WO1997024387A1 - Fluorine containing poly (beta-hydroxyorganoate) - Google Patents

Abstract

The present application provides fluorinated poly(β-hydroxyalkanoates), PHAs, of general formula (I), wherein R is an organic group containing 1-30 carbon atoms; m is about 0-7; n is about 0-10; x is at least about 10; and z is about 0-1,000; coating compositions and cured coatings containing these polymers.

Description

FLUORINE CONTAINING P0LY( BETA-HYDROXYORGANOATE)

Statement of Government Rights

This invention was made with government support from the National Science Foundation under Grant No. DMR-9057233. The government may have certain rights in this invention.

Background ofthe Invention

"PHAs", are a class of β-monoalkyi- substituted-poiy-β-esters that are naturally occurring in a wide variety of bacterial rmcroorgaiiisrns. These polyesters function as ϋrtπu^ular carbon ar i energy storage materials. They are biodegradable polymers. This family of microbial polyesters have the following general structure wherein the repeat unit chiral centers have [R]-stereochemical configurations.

β-linked PHA structure, R = side chain substituent

Thus, the polymers are isotactic and optically active. PHAs are normally built from 3-hydroxyalkanoates, although much work has been carried out in preparing PHAs ∞ntaining 4-hydroxybutyrate repeat units.

Various bacteria, e.g., Pseudomonas oleovorans, Pseudomonas putida, Pseudomonas aeruginosa, Alcaligenes eutrophus, Rhodospirilhon rubrum, Bacillus megaterium are capable of metabolizing alkanes, alkanols, alkanoic acids, alkenes, alkenols, alkenoic acids, and esters, for example, to poly(β-hydroxyorganoate)s when grown under riutaent-lixrriting conditions. For example, when P. oleovorans is grown under nitjogen-lύniting conditions on the substrates hexane, heptane, octane, etc., through dodecane, rχύ^β-hydπ>xyorganoate)s are formed which, depending on the growth substrate used, contain variable amounts ofthe monomer units. In fact, P. oleovorans is capable of producing very unusual poly(β-hydroxyorganoate)s, such as those containing relatively long w-alkyl pendant groups. By using combinations of feedstocks, e.g., a combination of octane and nonane or octanoic and nonanoic acids, copolymers can be obtained, e.g., copolymers of β-hydroxyoctanoates and β-hydroxynonanoates.

A wide range of 3-hydroxyalkanoate repeat unit structures having functional side groups have been incoφorated into product polyesters by bacterial polymerization systems. Examples of 3-hydroxyalkanoate functional side groups incoφorated by the medium side chain (n-propyl to n-nonyl) producing strain Pseudomonas oleovorans ATCC 29347 include vinyl, cyano, phenyl, and phenoxy. Pseudomonas putida (KT 2442) has been used to produce PHAs with medium chain length 3-hydroxyalkanoate repeat units that contain multiple double bonds. P. putida was also found useful for the incoφoration of 3-hydroxy-6- cyanophenoxy-hexanoate repeat units. Such investigations of carbon source incoφoration are of fundamental importance since they probe the flexibility of enzymes involved in PHA synthesis. Also, control over microbial polyester structure is of practical importance because this creates opportunities for the design of polvmer structures to meet specific performance requirements.

Fluorochemistry to prepare fluoropolymers has proven useful in modulating performance characteristics relative to their hydrocarbon analogues to achieve higher heat and oil resistant materials having superior surface properties with low surface tension, high lubricity (i.e., low friction coefficient), and non- wetting properties (e.g., low water absoφtion). More recently, there has been increasing interest in exploring fluorine containing compounds for use as bioactive agents and in the development of materials for diagnostic devices. Also, fluorinated amino acids have been used in cultivation media for the biosynthesis of designer fluorinated proteins. For example, fluorinated proteins containing para- fluoro-phenylalanine units have been synthesized using a strain of Escherichia coli that required phenylalanine in the growth medium.

Recently PHAs containing mono-halogenated side groups, specifically fluorine, bromine, and chlorine, have been produced in P. oleovorans from ω-mono-halogenated alkanes. P. oleovorans has been cultivated using various mixtures of nonane and 1-fluorononane in bulk one-stage fermentations to produce copolyesters containing 3-hydroxy-9-fluorononanoate with a terminal CH₂F moiety. See, for example, C. Abe, et al., Polymer Communications. 31. 404 (1990). Such compounds, however, do not have the desired properties of fluoropolymers where there is a terminal CF₃ group (e.g., low surface energy, high lubricity, and non-wetting properties). Thus, what is needed are additional fluoropolymers, particularly those having low surface tension, high lubricity, and non-wetting properties, preferably produced by microbial polyester producing organisms.

Summary ofthe Invention

The present invention provides a fluorine-containing poly(β- hydroxyorganoate) ofthe general formula:

wherein: R is an organic group containing 1-30 carbon atoms (preferably 1-20 carbon atoms); m is about 0-7; n is about 0-10; x is at least about 10; and z is about 0-1,000. A preferred embodiment ofthe invention is a fluorine-containing poly(β- hydroxyorganoate) ofthe above general formula wherein: m is about 0-7; n is about 0- 10; R is -(CH₂)_pCH₃ and p is about 0-15; x is about 50-500; and z is about 50-100. The present invention also provides a fluorine-containing poly(β- hyάroxyorganoate) coating composition and a cured coating comprising a fluorine- containing poly(β-hydroxyorganoate) ofthe general formula:

wherein: R is an organic group containing 1-30 carbon atoms (preferably 1-20 carbon atoms); m is about 0-7; n is about 0-10; x is at least about 10; and z is about 0-1000.

