiments, said fluorinated amino acidare selected from o-fluorophenylalanine, m-fluorophenylalanine and p-fluorophenylalanine.
[0242] In some embodiments, the compound comprising DOPA at one termini and a fluorinated aromatic amino acid selected from o-fluorophenylalanine, m-fluorophenylalanine and p-fluorophenylalanine at the other termini.
[0243] In some embodiments, the compound comprising DOPA at a mid-point amino acid along the peptide and a fluorinated aromatic amino acid selected from o-fluorophenylalanine, m-fluorophenylalanine and p-fluorophenylalanine at each of the peptide termini.
[0244] In some embodiments, the film or coat is provided for preventing or arresting or minimizing or diminishing one or more of the following:
[0245] (b) adsorption of organic and/or bio-organic materials to a surface;
[0246] (b) adsorption of proteins and/or (poly)saccharides and (poly)lipids to a surface;
[0247] (c) secretion from cells of multi-organism or of micro-organisms onto a surface; and
[0248] (d) adsorption of cells of multi-organism or micro-organisms to a surface.
[0249] In some embodiments, the compound has the structure:
##STR00017##
[0250] Peptide 1: (1S, 2S, 3S) A=B=D=E=H, CF
[0251] Peptide 2: (1S, 2S, 3R) A=B=D=E=H, CF
[0252] Peptide 3: (1S, 2R, 3S) A=B=D=E=H, CF
[0253] Peptide 4: (1S, 2R, 3R) A=B=D=E=H, CF
[0254] Peptide 5: (1S, 2S, 3S) A=B=C=D=E=F
##STR00018##
[0255] Peptide 6
##STR00019##
[0256] Peptide 7: (1S, 2S, 3S) A=B=C=D=H, E=F
[0257] Peptide 8: (1S, 2S, 3R) A=B=C=D=H, E=F
[0258] Peptide 9: (1S, 2R, 3S) A=B=C=D=H, E=F
[0259] Peptide 10: (1S, 2R, 3R) A=B=C=D=H, E=F.
##STR00020##
[0260] In some embodiments, the compound is selected from
[0261] NH.sub.2-L-DOPA-L-(4-F)-Phe-COOH Peptide 15
[0262] NH.sub.2-L-DOPA-D-(4-F)-Phe-COOH Peptide 16
[0263] NH.sub.2-L-DOPA-L-(4-F)-Phe-L-(4-F)-Phe-COOMe Peptide 17.
[0264] The invention also provides an article or a device comprising at least one surface region coated with a film or coat according to the invention. In some embodiments, the article or device is selected from a marine vessel, a hull of a marine vessel, a medical device, a contact lens, a food processing apparatus, a drinking water dispensing apparatus, a pipeline, a cable, a fishing net, a pillar of a bridge and a surface region of a water immersed article.
[0265] In some embodiments, the film or coat is provided for preventing biofouling caused by an organism selected from bacteria, diatoms, hydroids, algae, bryozoans, protozoans, ascidians, tube worms, asiatic clams, zebra mussels and barnacles.
[0266] In some embodiments, the organisms are bacteria. In some embodiments,the bacteria are selected from Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumonia, Chlamydia psittaci, Chlamydia trachomatis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheria, Enterococcus faecalis, Enterococcus faecium, Escherichia coli (E. coli), Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic E. coli, Francisella tularensis, Haemophilus influenza, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans Streptococcus pneumonia, Streptococcus pyogenes, Treponema pallidum, Vibrio cholera, Vibrio harveyi and Yersinia pestis.
[0267] In some embodiments, the bacteria are Escherichia coli (E. Coli). In some embodiments, the bacteria are P. aeruginosa.
[0268] The invention also provides a composition comprising a compound having at least one antifouling moiety and at least one surface-adsorbing moiety, wherein the at least one antifouling moiety is selected amongst fluorine (F) and a group comprising at least one fluorine atom and said at least one surface-adsorbing moiety being selected amongst 3,4-dihydroxy-L-phenylalanin (DOPA) and DOPA containing groups, for use in forming a self-assembled antifouling film or coat on a surface region of a device or an article.
[0269] The invention also a composition comprising a bifunctional compound comprising at least one antifouling moiety and at least one surface-adsorbing moiety (or group), wherein the at least one antifouling moiety is selected amongst fluorine (F) and at least one group comprising a fluorine atom and said at least one surface-adsorbing moiety being selected amongst dihydroxy-amino acids and dihydroxy-amino acid containing groups, said at least one antifouling moiety and said at least one surface-adsorbing moiety being associated to each other via a covalent bond or via a linker moiety.
[0270] In some embodiments, the composition comprises at least one antifouling moiety and at least one surface-adsorbing moiety, wherein the at least one antifouling moiety being selected amongst fluorine (F) and at least one group comprising a fluorine atom and said at least one surface-adsorbing moiety is selected amongst 3,4-dihydroxy-L-phenylalanin (DOPA) and DOPA containing groups, and wherein said at least one antifouling moiety and said at least one surface-adsorbing moiety being associated to each other via a covalent bond or via a linker moiety.
[0271] In some embodiments, the compound is of the general formula A-L-F, wherein A is a surface-adsorbing moiety, L is a covalent bond or a linker moiety linking A and F, and F is an antifouling moiety, and wherein each of A, L and F are associated to each other via a non-hydrolyzable bond.
[0272] The composition is antifouling for preventing or arresting adsorption of organic and/or bio-organic materials to said surface, or for preventing or arresting adsorption of secretion products of cells of multi-cellular organisms or of microorganisms to a surface.
[0273] In some embodiments, the surface-adsorbing moiety is DOPA being linked, associated or bonded to an atom on said linker moiety. In some embodiments, said linker moiety is a one-carbon chain. In some embodiments, the linker moiety is selected from substituted or unsubstituted carbon chain. In some embodiments, the linker moiety is selected from amino acids and peptides. In some embodiments, the linker moiety comprises between 1 to 40 carbon atoms. In some embodiments, the linker moiety is substituted by one or more functional groups selected from substituted or unsubstituted alkyl, substituted or unsubstituted alkenyl, substituted or unsubstituted alkynyl, substituted or unsubstituted cycloalkyl, substituted or unsubstituted cycloalkenyl, substituted or unsubstituted cycloalkynyl, substituted or unsubstituted aryl, substituted or unsubstituted heteroaryl, substituted or unsubstituted heterocyclyl, substituted or unsubstituted NR.sub.1R.sub.2, substituted or unsubstituted OR.sub.3, substituted or unsubstituted SR.sub.4, substituted or unsubstituted S(O)R.sub.5, substituted or unsubstituted alkylene-COOH, and substituted or unsubstituted ester.
[0274] In some embodiments, the linker moiety is of the general structure
##STR00021## [0275] wherein [0276] each * denotes a point of connectivity; [0277] n is between 0 and 40; and [0278] m is between 1 and 40.
[0279] In some embodiments, n is between 1 and 12. In some embodiments, n is between 1 and 8. In some embodiments, n is between 1 and 6. In some embodiments, m is between 1 and 20. In some embodiments, m is between 1 and 12. In some embodiments, m is between 1 and 8. In some embodiments, m is between 1 and 6.
[0280] In some embodiments, one or more of the (CH.sub.2).sub.n groups are substituted.
[0281] In some embodiments, the linker moiety is an amino acid comprising 2 or 3 or 4 or 5 or 6 or 7 or 8 or 9 or 10 or 11 or 12 or 13 or 14 or 15 or 16 or 17 or 18 or 19 or 20 or 21 or 22 or 23 or 24 or 25 or 26 or 27 or 28 or 29 or 30 or 31 or 32 or 33 or 34 or 35 or 36 or 37 or 38 or 39 or 40 amino acids.
[0282] In some embodiments, the compound is constructed of two amino acids bonded to each other via an amide bond, wherein one amino acid is DOPA and the other being a fluorinated amino acid. In some embodiments, the antifouling moieties are bonded to the linker at one end and the surface-adsorbing moieties at the other end of the linker moiety. In some embodiments, the antifouling moieties and the surface-adsorbing moieties are at alternating positions along the linker moiety.
[0283] In some embodiments, the linker moiety comprises or consists a peptide of two or more amino acids.
[0284] In some embodiments, the compound is a peptide having at least two amino acids, at least one DOPA and at least fluorinated group, which may or may not be a fluorinated amino acid. In some embodiments, the peptide comprises between 2 and 40 amino acids. In some embodiments, the peptide comprises 2, or 3, or 4, or 5, or 6, or 7, or 8 or 9 or 10 or 11 or 12 amino acids.
[0285] In some embodiments, said antifouling moiety is a fluorinated amino acid selected amongst natural or unnatural amino acid, an amino acid analog, - or -forms, and L- or D amino acids. In some embodiments, the amino acid is selected amongst alanine, arginine, asparagine, aspartic acid, cysteine, glutamic acid, glutamine, glycine, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, proline, serine, threonine, tryptophan, tyrosine valine, pyrrolysine and selnocysteine; and amino acid analogs such as homo-amino acids, N-alkyl amino acids, dehydroamino acids, aromatic amino acids and ,-disubstituted amino acids, cystine, 5-hydroxylysine, 4-hydroxyproline, a-aminoadipic acid, a-amino-n-butyric acid, 3,4-dihydroxyphenylalanine, homoserine, -methylserine, ornithine, pipecolic acid, ortho, meta or para-aminobenzoic acid, citrulline, canavanine, norleucine, d-glutamic acid, aminobutyric acid, L-fluorenylalanine, L-3-benzothienylalanine and thyroxine.
[0286] In some embodiments, the amino acid is selected amongst aromatic amino acids. In some embodiments, said aromatic amino acids are selected from tryptophan, tyrosine, naphthylalanine, and phenylalanine.
[0287] In some embodiments, the amino acids are selected from phenylalanine and derivatives thereof. In some embodiments, the phenylalanine derivatives are selected from 4-methoxy-phenylalanine, 4-carbamimidoyl-1-phenylalanine, 4-chloro-phenylalanine, 3-cyano-phenylalanine, 4-bromo-phenylalanine, 4-cyano-phenylalanine, 4-hydroxymethyl-phenylalanine, 4-methyl-phenylalanine, 1-naphthyl-alanine, 3-(9-anthryl)-alanine, 3-methyl-phenylalanine, m-amidinophenyl-3-alanine, phenylserine, benzylcysteine, 4,4-biphenylalanine, 2-cyano-phenylalanine, 2,4-dichloro-phenylalanine, 3,4-dichloro-phenylalanine, 2-chloro-penylalanine, 3,4-dihydroxy-phenylalanine, 3,5-dibromotyrosine, 3,3-diphenylalanine, 3-ethyl-phenylalanine, 3,4-difluoro-phenylalanine, 3-chloro-phenylalanine, 3-chloro-phenylalanine, 2-fluoro-phenylalanine, 3-fluoro-phenylalanine, 4-amino-L-phenylalanine, homophenylalanine, 3-(8-hydroxyquinolin-3-yl)-1-alanine, 3-iodo-tyrosine, kynurenine, 3,4-dimethyl-phenylalanine, 2-methyl-phenylalanine, m-tyrosine, 2-naphthyl-alanine, 5-hydroxy-1-naphthalene, 6-hydroxy-2-naphthalene, meta-nitro-tyrosine, (beta)-beta-hydroxy-1-tyrosine, (beta)-3-chloro-beta-hydroxy-1-tyrosine, o-tyrosine, 4-benzoyl-phenylalanine, 3-(2-pyridyl)-alanine, 3-(3-pyridyl)-alanine, 3-(4-pyridyl)-alanine, 3-(2-quinolyl)-alanine, 3-(3-quinolyl)-alanine, 3-(4-quinolyl)-alanine, 3-(5-quinolyl)-alanine, 3-(6-quinolyl)-alanine, 3-(2-quinoxalyl)-alanine, styrylalanine, pentafluoro-phenylalanine, 4-fluoro-phenylalanine, phenylalanine, 4-iodo-phenylalanine, 4-nitro-phenylalanine, phosphotyrosine, 4-tert-butyl-phenylalanine, 2-(trifluoromethyl)-phenylalanine, 3-(trifluoromethyl)-phenylalanine, 4-(trifluoromethyl)-phenylalanine, 3-amino-L-tyrosine, 3,5-diiodotyrosine, 3-amino-6-hydroxy-tyrosine, tyrosine, 3,5-difluoro-phenylalanine and 3-fluorotyrosine.
[0288] In some embodiments, said fluorinated amino acidare selected from o-fluorophenylalanine, m-fluorophenylalanine and p-fluorophenylalanine.
[0289] In some embodiments, the compound comprising DOPA at one termini and a fluorinated aromatic amino acid selected from o-fluorophenylalanine, m-fluorophenylalanine and p-fluorophenylalanine at the other termini.
[0290] In some embodiments, the compound comprising DOPA at a mid-point amino acid along the peptide and a fluorinated aromatic amino acid selected from o-fluorophenylalanine, m-fluorophenylalanine and p-fluorophenylalanine at each of the peptide termini.
[0291] In some embodiments, the film or coat is provided for preventing or arresting or minimizing or diminishing one or more of the following:
[0292] (c) adsorption of organic and/or bio-organic materials to a surface;
[0293] (b) adsorption of proteins and/or (poly)saccharides and (poly)lipids to a surface;
[0294] (c) secretion from cells of multi-organism or of micro-organisms onto a surface; and
[0295] (d) adsorption of cells of multi-organism or micro-organisms to a surface.
[0296] In some embodiments, the compound has the structure:
##STR00022##
[0297] Peptide 1: (1S, 2S, 3S) A=B=D=E=H, CF
[0298] Peptide 2: (1S, 2S, 3R) A=B=D=E=H, CF
[0299] Peptide 3: (1S, 2R, 3S) A=B=D=E=H, CF
[0300] Peptide 4: (1S, 2R, 3R) A=B=D=E=H, CF
[0301] Peptide 5: (1S, 2S, 3S) A=B=C=D=E=F
##STR00023##
[0302] Peptide 6
##STR00024##
[0303] Peptide 7: (1S, 2S, 3S) A=B=C=D=H, E=F
[0304] Peptide 8: (1S, 2S, 3R) A=B=C=D=H, E=F
[0305] Peptide 9: (1S, 2R, 3S) A=B=C=D=H, E=F
[0306] Peptide 10: (1S, 2R, 3R) A=B=C=D=H, E=F.
##STR00025##
[0307] In some embodiments, the compound is selected from
[0308] NH.sub.2-L-DOPA-L-(4-F)-Phe-COOH Peptide 15
[0309] NH.sub.2-L-DOPA-D-(4-F)-Phe-COOH Peptide 16
[0310] NH.sub.2-L-DOPA-L-(4-F)-Phe-L-(4-F)-Phe-COOMe Peptide 17.
[0311] In some embodiments, the composition is provided for preventing biofouling caused by an organism selected from bacteria, diatoms, hydroids, algae, bryozoans, protozoans, ascidians, tube worms, asiatic clams, zebra mussels and barnacles.
[0312] In some embodiments, the organisms are bacteria. In some embodiments,the bacteria are selected from Bordetella pertussis, Borrelia burgdorferi, Brucella abortus, Brucella canis, Brucella melitensis, Brucella suis, Campylobacter jejuni, Chlamydia pneumonia, Chlamydia psittaci, Chlamydia trachomatis, Clostridium botulinum, Clostridium difficile, Clostridium perfringens, Clostridium tetani, Corynebacterium diphtheria, Enterococcus faecalis, Enterococcus faecium, Escherichia coli (E. coli), Enterotoxigenic Escherichia coli (ETEC), Enteropathogenic E. coli, Francisella tularensis, Haemophilus influenza, Helicobacter pylori, Legionella pneumophila, Leptospira interrogans, Listeria monocytogenes, Mycobacterium leprae, Mycobacterium tuberculosis, Mycoplasma pneumonia, Neisseria gonorrhoeae, Neisseria meningitidis, Pseudomonas aeruginosa, Rickettsia rickettsii, Salmonella typhi, Salmonella typhimurium, Shigella sonnei, Staphylococcus epidermidis, Staphylococcus saprophyticus, Streptococcus agalactiae, Streptococcus mutans Streptococcus pneumonia, Streptococcus pyogenes, Treponema pallidum, Vibrio cholera, Vibrio harveyi and Yersinia pestis.
[0313] In some embodiments, the bacteria are Escherichia coli (E. Coli). In some embodiments, the bacteria are P. aeruginosa.
[0314] The invention further provides an antifouling formulation comprising a composition according to the invention. Also provided is an antimicrobial formulation comprising a composition of the invention. Further provided is an antibacterial formulation comprising a composition of the invention.
[0315] The invention further provides a kit comprising a composition according to the invention and instructions of use.
[0316] The invention also provides the use of a composition according to the invention for making an antifouling formulation or antimicrobial formulation or antibacterial formulation.
[0317] The invention also provides a method for forming a film or a coat of a plurality of compounds on a surface region, the compounds each comprising at least one antifouling moiety and at least one surface-adsorbing moiety, wherein the at least one antifouling moiety is selected amongst fluorine (F) and a group comprising at least one fluorine atom and said at least one surface-adsorbing moiety being selected amongst 3,4-dihydroxy-L-phenylalanin (DOPA) and DOPA containing groups, the method comprising contacting said said surface region with said compounds and permitting self assembly thereof on said surface region.
[0318] In some embodiments, said surface region is of a device or article. In some embodiments, the compound is provided as a formulation. In some embodiments, said film or coat having a property selected from antifouling, antimicrobial and antibacterial.
Materials and Methods
[0319] All chemicals, solvents, proteins and bacteria were purchased from commercially available companies and used as supplied unless otherwise stated. Fmoc-DOPA(ac)-COOH was obtained from Novabiochem/EMD chemicals (San-Diego, USA). L and D-4-fluoro phenylalanine, Boc-penta Fluoro phe-COOH were purchased from chem-impex Inc. (Wood Dale, USA). Solvents and TFA were purchased from Bio-lab (Jerusalem, Israel). NMR solvents (CDCl.sub.3 and DMSO-d.sub.6) were supplied by Sigma-Aldrich (Jerusalem, Israel). Piperidine used for deprotection of Fmoc group was obtained from Alfa-Aesar (UK). The proteins BSA, fibrinogen and lysozyme were obtained from Sigma-Aldrich (Jerusalem, Israel), Chem impex INC. (Wood Dale, USA) and Merck (Darmstadt, Germany) respectively. Pseudomonas aeruginosa (ATCC 27853) and Eschrichia coli (ATCC 1655) were purchased from ATCC (Virginia, USA). Crystal violet was obtained from Merck (Germany).
Peptide Synthesis
[0320] NMR spectra were obtained at 400.13 MHz (.sup.1H) using a Bruker DRX 400 spectrometer. The mass of the peptides was measured using Applied Biosystem Voyager-DE pro MALDI TOF mass spectrometer. The peptides were synthesized by a conventional solution-phase method using a racemization free strategy. The Boc group and Fmoc group were used for N-terminal protection and the C-terminus was protected as a methyl ester. Couplings were mediated by dicyclohexylcarbodiimide/1-hydroxybenzotriazole (DCC/HOBt). The intermediate compounds were characterized by .sup.1H NMR and MALDI-TOF mass spectroscopy and final peptides were fully characterized by .sup.1H NMR, .sup.13C NMR, .sup.19F NMR, MALDI-TOF.