These polymers can be used in a variety of applications. For example, those having a generally high degree of fluorination also have generally high release properties and a generally non-wetting behavior. Thus, preferred fluorine- containing PHAs ofthe present invention are suitable for use in films having release properties that can be useful as release liners, for example.

As used herein, the term "organic group" means a hydrocarbon group that is classified as an aliphatic group, cyclic group, or combination of aliphatic and cyclic groups (e.g., aralkyl and alkaryl groups). The term "aliphatic group" means a saturated or unsaturated, linear or branched hydrocarbon group. This term includes alkyl, alkoxy, alkenyl, vinyl, and alkynyl groups. The term "alkenyl group" means an urisaturated, linear or branched hydrocarbon group with one or more double bonds. The term "cyclic group" means a closed ring hydrocarbon group that is classified as an alicyclic group, aromatic group, or heterocyclic group. The term "alicyclic group" means a cyclic hydrocarbon group having properties resembling those of aliphatic groups. This term includes cycloalkyl, cycloalkenyl, and cycloalkynyl groups. The term "aromatic group" or "aryl group" means a mono- or polynuclear aromatic hydrocarbon group. The term "heterocyclic group" means a closed ring hydrocarbon in which one or more ofthe atoms in the ring is an element other than carbon (e.g., sulfur, nitrogen, oxygen, etc.).

Brief Description ofthe Drawings Figure 1. The 500 MHz H NMR spectrum in chloroform-d ofthe PHA containing 9.8 mole-% fluorinated repeat units produced by P. putida after a 2- day second stage cultivation time using NA/NFNA 7.5/7.5 (mM) as cosubstrates.

Figure 2. The 125 MHz C NMR spectrum in chloroform-d ofthe PHA containing 9.8 mole-% fluorinated repeat units produced by P. putida after a 2-day second stage cultivation time using NA NFNA 7.5/7.5 (mM) as cosubstrates. Figure 3. The 470 MHz ¹⁹F NMR spectrum in chloroform-d ofthe PHA containing 9.8 mole-% fluorinated repeat units produced by P. putida after a 2-day second stage cultivation time using NA/NFNA 7.5/7.5 (mM) as cosubstrates. Figure 4. DSC thermograms (first heating scans) of the PHAs isolated from P. oleovorans and P. putida using either NA or NA/NFNA mixtures as carbon sources during second stage cultivations: (a) 0 mole-% NF3HNA (from P. oleovorans), (b) 0 mole-% NF3HNA (from P. putida), (c) 8.8 mole-% NF3HNA (from P. oleovorans) and (d) 12.4 mole-% NF3HNA (from P. putida). Figure s. DSC thermograms (second heating scan after quenching from the melt) ofthe PHAs isolated from P. oleovorans and P. putida using either NA or NA/NFNA mixtures as carbon sources during second stage cultivations: (a) 0 mole-% NF3HNA (from P. oleovorans), (b) 0 mole-% NF3HNA (from P. putida), (c) 8.8 mole-% NF3HNA (from?, oleovorans) and (d) 12.4 mole-% NF3HNA (from P. putida).

Figure 6. The surface contact angle of PHAs produced by P. oleovorans and P. putida using NA/NFNA 7.5/7.5 (mM) cosubstrates as a function of NF3HNA contents.

Detailed Description

The present invention provides fluorine-containing poly(β- hydroxyorganoate)s, i.e., pory(hydroxyorganoate)s or poly(3-hydroxyorganoate)s, or a mixture of various types of such polymers that can be used for a variety of applications. Preferably, these polymers have a generally high degree of fluorination, which typically results in such desirable properties as a generally low surface energy, generally high lubricity or low friction coefficient such that they have generally high release properties, and a generally non-wetting behavior. Thus, preferred fluorme-containing PHAs ofthe present invention are suitable for use in films having release properties that can be useful as antigrafiti coatings, and as release liners in numerous applications, such as fog and dew resistant coatings. The polymers ofthe present invention, and the compositions and coatings containing such polymers, can be disposed of in a generally environmentally sound manner (e.g., in a municipal solid waste compost site). Although these polymers, compositions, and coatings are generally resistant to oxidative and photochemical degradation, they will undergo degradation upon exposure to biologically active environments.

The fluorine-containing PHAs ofthe present invention have the following general formula:

wherein: R is an organic group (i.e., an aliphatic group, a cyclic group, or a combination of aliphatic an cyclic groups), containing 1-30 carbon atoms (preferably 1-20 carbon atoms); m is about 0-7, preferably about 1-5, and more preferably about 2-4; n is about 0- 10, preferably about 0-6, more preferably about 2-4, and most preferably about 2-3 ; x (for the fluorine-containing repeat unit) is at least about 10, preferably about 10-1,000, more preferably about 30-500, and most preferably about 50-500; and z (for the hydrocarbon repeat unit) is about 0-1,000, preferably about 10-1,000, more preferably about 20-500, and most preferably about 50-100. Preferably R is an aliphatic group having 1-30 carbon atoms, more preferably an alkyl group having 1-20 carbon atoms, and most preferably a - (CH^_pdrk group wherein p is about 0-15, preferably about 3-7, and more preferably about 4-6. For fluor e-containing homoporymers, z is 0. As used herein, the term "copolymer" includes polymers containing more than one type of repeat unit. That is, the term "copolymer" includes teφolymers, tetraporymers, etc. In any one polymer ofthe present invention, the fluorine-containing repeat unit (i.e., fluorocarbon monomeric unit) will typically remain the same. For copolymers, however, the hydrocarbon repeat unit can (and typically will) vary. For example, when R is a -(CH₂)_pCH₃ group, p can vary from monomer unit to monomer unit within any one polymer chain. Thus, a preferred class of polymers ofthe present invention can include a mixture of monomeric units, wherein the side chain ofthe hydrocarbon repeat unit contains anywhere from one carbon to sixteen carbons. Also, polymers ofthe present invention could include hydrocarbon repeat units containing alkyl side chains and alkenyl side chains in the same polvmer. Furthermore, the fluorine-containing poryφ- hydroxyorganoate)s ofthe present invention can be either random or block copolymers, depending on the relative reactivities ofthe various monomers. However, they are generally random copolymers, particularty for those that are prepared by bacteria.