##STR00026##
A. Synthesis of Peptide 1
[0321] 1. Boc-L-(4F)Phe-COOH 7a: A solution of L-4F-Phe-COOH 1.97 g (10 mmol) in a mixture of dioxane (20 mL), water (20 mL) and 1 M NaOH (10 mL) was stirred and cooled in an ice-water bath. Ditert-butylpyrocarbonate 2.4 g (11 mmol) was added and stirring was continued at room temperature for 6 h. Then the solution was concentrated in vacuum to about 15-20 mL, cooled in an ice water bath, covered with a layer of ethyl acetate (about 30 mL) and a dilute solution of KHSO.sub.4 was added to acidify (pH 2-3). The aqueous phase was extracted with ethyl acetate and this operation was done three times. The ethyl acetate extracts were collected and dried over anhydrous Na.sub.2SO.sub.4 and evaporated in a vacuum. The pure material was obtained as a waxy solid.
[0322] Yield: 2.115 g (7.25 mmol, 72.5%)
[0323] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 12.60 [s, 1H COOH], 7.29-7.25 & 7.11-7.07 [m, 4H, Aromatic protons], 4.10-3.00 [m, 1H, CH 4F Phe], 3.03-2.77 [m, 2H, CH 4F Phe], 1.33 [s, 9H, Boc].
[0324] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H]+ 284.12 (calculated), 284.29 (observed), [M+Na]+ 306.11 (calculated), 306.25 (observed).
[0325] 2. Boc-L-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 8a: 500 mg (1.766 mmol) of Boc-L-(4F)Phe-OH was dissolved in 25 mL dry DCM in an ice-water bath. NH.sub.2-L-(4F)Phe OMe 697.13 mg (3.532 mmol) was isolated from the corresponding methyl ester hydrochloride by neutralization, subsequent extraction with ethyl acetate and solvent evaporation. It was then added to the reaction mixture, followed immediately by 365 mg (1.766 mmol) dicyclohexylcarbodiimide (DCC) and 239 mg (1.766 mmol) of HOBt. The reaction mixture was allowed to come to room temperature and stirred for 48 h. DCM was evaporated and the residue was dissolved in ethyl acetate (60 mL) and dicyclohexyl urea (DCU) was filtered off. The organic layer was washed with 2 M HCl (330 mL), brine (230 mL), 1 M sodium carbonate (330 mL) and brine (230 mL) and dried over anhydrous sodium sulfate; and evaporated in a vacuum to yield compound 8a, as a white solid. The product was purified by silica gel (100-200 mesh) using n hexane-ethyl acetate (4:1) as eluent.
[0326] Yield: 616.6 mg (1.334 mmol, 75.5%)
[0327] .sup.1H NMR (CDCl.sub.3, 400 MHz, .sub.ppm): 7.16-7.12 & 6.99-6.90 [m, 8H, Aromatic protons], 6.27-6.25 [d, 1H, NH 4F Phe(3)], 4.93 [b, 1H, NH 4F Phe(2)], 4.77-4.72 [m, 1H, CH 4F Phe(3)], 4.28-4.27 [m, 1H, CH 4F Phe(2)], 3.67 [s, 3H, OMe], 3.08-2.98 [m, 4H, CH 4F Phe(2) and 4F Phe(3)], 1.41 [s, 9H, Boc].
[0328] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na].sup.+ 485.18 (calculated), 485.45 (observed), [M+K].sup.+ 501.16 (calculated), 501.32 (observed).
[0329] 3. NH.sub.2-L-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 9a: 600 mg (1.298 mmol) compound 8a was dissolved in 16 mL of DCM in an ice bath. Then 4 ml of TFA was added and stirred for 2 h. The progress of reaction was monitored through TLC (Thin layer chromatography). After completion of reaction all the solvents were evaporated in rotary evaporator. The product was dissolved in water, neutralized with NaHCO.sub.3 solution and extracted with ethyl acetate, dried over anhydrous sodium sulphate, evaporated into rotary evaporator to get oily product 9a.
[0330] Yield: 435.3 mg (1.202 mmol, 92.6%)
[0331] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 9.06-9.05 [d, 1H, NH 4F Phe(3)], 7.32-7.26 & 7.17-7.04 [m, 8H, Aromatic protons], 4.57-4.51 [m, 1H, CH 4F Phe(3)], 4.04-3.96 [m, 1H, CH 4F Phe(2)], 3.61 [s, 3H, OMe], 3.18-2.91 [m, 4H, CH 4F Phe(2) and 4F Phe(3)].
[0332] MALDI-TOF (matrix: -cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+2H].sup.+ 364.14 (calculated), 364.34 (observed), [M+H.sub.2O].sup.+ 480.15 (calculated), 480.35 (observed).
[0333] 4. Fmoc-L-DOPA(ac)-L-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 10a: 430 mg (1.187 mmol) of compound 9a was dissolved in 25 mL dry DCM in an ice-water bath and 652.37 mg (1.42 mmol) of Fmoc-L-DOPA(ac)-COOH was added. Then 245 mg (1.187 mmol) dicyclohexylcarbodiimide (DCC) and 161 mg (1.187 mmol) of HOBt were added to reaction mixture. The reaction mixture was allowed to come to room temperature and stirred for 48 h. DCM was evaporated and the residue was dissolved in ethyl acetate (60 mL) and dicyclohexylurea (DCU) was filtered off. The organic layer was washed with water, extracted, dried over anhydrous sodium sulfate and evaporated in a vacuum to yield compound 10a, as a white solid. The product was purified by silica gel (100-200 mesh) using n hexane-ethyl acetate (4:1) as eluent.
[0334] Yield: 594.8 mg (0.74 mmol, 62.4%).
[0335] .sup.1H NMR (CDCl.sub.3, 400 MHz, .sub.ppm): 7.77-7.75, 7.54-7.50, 7.42-7.38, 7.33-7.29 [d & m, 8H, Fmoc aromatic protons], 7.05-6.86 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.62-6.55 [s & m, 3H, DOPA aromatic protons], 6.50 [b, 1H, NH 4F Phe(2)], 6.19 [b, 1H, NH 4F Phe(3)], 5.17 [b, 1H, NH DOPA], 4.68-4.66 [m, 1H, CH DOPA], 4.54-4.52 [m, 1H, CH 4F Phe(2)], 4.47-4.42 [m, 1H, CH 4F Phe(3)], 4.31 (b, 2H, CH Fmoc], 4.20-4.17 [m, 1H, CH Fmoc], 3.65 [s, 3H, OMe], 2.98-2.92 [m, 6H, CH 4F Phe(2) 4F Phe(3) & DOPA], 1.62 [s, 6H, 2COCH.sub.3].
[0336] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 804.31 (calculated), 804.70 (observed), [M+Na+2H].sup.+ 828.30 (calculated), 828.07 (observed), [M+K+H].sup.+ 843.27 (calculated), 843.60 (observed).
[0337] 5. NH.sub.2-L-DOPA(ac)-L-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 11a: 580 mg (0.721 mmol) of compound 10a was treated 15 mL with 20% Piperidine solution and stirred for 3 h in room temperature. The completion of reaction was monitored by TLC. Then the solution was lyophilized and purified with column chromatography to get pure sticky compound 11a.
[0338] Yield: 275.6 mg (0.474 mmol, 65.8%)
[0339] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 8.53 [b, 1H, NH 4F Phe(2)], 7.96 [b, 1H, NH 4F Phe(3)], 7.24-7.23, 7.10-7.04 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.69-6.65, 6.55-6.53 [m, 3H, DOPA aromatic protons], 5.56 [m, 1H, CH DOPA], 4.56 [m, 1H, CH 4F Phe(2)], 4.47 [m, 1H, 4F Phe(3)], 3.61 [s, 3H, OMe], 3.12-2.73 [m, 6H, CH 4F Phe(2) 4F Phe(3) & DOPA], 1.61-1.58 [d, 6H, 2COCH.sub.3].
[0340] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 582.23 (calculated), 582.25 (observed), [M+Na].sup.+ 604.22 (calculated), 604.37 (observed), [M+K].sup.+ 620.20 (calculated), 620.19 (observed).
[0341] 6. NH.sub.2-L-DOPA-L(4F)-Phe(2)-L(4F)-Phe(3)-COOMe 1: 260 mg (0.447 mmol) of compound 11a, was stirred in 10 mL of 95% TFA in water for 6 h. The progress of the reaction was monitored through TLC. After completion of reaction the solvent was evaporated in rotary evaporator. The product was washed with hexane, cold ether and water three times each to get final peptide 1.
[0342] Yield: 139.1 mg (0.257 mmol, 57.5%)
[0343] .sup.1H NMR (DMSO-d.sub.6, 500 MHz, .sub.ppm): 8.72-8.70 [d, 1H, NH 4F Phe(2)], 8.66-8.64 [d, 1H, NH 4F Phe(3)], 7.88 [b, 2H, OH DOPA], 7.29-7.23, 7.12-7.05 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.7-6.64, 6.5-6.47 [m, 3H, DOPA aromatic protons], 4.60-4.58 [m, 1H, CH 4F Phe(2)], 4.53-4.52 [m, 1H, CH 4F Phe(3)], 3.83 [m, 1H, CH DOPA], 3.58 [s, 3H, OMe], 3.08-2.75 [m, 6H, CH 4F Phe(2) 4F Phe(3) & DOPA]. .sup.13C NMR (DMSO-d.sub.6, 125 MHz, .sub.ppm): 171.9, 170.1, 168.5, 158.9, 158.54, 145.2, 144.5, 131.5, 125.2, 117.4, 115.5, 115.4, 115.3, 11.2, 114.5, 53.9, 52.3, 47.5, 36.2, 33.8, 25.8, 24.9. .sup.19F NMR (DMSO-d6, 470 MHz, .sub.ppm): 116.42, 116.71.