Specific examples of fluorine-containing pory(β-hydroxyorganoate)s of the present invention are identified by the formula listed above and contain the following fluorine-containing repeat units: trifluoro-3-hydroxyhexanoate (m = 0, n = 2, "TF3HHxA"); heptafluoro-3-hydroxyoctanoate (m = 2, n = 2, ^*ΗpF3HOA"); nonafluoro-3-hydroxynonanoate (m - 3, n = 2, "NF3HNA"); and tridecafluoro-3- hydroxyundecanoate (m = 5, n = 2, "TDF3HUDA"). Each of these polymers contains a mixture of hydrocarbon repeat units wherein R is a -(CH₂)pCH3 group and p = 5, 3, and 1 (hence, tetrapolymers, with a major amount of p - 5), and is identified by its fluorine- containing repeat unit. That is, although each of these polymers contains a number of different repeat units, such that they are tetrapolymers, they are identified by the fluorine- containing repeat units. Each of these specific examples of polymers are identified by the formula listed above wherein x = 50-500 and z = 50-100. The fluorine-containing PHAs ofthe present invention have at least about

1 mole percent (mole-%) monomeric units (i.e., repeat units) with fluorine therein, preferably, at least about 10 mole-%, and more preferably at least about 15 mole-%. For optimum release characteristics, these polymers have 100 mole-% monomeric units with fluorine therein. Preferably, the fluorme-coritaining PHAs ofthe present invention have a surface contact angle of at least about 94° (as measured according to the procedure described in the Examples). The molecular weights (number average) ofthe polymers of the present invention are preferably about 40,000-300,000. More preferably, the molecular weights are about 50,000-200,000, and most preferably, about 60,000- 180,000. The polydispersity (ratio of weight average molecular weight to number average molecular weight) ofthe polymers ofthe present invention is preferably about 1.6-3.4. The fluorine-containing PHAs ofthe present invention can be prepared chemically or chemo-enzymatically using microbial polyester-producing organisms. Specifically, achiral fluoroacids prepared by traditional chemical methods can be converted into chiral monomers and polymerized by microbial catalysts to form high molecular weight bacterial polyesters containing fluorocarbon side chain substituent groups. Suitable fluoroacid starting materials are those that have terminal CF₃ groups. A wide variety of such fluoroacids can be used. Preferably, the fluoroacids suitable for making the fluorine-containing PHAs ofthe present invention have the following general formula:

wherein m is about 0-7, preferably about 1-5, and more preferably about 2-4; n is about 0-10, preferably about 0-6, more preferably about 2-4, and most preferably about 2-3. A nonfluorine-containing carboxylic add can be used in combination with a fluoroadd to produce polymers containing both fluorine-containing moieties and nonfluorine-containing moieties. Suitable nonfluorine-containing carboxylic adds indude, for example, 5-phenyl valeric add, undecenoic add, linoldc add, oleic add, and those represented by the formula CH₃(CH₂)pCH₂CH₂CO₂H, wherein p is 0-15, preferably 3-7, and more preferably 4-6.

The fluorine-containing PHAs can be made, for example, as follows. After growth of P. oleovorans (ATCC 29347) and P. putida (KT 2442) on sodium citrate in a first stage, the carbon sources 6,6,6-trifluorohexanoic acid ("TFHxA"), 6,6,7,7,8,8,8-heptafluorooctanoic add (ΗpFOA"), 6,6,7,7,8,8,9,9,9- nonafluorononanoic acid ("NFNA"), or 6,6,7,7,8,8,9,9,10,10,11,11,11- tridecafluoroundecanoic acid ("TDFUDA") are co-fed along with nonanoic acid, for example, in the second or polymer producing stage.

The fluorine-containing PHAs ofthe present invention can also be synthesized chemically. This can be done by a two-step synthesis similar to that described in K. Teranishi, et al., Macromolecules. 7, 421 (1974), and M. Iida, et al., Macromolecules. 10, 275, (1977), as shown below. The appropriate fluorochemical aldehyde is reacted to give the beta-propiolactone. This lactone is then ring-open polymerized using the aluminum catalyst, (EtAlO)y, disclosed in K. Teranishi, et al., Macromolecules. 7, 421 (1974), and M. Iida, et al., Macromolecules. 10, 275, (1977).

CF₃(CF

(EtAlO)y

CF₃(CF₂ CH₂)n O

-[-CHCH₂CO-]-

m = 0 - 7 n = 0 - 10

Copolymers may be prepared when one uses two or more different beta-propiolactones as shown below:

catalyst

CF₃(CF₂)α CH2)n ₀ CH₃(CH₂)p ₀

- :HCH₂CO — — CHCH₂CO-]-

The films incorporating the fluorine-containing PHAs ofthe present invention can be crosslinked to improve their internal strength. They can be crosslinked by radiation, such as e-beam, for example, without the presence ofa crosslinking agent as a result of radical formation. Additionally, a crosslinking agent can be added to assist in crosslinking and/or become incoφorated into the crosslinked polymer. However, suitable crosslinking agents are radiation active crosslinkers. Other crosslinking agents indude thermal initiators, photoinitiators, and sensitizers. If desired, a crosslinking agent is used in an amount effective to cause crosslinking and improve the internal strength ofthe film. It should be understood that a mixture of crosslinking agents can be used to advantage, such as a photoinitiator and a sensitizer.