[0344] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 542.20 (calculated), 542.57 (observed), [M+Na].sup.+ 564.19 (calculated), 564.46 (observed), [M+K].sup.+ 580.16 (calculated), 580.32 (observed).
B. Synthesis of Peptide 2
[0345] 1. Boc-L-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 8b: The compound was synthesized with the same procedure as compound 8a.
[0346] .sup.1H NMR (CDCl.sub.3, 400 MHz, .sub.ppm): 7.13-7.10 & 6.98-6.91 [m, 8H, Aromatic protons], 6.51 [b, 1H, NH 4F Phe(3)], 4.91-4.89 [d, 1H, NH 4F Phe(2)], 4.82-4.77 [m, 1H, CH 4F Phe(3)], 4.33 [m, 1H, CH 4F Phe(1)], 3.68 [s, 3H, OMe], 3.09-2.93 [m, 4H, CH 4F Phe(2) and 4F Phe(3)], 1.38 [s, 9H, Boc].
[0347] MALDI-TOF (matrix: -cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+2H].sup.+ 464.21 (calculated), 464.15 (observed), [M+Na+2H].sup.+ 586.18 (calculated), 586.37, [M+K+H].sup.+ 502.16 (calculated), 502.25 (observed).
[0348] 2. NH.sub.2-L-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 9b: The compound was synthesized with the same procedure as compound 9a.
[0349] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 8.34 [d, 1H, NH 4F Phe(3)], 7.23-7.19 & 7.12-7.01 [m, 8H, Aromatic protons], 4.61-4.51 [m, 1H, CH 4F Phe(3)], 3.62 [s, 3H, OMe], 3.44-3.41 [m, 1H, CH 4F Phe(2)], 3.03-2.74 [m, 4H, CH 4F Phe(2) and 4F Phe(3)]. 2.35 (b, 2H, free NH.sub.2].
[0350] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+2H].sup.+ 364.14 (calculated), 364.41 (observed).
[0351] 3. Fmoc-L-DOPA(ac)-L-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 10b: The compound was synthesized with the same procedure as compound 10a.
[0352] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 8.68-8.55 [d, 1H, NH Phe(2)], 8.15-7.92 [d, 1H, NH 4F Phe(3)], 7.88-7.86, 7.61-6.96 [d & m, 16H, Fmoc aromatic protons, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.75 & 6.64 [s, 3H, DOPA aromatic protons], 5.83 [d, 1H, NH DOPA], 4.62-4.53 [m, 2H, CH 4F Phe(2) and Phe(3)], 4.14-4.02 [m, 3H, CH DOPA & CH Fmoc], 3.63 [s, 3H, OMe], 2.76-2.57 [m, 6H, CH 4F Phe(2), 4F Phe(3) & DOPA], 1.55 [s, 6H, 2COCH.sub.3].
[0353] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na].sup.+ 826.29 (calculated), 826.15 (observed), [M+K].sup.+ 842.27 (calculated), 841.94 (observed).
[0354] 4. NH.sub.2-L-DOPA(ac)-L-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 11b: The compound was synthesized with the same procedure as compound 11a.
[0355] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, 6.sub.ppm): 8.66-8.64 [b, 1H, NH 4F Phe(2)], 7.95 [b, 1H, NH 4F Phe(3)], 7.30-6.80 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.68-6.64, 6.56-6.53 [m, 3H, DOPA aromatic protons], 5.57-5.55 [m, 1H, CH DOPA], 4.56 [m, 1H, CH 4F Phe(2)], 4.47 [m, 1H, 4F Phe(3)], 3.63 [s, 3H, OMe], 3.05-2.67 [m, 6H, CH 4F Phe(2), 4F Phe(3) & DOPA]. 1.59-1.57 [s, 6H, 2COCH.sub.3].
[0356] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)): m/z=[M+Na].sup.+ 604.22 (calculated), 604.06 (observed), [M+K].sup.+ 620.20 (calculated), 619.88 (observed).
[0357] 5. NH.sub.2-L-DOPA-L-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 2: The peptide 2 was synthesized with the same procedure as peptide 1.
[0358] .sup.1H NMR (DMSO-d.sub.6, 500 MHz, .sub.ppm): 8.77-8.75 [d, 1H, NH 4F Phe(2)], 8.66-8.64 [d, 1H, NH 4F Phe(3)], 7.80 [b, 2H, OH DOPA], 7.27-7.24, 7.11-7.00 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.71-6.60 [m, 3H, DOPA aromatic protons], 5.15 [b, 2H, NH2], 4.62-4.60 [m, 1H, CH 4F Phe(2)], 4.52-4.49 [m, 1H, CH 4F Phe(3)], 3.83 [m, 1H, CH DOPA], 3.65 [s, 3H, OMe], 3.10-2.73 [m, 6H, CH 4F Phe(2), 4F Phe(3) & DOPA]. .sup.13C NMR (DMSO-d.sub.6, 125 MHz, .sub.ppm): 117.43, 170.42, 147.86, 146.63, 143.75, 143.69, 141.30, 135.47, 128.64, 127.75, 127.23, 127.13, 125.054, 121.81, 120.02, 118.04, 109.44, 108.25, 67.20, 53.02, 52.33, 47.09, 37.91, 31.94, 29.71, 25.89.
[0359] .sup.19F NMR (DMSO-d.sub.6, 470 MHz, .sub.ppm): 116.43, 116.91.
[0360] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 542.20 (calculated), 542.65 (observed), [M+Na].sup.+ 564.19 (calculated), 564.55 (observed), [M+K].sup.+ 580.16 (calculated), 580.57 (observed).
C. Synthesis of Peptide 3
[0361] 1. Boc-D-(4F)Phe-COOH 7b: The compound 7b was synthesized as compound 7a.
[0362] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 12.59 [s, 1H COOH], 7.29-7.26 & 7.12-7.08 [m, 4H, Aromatic protons], 4.10-3.57 [m, 1H, CH 4F Phe], 3.03-2.77 [m, 2H, CH 4F Phe], 1.32 [s, 9H, Boc].
[0363] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 284.12 (calculated), 284.36 (observed), [M+Na].sup.+ 306.11 (calculated), 306.28 (observed).
[0364] 2. Boc-D-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 8c: The compound was synthesized with the same procedure as compound 8a.
[0365] .sup.1H NMR (CDCl.sub.3, 400 MHz, .sub.ppm): 7.14-7.09 & 6.99-6.93 [m, 8H, Aromatic protons], 6.50 [b, 1H, NH 4F Phe(3)], 4.88 [b, 1H, NH 4F Phe(2)], 4.82-4.77 [m, 1H, CH 4F Phe(3)], 4.33 [m, 1H, CH 4F Phe(2)], 3.68 [s, 3H, OMe], 3.09-2.91 [m, 4H, CH 4F Phe(2) and 4F Phe(3)], 1.38 [s, 9H, Boc].
[0366] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na].sup.+ 485.18 (calculated), 485.88 (observed), [M+K].sup.+ 501.16 (calculated), 501.75 (observed).
[0367] 3. NH.sub.2-D-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 9c: The compound was synthesized with the same procedure as compound 9a.
[0368] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 8.71-8.67 [d, 1H, NH 4F Phe(3)], 7.25-7.21 & 7.12-7.03 [m, 8H, Aromatic protons], 5.49 [b, 2H, NH.sub.2], 4.56-4.54 [m, 1H, CH 4F Phe(2)], 3.77-3.70[m, 1H, CH 4F Phe(3)], 3.64 [s, 3H, OMe] 3.07-2.57 [m, 4H, CH 4F Phe(2) and 4F Phe(3)].
[0369] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+2H].sup.+ 364.14 (calculated), 364.26 (observed).
[0370] 4. Fmoc-L-DOPA(ac)-D-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 10c: The compound was synthesized with the same procedure as compound 10a.
[0371] .sup.1H NMR (CDCl.sub.3, 400 MHz, .sub.ppm): 7.79-7.72, 7.51-7.47, 7.42-7.38, 7.33-7.29 [d & m, 8H, Fmoc aromatic protons], 6.94-6.88 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.76-6.61[s & m, 3H, DOPA aromatic protons], 6.54 [b, 1H, NH 4F Phe(2)], 6.18 [b, 1H, NH 4F Phe(3)], 5.20 [b, 1H, NH DOPA], 4.76-4.68 [m, 1H, CH DOPA], 4.67-4.57 [m, 1H, CH 4F Phe(2)], 4.43-4.35 [m, 1H, CH 4F Phe(3)], ], 4.30-4.21 [m, 1H, CH Fmoc], 4.19-4.01 (b, 2H, CH Fmoc], 3.62 [s, 3H, OMe], 3.09-2.75 [m, 6H, CH 4F Phe(2), 4F Phe(3) & DOPA], 1.63 [s, 6H, 2COCH.sub.3].
[0372] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 804.31 (calculated), 804.74 (observed), [M+Na+H].sup.+ 827.30 (calculated), 827.32 (observed), [M+K+H].sup.+ 843.27 (calculated), 843.62 (observed).
[0373] 5. NH.sub.2-L-DOPA(ac)-D-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 11c: The compound was synthesized with the same procedure as compound 11a.
[0374] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 8.66-8.64 [b, 1H, NH 4F Phe(2)], 7.95 [b, 1H, NH 4F Phe(3)], 7.29-6.81 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.68-6.64, 6.54-6.53 [m, 3H, DOPA aromatic protons], 5.57-5.55 [m, 1H, CH DOPA], 4.60 [m, 1H, CH 4F Phe(2)], 4.48 [m, 1H, 4F Phe(3)], 3.63 [s, 3H, OMe], 2.88-2.73 [m, 6H, CH 4F Phe(2), 4F Phe(3) & DOPA]. 1.59-1.56 [s, 6H, 2COCH.sub.3].
[0375] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 582.23 (calculated), 581.93 (observed), [M+Na].sup.+ 604.22 (calculated), 604.01 (observed), [M+K].sup.+ 620.20 (calculated), 619.85 (observed).
[0376] 6. NH.sub.2-L-DOPA-D-(4F)Phe(2)-L-(4F)Phe(3)-COOMe 3: The peptide 3 was synthesized with the same procedure as peptide 1.
[0377] .sup.1H NMR (DMSO-d.sub.6, 500 MHz, .sub.ppm). 8.72-8.71 [d, 1H, NH 4F Phe(2)], 8.65-8.64 [d, 1H, NH 4F Phe(3)], 7.89 [b, 2H, OH DOPA], 7.28-7.23, 7.12-7.06 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.67-6.64, 6.49-6.47 [m, 3H, DOPA aromatic protons], 4.61-4.50 [m, 1H, CH 4F Phe(2) & Phe(3)], 3.85-3.80 [m, 1H, CH DOPA], 3.58 [s, 3H, OMe], 3.05-2.72 [m, 6H, CH 4F Phe(2), 4F Phe(3) & DOPA]. .sup.13C NMR (DMSO-d.sub.6, 125 MHz, .sub.ppm). 171.9, 170.9, 168.6, 162.5, 160.6, 158.5, 158.23, 145.7, 145.1, 133.8, 133.7, 133.5, 131.5, 125.8, 120.7, 117.3, 116.1, 115.5, 115.4, 115.3, 115.2, 54.3, 53.9, 52.4, 46.2, 37.3, 37.0, 36.2, 26.7, 25.3, 24.7.
[0378] .sup.19F NMR (DMSO-d.sub.6, 470 MHz, .sub.ppm). 116.31, 116.53.
[0379] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 542.20 (calculated), 542.51 (observed), [M+Na].sup.+ 564.19 (calculated), 564.53 (observed), [M+K].sup.+ 580.16 (calculated), 580.43 (observed).
D. Synthesis of Peptide 4
[0380] 1. Boc-D-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 8d: The compound was synthesized with the same procedure as compound 8a.
[0381] .sup.1H NMR (CDCl.sub.3, 400 MHz, .sub.ppm): 7.18-7.15 & 7.01-6.925 [m, 8H, Aromatic protons], 6.25-6.23 [d, 1H, NH 4F Phe(3)], 4.93 [b, 1H, NH 4F Phe(2)], 4.77-4.76 [m, 1H, CH 4F Phe(2)], 4.30-4.28 [m, 1H, CH 4F Phe(3)], 3.7 [s, 3H, OMe], 3.10-3.00 [m, 4H, CH 4F Phe(2) and 4F Phe(3)], 1.40 [s, 9H, Boc].
[0382] MALDI-TOF (matrix:-cyano-4 -hydroxy cinnamic acid (CHCA)):m/z=[M+Na+H].sup.+ 486.18 (calculated), 485.93 (observed), [M+K+H].sup.+ 502.16 (calculated), 502.00 (observed).
[0383] 2. NH.sub.2-D-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 9d: The compound was synthesized with the same procedure as compound 9a.
[0384] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 8.36-8.34 [d, 1H, NH 4F Phe(3)], 8.02 [b, 1H, NH 4F Phe(2)], 7.22-7.17 & 7.11-7.01 [m, 8H, aromatic protons], 4.55-4.50 [m, 1H, CH 4F Phe(3)], 4.08-3.92 [m, 1H, CH 4F Phe(2)], 3.60 [s, 3H, OMe], 3.04-2.84 [m, 4H, CH 4F Phe(2) and 4F Phe(3)].
[0385] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+2H].sup.+ 364.14 (calculated), 364.29 (observed), [M+Na+H].sup.+ 486.13 (calculated), 486.33 (observed).
[0386] 3. Fmoc-L-DOPA(ac)-D-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 10d: The compound was synthesized with the same procedure as compound 10a.
[0387] .sup.1H NMR (CDCl.sub.3, 400 MHz, .sub.ppm): 7.77-7.75, 7.55-7.53, 7.42-7.40 [d & m, 8H, Fmoc aromatic protons], 6.94-6.55 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.71-6.52 [m, 3H, DOPA aromatic protons], 6.52-6.45 [b, 1H, NH 4F Phe(2)], 6.15 [b, 1H, NH 4F Phe(3)], 5.31 [b, 1H, NH DOPA], 4.73-4.65 [m, 1H, CH DOPA], 4.64-4.56 [m, CH 4F Phe(2)], 4.51-4.42 [m, 1H, CH 4F Phe(3)], 4.24-4.11 [m, 1H, CH Fmoc], 4.19 (b, 2H, CH Fmoc], 3.61 [s, 3H, OMe], 3.08-2.72 [m, 6H, CH 4F Phe(2) 4F Phe(3) & DOPA], 1.62 [s, 6H, 2COCH.sub.3].
[0388] MALDI-TOF (matrix:-cyano-4 -hydroxy cinnamic acid (CHCA)):m/z=[M+Na+2H].sup.+ 828.30 (calculated), 828.03 (observed), [M+K+2H].sup.+ 844.27 (calculated), 844.12 (observed).
[0389] 4. NH.sub.2-L-DOPA(ac)-D-(4F)-Phe(2)-D-(4F)-Phe(3)-COOMe 11d: The compound was synthesized with the same procedure as compound 11a.
[0390] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 8.58-8.53 [d, 1H, NH 4F Phe(2)], 8.12 [d, 1H, NH 4F Phe(3)], 7.31-7.09 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.69-6.68, 6.61-6.60 [m, 3H, DOPA aromatic protons], 5.63-5.61 [m, 1H, CH DOPA], 4.61 [m, 1H, CH 4F Phe(2)], 4.52 [m, 1H, 4F Phe(3)], 3.64 [s, 3H, OMe], 3.15-2.65 [m, 6H, CH 4F Phe(2) 4F Phe(3) & DOPA]. 1.54 [d, 6H, 2COCH.sub.3].
[0391] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na].sup.+ 604.22 (calculated), 604.23 (observed), [M+K].sup.+ 620.20 (calculated), 620.12 (observed).
[0392] 5. NH.sub.2-L-DOPA-D-(4F)Phe(2)-D-(4F)Phe(3)-COOMe 4: The peptide 4 was synthesized with the same procedure as peptide 1.
[0393] .sup.1H NMR (DMSO-d.sub.6, 500 MHz, .sub.ppm): 8.80-8.77 [d, 1H, NH 4F Phe(2)], 7.95 [b, 2H, OH DOPA], 7.31-7.20, 7.12-7.03 [m, 8H, 4F Phe(2) and 4F Phe(3) aromatic protons], 6.59-6.57, 6.22-6.20 [m, 3H, DOPA aromatic protons], 5.58 [ b, 2H, free NH2)], 4.75-4.62 [m, 1H, CH 4F Phe(2)], 4.51-4.45 [m, 1H, CH 4F Phe(3)], 3.91-3.82 [m, 1H, CH DOPA], 3.62 [s, 3H, OMe], 3.08-2.62 [m, 6H, CH 4F Phe(2), 4F Phe(3) & DOPA]. .sup.13C NMR (DMSO-d.sub.6, 125 MHz, .sub.ppm): 172.01, 171.20, 168.27, 162.77, 158.59, 158.27, 157.09, 145.65, 145.02, 133.58, 133.71, 131.46, 131.45, 131.37, 125.74, 120.65, 117.37, 115.95, 115.63, 115.42, 115.31, 115.09, 54.14, 52.44, 47.97, 33.80, 25.78, 24.92. .sup.19F NMR (DMSO-d6, 470 MHz, .sub.ppm): 116.08, 116.42.
[0394] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H].sup.+ 542.20 (calculated), 542.85 (observed), [M+Na].sup.+ 564.19 (calculated), 564.55 (observed), [M+K].sup.+ 580.16 (calculated), 580.40 (observed).
E. Synthesis of Peptide 5
[0395] 1. Boc-L-(F5)Phe(2)-L-(F5)Phe(3)-COOMe 8e.
[0396] We have purchased Boc-L-(F5)Phe-COOH. We first deprotected the Boc group by treatment of TFA/DCM, then evaporate all the solvents and esterification of NH.sub.2-Phe(F5)-COOH was done by treating with thionyl chloride and methanol. Then the compound 8e was synthesized by coupling of Boc-L-(F5)Phe-COOH with NH.sub.2-L-(F5)Phe-COOMe as described for compound 8a.
[0397] .sup.1H NMR (CDCl.sub.3, 400 MHz, .sub.ppm): 6.52 [b, 1H, NH Phe(3)], 4.93 [b, 1H, NH 4F Phe(2)], 4.92-4.85 [m, 1H, CH Phe(3)], 4.42-4.29 [m, 1H, CH Phe(2)], 3.81 [s, 3H, OMe], 3.42-2.95 [m, 4H, CH Phe(2) and Phe(3)], 1.44 [s, 9H, Boc].
[0398] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na+H].sup.+ 630.11 (calculated), 630.08 (observed), [M+K+H].sup.+ 646.08 (calculated), 646.13 (observed).
[0399] 2. NH.sub.2-L-(F5)Phe(2)-L-(F5)Phe(3)-COOMe 9e.
[0400] The compound 9e was prepared as described for compound 9a.
[0401] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 8.93-8.90 [d, 1H, NH Phe(3)], 8.40 [b, 1H, free NH.sub.2], 4.72-4.70 [m, 1H, CH Phe(3)], 3.90 [m, 1H, CH Phe(2)], 3.61 [s, 3H, OMe], 3.17-2.99 [m, 4H, CH Phe(2) and Phe(3)].