Suitable photoinitiators include, but are not limited to: aldehydes, such as benzaldehyde, acetaldehyde, and their substituted derivatives; ketones such as acetophenone, benzophenone, bisbenzophenone, porybenzophenone, and their substituted derivatives such as SANDORAY 1000 (Sandoz Chemicals, Inc., Charlotte, NC); quinones photoinitiator such as the benzoquinones, anthraquinone and their substituted derivatives; thioxanthones such as 2-isopropytthioxanthone and 2-dodecylthioxanthone; and certain chromophore-substituted vinyl halomethyl-sym-triazines such as 2,4-bis-

(tricMoromethyl)-6-(3\4'-ό^ethoxyphenyl)-sym-triazine. Certain of these photoinitiators are also radiation crosslinkers, such as bisbenzophenone and triazines. Preferably, a photoinitiator, or mixture of photoinitiators, (or alternatively, radiation active crosslinker(s)) can be present in the compositions in an amount of about 0.05-11 parts, more preferably about 0.1-5.3 parts, and most preferably about 0.1-3.1 parts, by wtight of 100 parts ofthe polymer. In corresponding wdght percentages (based on the total wdght ofthe composition), the total amount of photoinitiators) in the compositions ofthe present invention is preferably in a range of about 0.05-10 wt-%, more preferably about 0.1-5.0 wt-%, and most preferably about 0.1-3.0 wt-%.

Suitable sensitizers indude, but are not limited to, xanthone, acetophenone, benzaldehyde, o-dibenzoylbenzene, benzophenone, 2-acetylfluorenone anthraquinone, flavone, Michder's ketone, 4-acetylbiphenyl, β-naphthyl phenyl ketone, β- naphthaldehyde, β-acetonaphthone, α-acetonaphthone, α-naphthaldehyde, biacetyle, benzil, fiurorenone, and duroquinone. Preferably, a sensitizer, or mixture of sensitizers, can be present in the compositions in an amount of about 0.05-11 parts, more preferably about 0.1-5.3 parts, and most preferably about 0.1-3.1 parts, by wdght of 100 parts ofthe polymer. In corresponding wdght percentages (based on the total wdght ofthe composition), the total amount of sensitizer(s) in the compositions ofthe present invention is preferably in a range of about 0.05-10 wt-%, more preferably about 0.1-5.0 wt-%, and most preferably about 0.1-3.0 wt-%.

The fluorine-containing PHA compositions containing radiation crosslinkers, photoinitiators, and sensitizers, can be cured using a source of radiation of sufficient energy (i.e., wavdength range) to generate free radicals when in dent upon the particular crosslinking agent sdected for use in the fluorine-containing PHA composition. The preferable wavdength range for the crosslinking agents disclosed above is about 400- 250 nm. The radiant energy in this preferred range of wavdengths required to crosslink the film ofthe invention is about 50-5000 millijoules/cm² and more preferably about 100- 1000 rnillijoules cm².

Crosslinked films prepared from the compositions ofthe present invention preferably have a percent gd in the range of about 2-95 weight percent, more preferably about 30-80 wdght percent, and most preferably about 50-70 wdght percent. As used herein, the percent gd is corrected for other additives as hereinafter described. Minor amounts, i.e., less than about 50 wt-%, of additives can also be included in the composition for particular advantage and for special end uses. Such additives may include pigments, dyes, fillers, stabilizers, ultraviolet absorbers, antioxidants, processing oils, and the like. Antioxidants can be used to provide stabilized compositions. Plasticizers can also be used to soften the composition if it is too crystalline. Examples of suitable plasticizers indude phthalate esters, dtrate esters, lactate esters, and low molecular weight (e.g., less than about 20,000 wdght average molecular wdght) polyesters. Preferred additives are those that are degradable. Preferably, the amount of additives used can vary from 0.1 to 50 wdght percent depending on the end use desired.

The fluorine-containing PHA compositions ofthe present invention are easily coated on suitable flexible or inflexible backing materials, preferably flexible backing materials, by conventional coating techniques to produce coated sheet materials in accord with the present invention. The flexible backing material can be any material conventionally utilized as a tape backing, as well as other flexible materials. Examples of substrate materials, i.e., backing materials, include, but are not limited to: polymer films such as polyester (e.g., polyethylene terephthalate), polypropylene (e.g., biaxially oriented polypropylene), polyethylene, polyvinyl chloride, polyurethane, cellulose acetate, and ethyl cellulose; woven and nonwoven fabrics formed of threads or fibers of synthetic or natural materials such as cotton, nylon, rayon, glass, or ceramic material; metals and metal foils such as aluminum, copper, lead, gold and the like; paper, glass; ceramics; and composite materials comprised of laminates of one or more of these materials.

The fluorine-containing PHA compositions ofthe present invention can be coated from solution by any ofthe coating processes well known in the art, such as knife coating, roll coating, gravure coating, curtain coating, spray coating, etc. Furthermore, the fluorine-containing PHA compositions ofthe invention can be applied by extrusion coating, coextrusion coating, thermal coating, and the like, with no solvent present, thereby eliminating environmental and toxiαty problems assodated with solution coating processes. Useful cured coating thicknesses for the present invention are in the range of about 12-2500 μm, preferably in the range of about 25-250 μm, and more preferably, in the range of about 25-125 μm. Examples

Objects and advantages of this invention are further illustrated by the following examples. The particular materials and amounts thereof recited in these examples as well as other conditions and details should not be construed to unduly limit this invention. All materials are commercially available except where stated or otherwise made apparent.

The polymers formed were analyzed by Η and ¹⁹F nuclear magnetic resonance (NMR) to establish the mole-% of fluorinated repeat units. ¹³C NMR analysis was also carried out to gain information on the repeat unit sequence distribution of fluorinated products. Assignments of NMR signals were made using information obtained from 2-D experiments. Polymer molecular weights were measured by gel permeation chromatography (GPC). The effects of PHA fluorine content on material thermal transitions and surface properties was investigated by differential scanning calorimetry (DSC) and surface contact angle measurements.

Materials and Methods

Synthesis of Carbon Sources. The general synthetic procedure used for the preparation of 5-fluorocarbon substituted pentanoic acid carbon sources where the fluorocarbon chain length was varied from 1 to 6 is summarized below. To a 50 mL round-bottom flask, 0.1 mole of 4-pentenoic acid (Aldrich Chem. Co., Milwaukee, WI, 97%), a slight excess of perfiuoroalkyl iodide (0.11 mole, Aldrich Chem. Co., 98%) and 0.2 g of azo-isobutyronitrile ("AIBN", Eastman Kodak Co., Rochester, NY) were added. This solution was stirred and heated at 80°C for 2 hours. T e reaction was followed by observing the olefin stretching band at 1653 cm^'1 in the infrared. After removal of volatiles on a rotary evaporator the yields ofthe corresponding iodo acids were greater than 95%. These products were hydrogenated over 5% Pd/C (Aldrich Chemical Co.) in ethanol with two equivalents of sodium acetate. The hydrogenation was conducted at room temperature (20-25°C) and about 13.79 Pa of hydrogen for 16- 18 hours. The reaction mixture was filtered through diatomaceous earth and rinsed with ethanol. The ethanol was removed using a rotary evaporator and the residue was partitioned between ether and dilute aqueous HCl. The ether layer was washed with a saturated NaCl solution and dried over anhydrous sodium sulfate. The structures, melting points (peak values measured using a Seiko DSC Model DSC220C), full and abbreviated names ofthe synthesized fluorinated acids are shown below. Spectra recorded by fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectroscopy (*H, ¹³C, and ¹⁹F) of these products were consistent with that expected based on their structures.

O

CF₃-(CF₂^CH₂-τ :H₂^-C— OH

m=0; 6,6,6-Trifluoroheιanoic acid ("TFHxA", liquid) m=2; 6,6,7,7,8,8,8-Heptafluorooctanoic acid ("HpFOA", liquid) m=3; 6,6,7,7,8,8,9,9,9-Nonafluorononanoic acid ("NFNA", m.p. 37 °C) m=5; 6,6,7,7,8,8,9,9,10,10,11,11,11-Tridecafluoroundecanoic acid

("TDFUDA", m.p. 55 °C)

Preparation of Growth Medium. The growth medium, medium E*, for Pseudomonas oleovorans (ATCC 29347) and Pseudomonas putida (KT 2442), was prepared using nanopure distilled water and contained the following (per liter):

5.8 g; KH/O,, 3.7 g; 15 mL of 0.1 M MgSO₄ and 3 mL of a micro-element solution. The micro-element solution was as follows (per liter of 1 N HCl): FeSO/TH_jO, 2.78 g; MnCL/411 ), 1.98 g; CoS0₄'lKp, 2.81 g;

1.67g;

0.17 g;

0.29 g. The pH of medium E* was adjusted to 7.0 using 1 N HCl and the medium was sterilized by autoclaving at 121°C for 20 minutes. Strain Infoπnation and Long-Term Preservation.

Pseudomonas oleovorans ATCC 29347 was obtained from the U.S. Department of Agriculture (Agricultural Research Service, National Center for Agricultural Utilization Research, Peoria, IL 61604) and Pseudomonas putida KT2442 was obtained from Professor G. Eggink at the Agrotechnological Research Institute (ATO-DLO, Wageningen, the Netherlands). For long term cell preservation (up to 2 years), liquid broth cultures of medium E* containing 40 mM of sodium citrate as the sole carbon source were inoculated by transfer of either P. oleovorans or P. putida from agar plates. The cells were grown aerobically in 500 mL flasks (100 mL culture volumes) at 30°C for 24 hours in a shaker incubator (New Brunswick Scientific Co., Inc.; 250 φm). Cultures were then diluted 1 : 1 with 20% sterile glycerol, 1 mL volumes were transferred to cryogenic vials, the vials were frozen in a dry ice/ethanol bath and then transferred to a liquid nitrogen container for long term storage. These preparations of P. oleovorans and P. putida cells served as inocula for all ofthe experiments described below. Inoculation, Cell Growth and PHA Production. To avoid inhibition of cell growth by fluorinated carboxylic add carbon sources, a two-stage batch culture process was used as described by O. Kim et al, Polvmer Preprints (Am. Chem. Soc. Div. Polvm. ChemΛ 25, 627 (1994), the disclosure of which is incoφorated herein by reference. In the first or cell growth stage, sterile medium E* amended with 40 mM of sodium dtrate as the sole carbon source was inoculated (0.4 % vol/vol) using the contents of rapidly thawed cryovials containing one ofthe above organisms. First stage P. oleovorans and P. putida cultivations were carried out to the early stationary phase (17- and 20-hour culture times for P. oleovorans and P. putida, respectively) in 500 mL flasks (100 mL culture volumes) at 30°C in a shaker incubator (New Brunswick Scientific Co., Inc.; 250 φm). For the second or PHA production stage, fluorinated acid/nonanoic acid 1 : 1 molar mixtures (total carbon source concentration equaled 15 mM) were added. Periodic monitoring of culture viable cell numbers (C.F.U./mL) was carried out by removing 1 mL aliquots from cultures under aseptic conditions and using the spread plate method as described by R. M. Atlas et al., Experimental Microbiology - Fundamentals and Applications: Exercise 16- Enumerization of Microorganisms. Macmillan Pub. Co., New York: pp. 109-112 (1984). Cultures were terminated at 1-, 2-, 3-, 4-, and 5-day second stage cultivation periods. The cells were harvested by centrifugation (Sorvall; 4°C, 8,000 φm), washed with 0.02 M of sodium phosphate buffer solution (pH = 7.2) and lyophilized. Polymer Isolation. Intracellular PHAs were isolated from the lyophilized cells by extraction with an excess of chloroform (15 mL per 1 g of biomass, 25°C, 48 hours). Residual cell material was then removed by filtering. The polymers formed were precipitated by addition of chloroform solutions into cold methanol (1:10 volvol), and washed with methanol and dried in-vacuo (30°C, 5 mm Hg, 24 hours).