[0402] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na+H].sup.+ 530.05 (calculated), 530.16 (observed), [M+K+H].sup.+ 546.03 (calculated), 646.53 (observed).
[0403] 3. Fmoc-DOPA(ac)-L-(F5)Phe(2)-L-(F5)Phe(3)-COOMe 10e.
[0404] The compound 10e was prepared as described for compound 10a.
[0405] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 8.75-8.72 [d, 1H, NH Phe(2)], 8.36-8.34 [b, 1H, NH Phe(3)], 7.88-7.26 [m, 8H, Fmoc aromatic protons], 6.79-6.67 [m, 3H, DOPA aromatic protons], 5.57-5.55 [b, 1H, NH DOPA], 4.66-4.63 [m, 2H, CH Fmoc], 4.14-4.09 [m, 3H, CH DOPA, CH Phe(2), CH Phe(3)], 3.62 [s, 3H, OMe], 3.05-2.90 [m, 6H, CH Phe(2), Phe(3) & DOPA], 1.56 [s, 6H, 2COCH.sub.3].
[0406] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na].sup.+ 970.21 (calculated), 970.22 (observed), [M+K].sup.+ 986.19 (calculated), 986.04 (observed).
[0407] 4. NH.sub.2-DOPA(ac)-L-(F5)Phe(2)-L-(F5)Phe(3)-COOMe 11e
[0408] The compound 11e was prepared as described for compound 11a.
[0409] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 8.73-8.71 [d, 1H, NH Phe(2)], 6.69-6.55 [m, 3H, DOPA aromatic protons], 5.57-5.55 [d, 1H, NH Phe(3)], 4.64-6.63 [m, 1H, CH DOPA], 4.54 [m, 1H, CH Phe(2)], 4.13-4.08 [m, 1H, CH Phe(3)], 3.61 [s, 3H, OMe], 3.15-2.67 [m, 6H, CH Phe(2), Phe(3) & DOPA], 1.60 [s, 6H, 2COCH.sub.3].
[0410] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na].sup.+ 748.15 (calculated), 748.23 (observed), [M+K].sup.+ 764.12 (calculated), 764.06 (observed).
[0411] 5. NH.sub.2-DOPA-L-(F5)Phe(2)-L-(F5)Phe(3)-COOMe 5
[0412] The compound 5 was prepared as described for compound 1.
[0413] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 9.46 [b, 1H, NH Phe(2)], 9.25 [b, 1H, NH Phe(2)], 8.39 [b, 2H, free NH.sub.2), 6.68-6.54 [m, 3H, DOPA aromatic protons], 4.69-4.65 [m, 2H, CH Phe (1) & Phe(2)], 4.55 [m, 1H, CH DOPA], 3.61 [s, 3H, OMe], 3.01-2.95 67 [m, 6H, CH Phe(2) Phe(2) & DOPA]. .sup.13C NMR (DMSO-d.sub.6, 100 MHz, .sub.ppm): 193.6, 158.5, 158.2, 144.3, 140.8, 139.5, 133.7, 129.9, 128.5, 127.8, 124.4, 53.8, 44.2, 33.8, 30.5, 29.4, 22.6, 17.6. .sup.19F (DMSO-d.sub.6, 470 MHz, .sub.ppm): 141.7, 142.4, 157.6, 163.1, 163.4.
[0414] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na].sup.+ 748.15 (calculated), 748.23 (observed), [M+K].sup.+ 764.12 (calculated), 764.06 (observed).
F. Synthesis of Peptide 6
[0415] 1. Boc-L-DOPA-COOH:
[0416] The compound was synthesized as compound 7a.
[0417] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 9.13 (b, 2H, 2OH], 7.35-7.33 [d, 1H, NH DOPA], 7.03-6.88[m, 3H, DOPA aromatic protons], 4.45-4.37 [m, 1H, CH DOPA], 3.22-2.92 [m, 1H, CH DOPA], 1.75 [s, 9H, OMe].
[0418] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na].sup.+ 320.11(calculated), 320.51 (observed), [M+K].sup.+ 336.08 (calculated), 336.29 (observed).
[0419] 2. Boc-L-DOPA-L-(4F) Phe-COOMe:
[0420] The compound was synthesized as compound 8a.
[0421] .sup.1H NMR (CDCl.sub.3, 400 MHz, .sub.ppm): 7.26-7.24 [d, 1H, NH Phe], 6.90-6.50 [m, 7H, all aromatic protons], 5.24 [b, 1H, NH DOPA], 4.82-4.77 [m, 1H, CH DOPA], 4.36 [b, 1H, CH Phe], 3.64 [s, 3H, OMe], 2.99-2.87 [m, 4H, CH DOPA & Phe], 1.42 [s, 9H, Boc].
[0422] MALD I-TO F (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+Na+H].sup.+ 500.18 (calculated), 500.02 (observed), [M+K+H].sup.+ 516.16 (calculated), 516.24 (observed).
[0423] 3. NH.sub.2-L-DOPA-L-(4F) Phe-COOMe 6:
[0424] This compound was synthesized as described for 9a.
[0425] .sup.1H NMR (DMSO-d.sub.6, 400 MHz, .sub.ppm): 8.92 & 8.81 [s, 2H, 2OH], 8.02 [b, 2H, free NH.sub.2], 7.27-7.09 [m, 4H, aromatic proton Phe], 6.67-6.48 [m, 3H, aromatic protons DOPA], 4.58-4.52 [m, 1H, CH DOPA], 3.90-3.86 [b, 1H, CH Phe], 3.61 [s, 3H, OMe], 3.08-2.67 [m, 4H, CH DOPA & Phe]. .sup.13C NMR (DMSO-d.sub.6, 100 MHz, .sub.ppm): 171.4, 168.7, 162.7, 160.4, 158.5, 145.6, 145.0, 133.3, 133.2, 131.4, 125.6, 120.6, 117.2, 115.9, 115.5, 115.3, 54.1, 53.9, 52.4, 41.0, 36.8, 36.2, 23.6.]. .sup.19F NMR (DMSO-d.sub.6, 470 MHz, .sub.ppm): (116.25-116.29).
[0426] MALDI-TOF (matrix:-cyano-4-hydroxy cinnamic acid (CHCA)):m/z=[M+H] 377.15 (calculated), 377.25 (observed), [M+Na].sup.+ 399.13 (calculated), 399.24 (observed).
[0427] Another two exemplary peptide derivatives have been synthesized using solid or solution phase synthesis. The purity and identify of the peptides was determined using HPLC and MS spectrometer.
##STR00027##
Substrates
[0428] The following substrates were coated with the peptides in the course of the research: a silicon wafer, a silicon wafer with a 100 nm titanium layer, a 400 mesh Copper-formvar/carbon grids.
Surface Modification 1010 mm Ti surfaces were sonicated 5 minutes in ethanol, washed with TDW and dried under nitrogen. The clean surfaces were dipped in a peptide solution (0.5 mg/mL in methanol) and left for overnight at RT. Then, they were rinsed extensively with methanol and dried under nitrogen.
[0429] The desired substrate was cut into a 1 cm.sup.2 square and cleaned by sonication (5 min in acetone and 5 min in isopropanol). Then, the substrate was immersed in a peptide solution at a concentration of 0.1 mg/ml and incubated over night at room temperature.
[0430] Following incubation, the substrate was rinsed by immersion in water, dried and store in a dissector until use.
Contact Angle Measurements
[0431] Contact angle measurements were carried out using a Theta Lite optical tensiometer (Attension, Finland). Each experimental measurement consisted of three repeats, and the reported angles were averaged.
AFM Analysis
[0432] Freshly cleaved mica surfaces were dipped overnight in different peptide solution at a concentration of 0.5 mg/mL in methanol. Then, the surfaces were washed with fresh methanol and dried under N.sub.2. AFM images were taken in AC mode with Si.sub.3N.sub.2 tip with spring constant 3N/m in JPK instrument (NanoWizard 3).
ATR-FTIR
[0433] ATR spectra were recorded using FT-IR (Thermo scientific, Model Nicolet 6700) with Ge-ATR arrangement (Harrick Scientific's VariGATR). For all the surfaces spectra were collected with applied force of 350 N, at 4 cm.sup.1 resolution with 3000 scans averaged signal and an incident angle of 65.
QCM-D
[0434] QCM-D (Q-sense, Biolin Scientific) was used for the study of peptide adhesion onto Ti surface. Measurements were performed in a flow module E1 system. Ti sensors with a fundamental resonant frequency of 5 MHz were also purchased from Q-sense and used as supplied. Prior to each experiment Ti sensors were cleaned with Oxygen/Plasma (Atto, Diener Electronic), followed by rinsing with 2% SDS and TDW and finally dried under N.sub.2. All QCM-D experiments were performed under flow-through conditions using a digital peristaltic pump (IsmaTec Peristaltic Pump, IDEX) operating in pushing mode. The studied solutions were injected to the sensor crystal chamber at a rate of 0.1 mL/min. Organic solvent compatible tube and O-ring were used for the flow system. Peptides were dissolved in MeOH to a concentration of 0.5 mg/mL.
[0435] The data were fitted with Sauerbrey model. According to this model the mass of adhering layer is calculated as
[0436] where C=17.7 ng Hz-1 for 5 MHz quartz crystal and n=1,3,5,7,9,11,13 overtone number.