Instrumental Procedures. A UNITY-500 NMR Spectrometer was used for all ofthe NMR experiments described below. Proton (*H) NMR were recorded at 500 MHz. Chemical shifts in parts per million (ppm) were reported downfidd from 0.00 ppm using tetramethylsilane (TMS) as an internal reference. The experimental parameters were as follows: 0.5% (wt/vol) polymer in chloroform-d, temperature 298K, 2.4 μsecond (14°) pulse width, 3 second acquisition time, and 8,000 Hz spectral width. Carbon (¹³C) NMR spectra were recorded at 125 MHz and the following parameters: 2.0% (wt/vol) polymer in chloroform-d, 298K, 7.4 millisecond (67°) pulse width, 0.4 second acquisition time, 26,400 Hz spectral width and continuous Waltz modulated proton decoupling. The observed ¹³C NMR chemical shifts in ppm were referenced relative to chloroform-d at 76.91 ppm. Fluorine (¹⁹F) NMR spectra were recorded at 470 MHz on 2.0% (wt/vol) solutions in chloroform-d at 298K, 4.6 μsecond pulse width, 0.64 second acquisition time, and 100,000 Hz spectral width.

Trifluorotoluene was added to the sample as a cross integration reagent. For the COSY experiment (0.5% (wt/vol) polymer in chloroform-d) the data were collected in a 1024 x 256 data matrix and zero-filled to 1024 x 1024 using 8 scans per increment, a 4260 Hz sweep width, and a 1.1 second delay between transients. The data was processed using sinebell weighting. Two-dimensional reverse- detected heteronuclear multiplet quantum correlation (HMQC) spectra were obtained (2.0% (wt/vol) polymer in chloroform-d) with spectral windows of 4260 Hz (1H) and 12771 Hz (¹³C). The data were optimized for a one-bond scalar coupling constant of 140 Hz and used 90° pulses of 9.8 milliseconds (¹³C) and 14.8 milliseconds (^lH). Delay time between scans was 1.0 second. The data matrix was zero-filled to 1024 x 1024 and processed with a Gaussian weighting function. Fourier transform irifrared (FT-IR) spectra ofthe fluorinated add carbon sources were carried out by preparation of KBr pellets and recording spectra at 25°C. FT-IR spectra were recorded using a Perkin-Elmer 1600 Series FT-IR. To determine the mole-% of 3-hydroxyheptanoate ("HH"), 3- hydroxynonanoate ("HN") and other 3-hydroxyalkanoate repeat units in the products formed, the isolated PHAs were subjected to acid-catalyzed methanolysis to obtain the corresponding β-hydroxyalkanoic acid methyl esters. The method used for methanolysis and isolation ofthe methyl esters was previously described by R. A. Gross et al., Macromolecules. 22. 1106 (1989), the disclosure of which is incoφorated herein by reference. Briefly, the methanolysis reaction involved reaction ofthe isolated PHAs in chloroform/methanol/sulfuric acid (1 mL/0.85 mL/0.15 mL) at 100°C for 140 minutes. The volatile methyl esters synthesized by the methanolysis reaction were identified by comparison of their retention time with those of standard methyl β-hydroxyalkanoates. A Perkin Elmer 8500 gas chromatograph (GC) equipped with a Hewlett Packard Ultra-2 capillary column (25 m x 0.2 mm x 0.33 mm) and a flame ionization detector was used. The chromatographic conditions used were as follows: temperature of injector and detector were 230°C and 275°C, respectively; a temperature program was used that separated the different methyl β-hydroxyalkanoates (80°C for 5 minutes, temperature ramp of 7°C per minute, 130°C for 14 minutes).

Elemental analyses (C, H, and F) of fluoro-PHAs were carried out on samples which were dried to constant weight (48 hours, 55°C, 0.03 mm Hg) in the presence of phosphorous pentoxide and then carefully stored in screw cap glass vials in a dessicator prior to analyses. The work was performed at Galbraith Laboratories Inc., Knoxville, TN 37950.

Molecular weights of PHAs were measured by gel permeation chromatography (GPC) using a Waters Model 510 pump, Model 410 refractive index detector and Model 730 data module with 500, 10³, 10⁴ and 10^s A ultrastyragel columns in series. Chloroform (HPLC grade) was used as an eluent at a flow rate of 1.0 mL/minute. The sample concentrations and injection volumes were 0.3% (wt/vol) and 150 μL, respectively. Polystyrene standards with a low polydispersity (Polysciences) were used to generate a calibration curve.

Thermal characterizations were carried out using a Dupont 2910 differential scanning calorimeter (DSC) equipped with a TA 2000 data station. Between 3.0 mg and 10.0 mg of sample were sealed in aluminum pans and analyses were carried out mamtaining a dry nitrogen purge. The polymer samples were heated at a rate of 10°C/minute from 25-100°C (first heating scan), cooled rapidly by quenching in liquid nitrogen, and then analyzed again during a second heating scan from -120°C to +100°C. Data reported for the melting temperature (T_m) and enthalpy of fusion (ΔHV) were taken from the first heating scan. Where multiple melting transitions were observed, the reported T_m was the peak melting temperature ofthe largest endotherm transition. ΔHf values were taken as the cumulative value over the entire melting transition range. The reported glass transition temperatures (T,) were the midpoint values measured during the second heating scans.

To characterize the surface properties of fluorinated PHAs, the surface contact angle through the profile ofa liquid drop (water) placed on a polymer film surface was measured by the direct observation-tangent method (i.e., direct tangent-water drop method) at room temperature as described by S. Wu, Experimental Methods for Contact Angles and Interfacial Tensions. Chapter 8, Marcel Dekker, Inc.: New York (1982), which is incoφorated herein by reference. To prepare highly uniform polymer films, polymer solutions (2 mg/mL chloroform) were filtered (0.45 mm) and then spin-coated onto glass slides (1,000 rpm, 30 second spinning, 25°C). Reported surface contact angle values were obtained from the average of duplicate measurements at three different sites on polymer films.

Results

General Strategy for the Production of Fluoro PHAs. The fluorinated adds used were prepared by reaction of 4-pentenoic acid with a series of perfluoroalkyl iodides to form iodoflouroacid intermediates which were then dehalogenated by hydrogenation. The fluoroacids contain fluorocarbon segments of variable length and hydrocarboxylic acid segments of fixed length (terminally substituted pentanoic acid). Spectral analyses ofthe synthesized flouroacids were consistent with that expected. A chemo-enzymatic route using the biocatalysts P. oleovorans and

P. putida were selected for the enzymatic conversion of fluoroacids to fluoro- PHAs ofthe following general formula wherein p - 5, 3, and 1:

CF₃(CF₂) CH₂CH₂ o CH₃(CH₂)p Q

- CHCH₂— C ] [ θ-CHCH₂-C-j-

m-0, TF3HHxA m«=2, HpF3HOA m-3, NF3HNA m=5, TDF3HUDA

The fluoroacids prepared herein were specifically designed with a spacer group (two methylene units) between carbon 3 (site of β-oxidation to a 3 -hydroxyl functionality) and the functional fluorinated side group.

Attempts to produce fluoro-PHAs in second stage cultivations using only the fluoroacids TFHxA, HpFOA, NFNA and TDFUDA as carbon sources gave no observable PHA formation over extended second stage cultivation times (up to 5 days). Efforts were then directed towards the formation of fluoro- PHAs in second stage cultivations by cometabolism. Equimolar mixtures (total of 15 mM) of nonanoic add ("NA"), a known PHA producing substrate, with the fluorinated adds were prepared. Prior to the second or polymer producing stage, P. oleovorans and P. putida were first cultivated using 40 mM of sodium citrate as the carbon source. This first stage ofthe fermentation, carried out to the early stationary growth phase, allowed the accumulation of 0.9 g/L and 1.2 g/L cell biomass for P. oleovorans and P. putida, respectively. By carrying out cell growth in the first stage, this circumvented problems that would likely have arisen due to differences in growth as a function ofthe fluoroacid NA mixture used.

Cell Viability of P. oleovorans and P. putida in the Second Stage Cultivations. P. oleovorans and P. Putida viability in colony forming units (C.F.U.) per mL as a function ofthe second stage culture time and the carbon source used were measured. For both organisms and cultivation periods up to 120 hours (5 days), the introduction of HpFOA, NFNA and TDFUDA as cosubstrates resulted in no substantial loss in cell viability relative to cultures containing only NA. In fact, the former substrate mixtures showed slightly enhanced C.F.U./mL values to 72 hours for P. oleovorans cultures. Both P. oleovorans and P. Putida show substantial losses in cell viability relative to 15 mM nonanoic add cultivations at prolonged culture times (96 and 120 hours) forNA/TFHxA. Thus, with the exception of TFHxA, the introduction ofthe fluoroacids as cosubstrates with NA did not result in any notable cellular toxicity as measured by C.F.U./mL for second stage culture times to 120 hours (5 days).

PHA Formation on NA/Fluoroacid Mixtures. Since significant quantities ofthe provided carbon sources remained as insoluble particles after 5- day cultivations, values of cell yields (g L) and percentage PHA in cells were not obtained. The solubility ofthe carbon sources in methanol facilitated their removal upon isolation of the products. PHA formation is therefore reported herein as the volumetric yield or weight of isolated product corrected for a 1 L culture volume (g/L). Moreover, it is important to note that at the onset ofthe second stage, both microorganisms showed no evidence of PHA granule inclusions and, therefore, polymer formation. This was based on careful inspection of cells using a phase contrast microscope (x 1000). Thus, polymer yields reported herein correspond to the polyester formed in the second stage ofthe fermentation after the addition of NA/fluorinated add mixtures.

In general, PHA volumetric yields for both P. oloevorans and P. putida decreased relative to 15 mM NA fermentations with the addition of fluorinated acid cosubstrates. The magnitude of the decrease in PHA yield was a function ofthe organism, cultivation time and the NA/fluorinated acid mixture (see Tables I and IT). Such a decrease in yield would be anticipated upon the addition of an unusual carbon source which deviates significantly from that of linear hydrocarbons, n-alkanoic adds, and other oil related substrates. Interestingly, NA/TDFUDA proved to be a striking exception to the above. Specifically, P. oleovorans PHA volumetric yields were similar in value (within 20% of the mean) when using either NA or NA/TDFUDA at cultivation times of 3, 4, and 5 days (approximately 0.20 g/L). P. putida also showed similar behavior for extended cultivation times (5 days). PHA yields formed by P. oleovorans using NA/HpFOA and NA/NFNA were comparatively similar throughout the 5-day cultivation period. In contrast, PHA yields formed by P. putida on NA/HpFO A were largest (0.11 g/L) at day 1 and then dropped rapidly to 0.01 g/L at day 2 while cultivations carried out using NA/NFNA showed a distinct maximum PHA yield at day 2 (0.16 g/L). For both organisms, PHA yields on NA TFHxA were similar to those from NA/HpFOA to day 2, after which, PHA yields on the former were generally lower. It should be noted that the low PHA yields at extended cultivation times using NA/TFHxA is consistent with the observed substantial decrease in cell viability (cell death) at corresponding culture times.