X-Ray Photoelectron Spectroscopy (XPS)
[0437] The X-ray Photoelectron Spectroscopy (XPS) measurements were performed using a Kratos AXIS Ultra X-ray photoelectron spectrometer (Kratos Analytical Ltd., Manchester, UK). Spectra were acquired using the A1-K monochromatic X-ray source (1,486.7 eV). Sample take-off angle was 90 (i.e. normal to the analyzer). The vacuum pressure in the analyzing chamber maintained to 2.Math.10.sup.9 Torr. High-resolution XPS spectra were collected for F 1 s, O 1 s, C 1 s and Ti 2 peaks with pass energy 20 eV and 0.1 eV step size. Data analyses were done using the Kratos Vision data reducing processing software (Kratos Analytical Ltd.) and Casa XPS (Casa Software Ltd.).
Evaluation of the Layer Thickness by XPS
[0438] Using the XPS measurements, it is possible to calculate the thickness of the assembled layers. We have done so using the standard attenuation relations of the photoelectrons emerging from different sample depths. The thickness calculation is based on the Briggs et al. method and others. For the Au substrate, the overlay thickness d (nm) expressed as:
[0439] where I.sub.s and I.sub.o are the intensities of the peaks from the substrate and the overlayer respectively, the substrate is the Ti 2p signal, and layer is the sum of the intensities of C 1 s, O 1 s, N 1 s and F 1 s peaks, is the takeoff angle (in our case sin =1) and N.sub.s and N.sub.o are the volume densities. The inelastic mean free paths (IMFPs) parameters for substrate (.sub.s) and for the overlayer (.sub.o) assumed as 2.18 nm and 3.3 nm respectively. Calculated, using S. Tougard QUASES-IMFP-TPP2M software (http://www.quases.com). Inelastic electron mean free path calculated from the Tanuma, Powell and Penn algorithm [Penn, 1994].
Ellipsometry
[0440] The thickness of the peptide-based coating was measured using -SE spectroscopic ellipsometer (J. A. Woollam, Lincoln, Nebr., USA). Measurements were performed at wavelengths from 380 to 900 nm, at a 70 angle of incidence. The optical properties of the substrate were fitted using standard Si with a 50 nm Ti. The thickness of the layers and refractive indices were fitted according to the Cauchy model. The coefficients of the Cauchy equation were initially fixed for organic layers (A.sub.n=1.45, B.sub.n=0.01 and C.sub.n=0), and an angle offset was permitted. Then, the parameters were allowed to be fitted to determine more accurate values.
Protein Adsorption
[0441] 50 L of single protein solution of BSA, lysozyme and fibrinogen (150 M in PBS) were pipetted onto the substrate in a petri dish. The plate was placed in a humidified incubator at 37 C. for 2 hours. The substrates were then rinsed 3 times with PBS (pH=7.43, 10 mM Nacl, 150 mM), and transferred into eppendorfs containing 1 mL of 2% (w/w) SDS. The samples were shaken for 60 minutes and sonicated for 20 minutes at room temperature to detach the adsorbed proteins. Protein concentrations in the SDS solution were determined using the Non-interfering protein assay (Calbiochem, USA) according to the instructions of the manufacturer, using a microplate reader (Synergy 2, BioTek) at 480 nm. All measurements were performed in triplicates and averaged.
[0442] To determine if the coated surfaces prevents protein adsorption, each of the examined substrates was incubated with a fluorescently-labeled protein (FITC-BSA) for 1 hour. After incubation, the substrate was rinsed exhaustively to wash access protein and the fluorescent signal was recorded using a fluorescent microscope.
Biofilm Growth
[0443] Pseudomonas aeruginosa and Eschrichia coli were grown in TSB medium (Fluka) and LB medium (BD Difco) respectively overnight at 37 C. in loosely capped tubes with agitation (120 rpm) to the stationary phase. Then, cultures were diluted to 10.sup.8 CFU/mL with TSB, and 3 mL of each culture were transferred to a Petri dish. Substrates were placed horizontally in the plate and incubated at 37 C. for 9 hours for the formation of biofilm by P. aeruginosa and 96 hours for the formation of biofilm by E. coli. Every 4.5 hours the medium was replaced with a fresh one to ensure sufficient supply of nutrients.
[0444] BL21 E. Coli strain was grown in LB broth to a steady state. The examined surfaces were immersed in the bacterial growth culture. After 1 hour of incubation the substrates were thoroughly rinsed with sterile PBS buffer (at least 20 ml for 1 cm.sup.2 surface), in order to get rid of non adherent cells, and placed in test tube containing a clean buffer. In order to determine the number of bacteria adsorb onto the substrate, the test tube was placed in an ultrasonic bath for 5 min. The buffer was then diluted 10 and 100, spread on LB agar plates and incubate over night at 37 C. The number of cell forming units (CFU)/colonies was counted.
Crystal Violet Assay
[0445] After incubation, the substrates were gently rinsed 3 times with di-ionized water, and stained with 0.2% crystal violet for 15 minutes. The stained samples were washed with running water and left to dry in air. Eventually the bound dye was eluted with 30% acetic acid. Absorbance values were recorded at 590 nm in a microplate reader (Synergy 2, BioTek). All measurements were performed in triplicates and averaged.
Results
[0446] The molecular structure of the studied peptides 1 to 6 is presented below. We chose to explore two variation of the peptide: one contains only one fluorine atom on each of the benzene rings and the other contains five. In addition, we studied peptides with either L or D amino acids, since L amino acids are more abundant in natural systems and D amino acid resist common proteases and can present an additional stability. The third amino acid of the peptide is 3,4-dihydroxy-L-phenylalanin (DOPA) (
[0447] To coat a substrate (e.g. gold, silicon, titanium, glass or polystyrene) with the peptide, we cleaned a bare substrate (11 cm.sup.2) by sonication in ethanol, washing with water and drying under nitrogen. We incubated the substrates for several hours (3-10 hours) in a 0.5 mg/mL peptide in methanol. We chose this concentration of peptide since it formed a substantial coating that gave a good signal in various characterization methods. After incubation, we thoroughly washed the substrate with methanol and dried it under nitrogen. Due to the hydrophobic moieties of the peptides, water could not be used as a solvent system despite its high polarity. We used methanol as the solvent since it dissolved the peptide completely, and at the same time allowed it to adhere the substrate. Since methanol is a toxic solvent, we also examined other solvents with different polarities. When we used solvents, such as acetone, ethanol and isopropanol, with polarities that resemble the polarity of methanol, the peptide-based coating self-assembled in a similar manner to the methanol solvent system (
[0448] In order to determine if the peptide indeed generated a Teflon-like layer on the substrates and increased their hydrophobicity we measured their contact angle. As we assumed, the modified surfaces (i.e. gold, silicon, titanium and stainless steel) exhibited an increase in the contact angle indicating an increase in the substrate hydrophobicity (
[0449] To characterize the morphology of the modified surfaces we performed AFM topography analysis to mica and Ti surfaces coated with the different peptides (
[0450] We also studied if the peptides indeed present on the substrate using ATR-FTIR spectroscopy. An informative IR frequency range is 3500-3200 cm.sup.1 as it corresponds to the NH stretching vibrations and can indicate on the formation of a peptide film on the substrate. For a titanium surface modified with peptide 1, the NH stretching frequency occurred at 3330 cm.sup.1. This IR frequency suggests the binding of the peptide to the substrate (
[0451] The IR region between 1800 cm.sup.1 and 1500 cm.sup.1 is related to the stretching band of amide I and can indicate on the secondary structure of the peptides. The ATR-FTIR spectra of a substrate coated with peptide 1 appeared at 1685 cm.sup.1 and 1629 cm.sup.1 indicating an anti parallel sheet secondary structure. For peptide 2, 3 4 and 5 the amide I peak appeared at (1687 cm.sup.-31 1, 1616 cm.sup.1), (1687 cm.sup.1, 1612 cm.sup.1) (1686 cm.sup.1, 1619 cm.sup.1) and (1679 cm.sup.1, 1605 cm.sup.1) respectively indicating the same type of peptide secondary structures on the substrates (
[0452] Using quartz crystal microbalance with dissipation mode (QCM-D) we studied the real-time adhesion of the peptides to titanium substrates. Each of the peptides dissolved in Me0H were injected into a flow cell containing a Ti coated sensor. The injection of peptide 1 resulted in changes in both frequency (f) and dissipation (D), this indicates on the peptide binding to the titanium substrates. Upon washing with MeOH, we only observed small changes in the frequency and dissipation; this indices the formation of a stable film on the surface (
TABLE-US-00001 TABLE 1 Quantitative analysis of peptides 1-6, according to the Sauerbrey model. Peptide Peptide Peptide Peptide Peptide Peptide 1 2 3 4 5 6 Thickness 9.11 0.05 7.3 0.3 5.4 0.5 5.6 0.3 3.4 1.7 () 0.5 0.3 Mass/ 72.1 0.4 57 3 43 4 45 2 27 3 13 2 Area (ng/cm.sup.2) Density 767 4 824 760 769 717 805 (Kg/m.sup.3) 13 37 30 44 15
[0453] It should be noted that the QCM-D experiments lasted 40 minutes and therefore measured only the beginning of the coating process. Using X-ray Photoelectron Spectroscopy analysis we were able to characterize surfaces that underwent a prolonged incubation with the peptide to ensure the complete modification of the Ti substrates. In comparison to a bare Ti, the signals resulted from the modified substrates indicated the presence of carbon, nitrogen and fluorine. (
[0454] We also determined the thickness of the coating using ellipsometry. By fitting the measurement to Cauchy film model, which is suitable for organic coatings, we evaluated a thickness of 3.410.05 nm, 3.460.04, 3.480.03 nm, 3.360.05 nm , 5.20.1 nm and 3.660.04 nm for peptides 1-6 respectively. These findings are with agreement with the results obtained by XPS analysis.
[0455] The process of biofouling initiates by the adsorption of bioorganic molecules, in the form of polysaccharides or proteins, onto a substrate. These bioorganic molecules mediate the subsequent attachment of organisms. We, therefore, investigated the resistance of the peptide-based coating to protein adsorption. A bare Ti surface and a coated Ti substrate were incubated in a protein (either Bovine Serum Albumin (BSA), or lysozyme) solution at a concentration of 150 M for 2 hours at 37 C. To determine the adsorbed amounts of the proteins on the substrates we used the non-interfering protein assay kit. The adsorbed amounts of BSA and lysozyme on the peptide coated substrates were negligible and below the detection limit of the kit (
[0456] To assess the bacterial attachment to the surface, bare and peptide coated substrates were incubated in inoculums of P. aeruginosa and E. coli for 9 and 96 hours respectively. These incubation times allowed the formation a biofilm by the different bacterial strains. After incubation, we washed and dried the substrates, and stained them with 2% (w/w) crystal violet. Crystal violet dye is part of the gram staining of bacteria and stains bacteria in purple. Using an optical microscope we observed a thick and dense purple layer on the bare titanium surface which indicated a thick bacterial coverage of the substrate, while on the coated titanium we only detected sparse bacteria (
Morphological Characterization of the Coated Substrates
[0457] The peptide films were prepared by the dip-coating. Unless noted otherwise, all experiments were carried out with a peptide concentration of 0.01 mg/mL. The films were deposited on either silicon wafers, silicon wafers coated with a 100 nm of titanium layer or 400 mesh copper-formvar/carbon grids. Using electron microscopy, the folds and defects in the film were identified, indicating the formation of a film on the substrate (
Proteins Adsorption to the Peptide-Coated Surfaces
[0458] In order to determine if the peptide-based coating indeed resisted protein adsorption, the modified surfaces were incubated with FITC-BSA (a fluorescently-labeled protein). After a thorough washing, the presence of the adsorb protein to the surface was analyzed using fluorescence microscopy. Results from this experiment clearly showed a strong fluorescence signal indicating on an extensive protein adsorption on the bare silicon substrate when compared to the weaker signal from the modified surface.
Antifouling Activity of the Peptide-Coated Surfaces
[0459] To determine the antifouling activity of the peptides, the modified silicon surfaces were placed in BL21 E. Coli bacterial culture. The surfaces were then rinsed, sonicated in a buffer and the buffer was spread on agar plates and cultivated. The colonies were counted and the number of colonies forming units (CFUs) was calculated.
[0460] As indicated in Table 1, the number of CFU on the modified surface was lower by two orders of magnitudes when compared to the bare silicon surface.
TABLE-US-00002 TABLE 2 CFU per cm.sup.2 of Si Bacterial strain Bare Si Si coated with the peptide E. coli 1.1 10.sup.5 1.3 10.sup.3
Surface Coverage
[0461] In order to establish the ability of the peptide to cover a substrate, peptide 8 was synthesized in such a fusion that an amine group would be located in a non-adjacent position to DOPA. Then, the peptide was conjugated to Fluorescein through its amine termini and deposited on a titanium substrate by dip coating. Results indicated the absence of florescent signal from a bare titanium substrate and a strong signal from the modified surface. This indicated that the peptide indeed coat the substrate.
[0462] Perfluorinated Derivatives
[0463] A perfluorinated DOPA (herein referred to as f-DOPA) demonstrated substrate-independence and superhydrophobicity as a coating. The in situ superhydrophobic, selfcleaning coating composed of the material was applied to various substrates, such as gold, glass, polydimethylsiloxane (PDMS), PET, vanadium foil (V foil), zinc foil (Zn foil), and titanium dioxide (TiO.sub.2). The manipulation of the water flow was also made possible by this coating approach.
[0464] As shown in the schematic below, the perfluoro group was attached onto the carboxylic acid in L-DOPA to increase the hydrophobicity of the resulting polymer films. Briefly, L-DOPA was coupled with 1H,1H,2H,2H-heptadecafluoro-1-decanol via esterification with protection/deprotection of the hydroxy and amine groups. Prior to substrate coating, the oxidation process of f-DOPA in solution was investigated by UV-Vis spectroscopy. The acetonitrile stock solution of f-DOPA (4 mg mL.sup.1) was diluted to 0.05 mg mL.sup.1, and 0.5 mL of the diluted solution was used for reliable UV-Vis analysis.
##STR00028##
[0465] The UV-Vis spectrum of f-DOPA showed a characteristic peak at 283 nm, corresponding to the symmetry forbidden transition (La-Lb) of the catechol moiety in f-DOPA. The peak intensity at 283 nm decreased upon the addition of the aqueous sodium periodate (NaIO.sub.4) solution (1.25 mM; 0.13 mL). As the reaction progressed, a new peak appeared as a shoulder over the peak at 283 nm and was observed clearly at 330 nm after 2 h. The peak at 330 nm indicated the oxidation of the catechol moiety in f-DOPA to o-quinone. In addition, the formation of dopaminechrome was evidenced by a weak, broad peak at around 480 nm. The UV-Vis spectra, therefore, confirmed that the reaction conditions employed were suitable for f-DOPA coating.
[0466] Various substrates were coated with polymerized f-DOPA, such as gold, glass, PDMS, PET, V foil, Zn foil, and TiO.sub.2. To the acetonitrile solution of f-DOPA (4 mgmL.sup.1) containing a substrate an aqueous solution of NaIO.sub.4 (100 mM) at the final ratio of 15:2 (v/v) was added. After 12 h, the substrate was washed with acetonitrile, and the coating process was repeated with a fresh f-DOPA solution. After coating, all the substrates became brownish or dark-colored, indicating the formation of f-DOPA films, except for the V foil that was black before coating.
[0467] X-ray photoelectron spectroscopy (XPS) analysis also confirmed the successful coating of all the substrates tested. For example, the characteristic peaks of f-DOPA at 688.0 (F 1 s) and 290.7 eV (C 1 s) were observed after coating on gold, and the surface elemental ratio (C/F) was 1.03, which was nearly consistent with that of f-DOPA (1.12). In addition, Au peaks at 83.6 (Au 4f7/2) and 87.3 eV (Au 4f5/2) disappeared after f-DOPA coating, indicating the formation of thick f-DOPA films with a thickness of 10 nm or more.
[0468] The intensity of the characteristic XPS peak(s) for glass, PDMS, V foil, Zn foil or TiO.sub.2 also decreased significantly after coating. In addition to the incorporation of fluorine, the f-DOPA films were structurally heterogeneous (i.e., rough), fulfilling the basic characteristics of superhydrophobic surfaces. The scanning electronmicroscopy (SEM) images showed that the substrate was coated with f-DOPA microparticles, ranging from 1.0 to 2.0 mm in diameter, which were hierarchically composed of smaller nanoparticles. The root-mean-square roughness was measured to be 533.92 nm in the atomic force microscopy (AFM) image.
[0469] Without wishing to be bound by theory, it is believed that the coating of the polymerized f-DOPA involved the same processes as the polydopamine coating, which was thought to result from the presence of the catechol and amine groups although the adhesion strength would be lower than that of polydopamine due to the perfluorinated group in f-DOPA.
[0470] The static water contact angles before and after coating were also tested, confirming that all the f-DOPA-coated substrates were superhydrophobic. Regardless of different contact angles before coating, the coating made the contact angle of all the substrates to be about 1551. Interestingly, PTFE, which exhibits the low surface energy (19.1 mJ m.sup.2) and is non-sticky, also became a self-cleaning, superhydrophobic surface with a static water contact angle of 1491 after f-DOPA coating.
[0471] The wetting properties were further investigated by the tilting-plate method that measured the dynamic contact angles, because it was essential in the confirmation of self-cleaning properties to investigate the dynamic water contact angles and surface free energies. The advancing (y.sub.adv) and receding (y.sub.rec) water contact angles of f-DOPA-coated substrates were measured, and the contact angle hysteresis (i.e., (y.sub.adv-y.sub.rec)) of each substrate was calculated. For example, the gold substrate, after coating, showed a low contact angle hysteresis of 9.91. A water droplet on the substrate easily rolled off at a tilt angle of 5.31, which is clear evidence of self-cleaning properties. All other f-DOPA-coated substrates also showed the low contact angle hysteresis and self-cleaning properties with low sliding angles of 2.51 to 6.71. In addition, the surface free energy (gS) was calculated based on the Owens-Wendt geometric mean equation that divides the surface free energy into the dispersive (gDS) and polar (gPS) ones.
[0472] The surface free energy (gS; gS=gDS+gPS) of each surface was determined by measuring the contact angles with water and diiodomethane (CH.sub.2I.sub.2). The surface free energy of the f-DOPA-coated gold surface was calculated to be 0.279 mJ m.sup.2, and the other substrates have the surface free energies between 0.2 and 0.9 mJ m.sup.2. These values are extremely low, probably because of both the structural roughness and the incorporated perfluoro groups. For comparison, the surface free energy of a smooth surface modified with CF.sub.3 groups in hexagonal close packing was reported to be 6.7 mJ m.sup.2. In this system, the simple f-DOPA coating, therefore, led to structurally heterogeneous rough films of perfluorinated materials without any further treatments, which definitely contributed to reduction of surface free energy and realization of superhydrophobic, selfcleaning properties.
[0473] Interestingly, the wetting characteristic of the f-DOPA films was changed to non-superhydrophobic by simple O.sub.2-plasma treatment: after 1 min of treatment, the static water contact angle of the f-DOPA-coated gold substrate was changed from 154.451 to 124.181. The spatio-selective oxidation of the film could be utilized for manipulation of water droplets and flow. For example, when a small square area of the film was made relatively hydrophilic by plasma treatment, a water droplet was captured at that hydrophilic area after fast rolling on superhydrophobic area with slight tilting. Droplet-based microfluidic channels could be fabricated with ease, demonstrated by a hydrophilic line on the superhydrophobic surface.