Table I shows the volumetric polymer yields, molecular weights, and mole-% of fluorinated repeat units in PHAs formed by P. oleovorans in second stage cultivations using NA and 1 : 1 mixtures of NA with TFHxA, HpFOA, NFNA and TDFUD A as the carbon sources. Table II shows the volumetric polymer yields, molecular weights, and mole-% of fluorinated repeat units in PHAs formed by P. putida in second stage cultivations using NA and 1 : 1 mixtures of NA with TFHxA, HpFOA, NFNA and TDFUDA as the carbon sources.

Table I.

a) Measurements by 1H and ¹⁹F NMR spectral integration except those given in parenthesis which are by elemental analysis. b) nd = not determined. c) Cultivation period for the second or polvmer producing stage ofthe fermentation.

Table IL

a) Measurements by 1H and ¹⁹F NMR spectral integration except those given in parenthesis which are by elemental analysis. b) nd = not determined. c) Cultivation period for the second or polymer producing stage ofthe fermentation.

NMR Characterization of Fluoro-PHAs. A detailed NMR study was undertaken to characterize the fluoro-PHA formed by P. putida after a 2-day cultivation period using NA/NFNA for polymer formation (see Table π). The one dimensional 500 MHz ^XH NMR spectrum (see Figure 1) showed signals at 1.85, 1.92, 2.08 and 2.62 which are not seen in spectra of PHAs formed fromNA. Observation ofthe 2D 'H-'H COSY spectrum showed that the signals at 1.85 and 1.92 have crosspeaks with that at 5.18 ppm. Furthermore, there was a crosspeak between the signal at 2.62 and the signal at 5.18 ppm. Also, the signals at 1.85 and 1.92 were of almost equal intensity and the additive spectral integration of these two signals was approximately equal and 2x that ofthe peak areas corresponding to the signals at 2.08 and 2.62, respectively. Based on the above, the signals at 1.85/1.92, 2.08 and 2.62 are assigned to protons 17 (H-17), H-18, and H-15, respectively. It should be noted that protons H-17 are diastereotopic and, therefore, chemically non-equivalent. This non-equivalence results in two partially resolved signals with peaks at 1.85 and 1.92 which each correspond to one ofthe diastereotopic protons. The ¹³C NMR spectrum of this product is shown in Figure 2. ¹³C NMR signals due to 3-hydroxynonanoate (HN) and 3-hydroxyheptanoate (HH) repeat units were assigned based on a previous literature report for poly (HN-co-HH) produced from NA by P. oleovorans. It is believed that the two weak ¹³C signals at 8.95 and 71.80 ppm can be assigned to the methyl and methine carbons of 3-hydroxyvalerate repeat units present at low concentrations. The 2D 1H-¹³C heteronuclear multiple quantum correlation (HMQC) spectrum of this product revealed that protons H-17 at 1.92 and 1.85 ppm show correlations to the ¹³C signal at 24.6 ppm which is therefore assigned to carbon 20 (C-20). Also, the 2.08 ppm proton signal correlates with a carbon signal at 26.88 and this was assigned to C-21. However, fluorinated carbons C-22 through C-25 were not detected in the ¹³C NMR spectrum. Based on model compounds such as 4,4,5,5,6,6,6-heptafluoro-l-(2-thienyl)-l,3-hexane-dione and 2,2,2- trifluoroethanol, ¹³C NMR signals for C-22 through C-25 should be observed in the spectral region 100-140 ppm. However, fluorinated carbons C-22 through C-25 are complex multiplets due to one bond and long range coupling between ¹⁹F and ¹³C nuclei and the sample concentration was insufficient to observe these fluorines. In the carbonyl region ofthe ¹³C NMR spectrum two signals were observed at 169.25 and 168.61 that have relative signal integration ratios of 10:1. Since the HH + HN : NF3HNA ratio is approximately 10:1, the minor resonance at 168.61 ppm was assigned to the carbonyl in NF3HNA (C-17). The carbonyls of NF3HNA repeat units are sufficiently far from the C4F9 side chain that there should be no perturbation ofthe chemical shift due to long range substitution effects. Therefore, chemical shift differences most likely result from changes in the main chain conformation due to the presence of fluorinated relative to hydrocarbon side chains. The above analyses by both *H and ¹³C NMR support that PHAs formed from NFNA/NA contain NF3HNA repeat units.

To determine the mole-% of PHA repeat units which contain fluorinated side chains, trifluorotoluene was added to the samples as an internal reference and was used as a cross integration reagent for 500 MHz H and 470 MHz ¹⁹F NMR spectra of products. Figures 1 and 2 show the phenyl and trifluoromethyl resonances, respectively, due to trifluorotoluene. The ¹⁹F spectrum ofthe product also shows four additional ¹⁹F signals at -126.5, -124.8, -115.1 and -81.4 ppm that were assigned to fluorine nucld 3 (F-3), F-2, F-l and F-4 of NF3HNA repeat units (see Figure 3). The moles of repeat units with fluorinated side groups was determined from the ¹⁹F spectra by measuring the relative integration ofthe trifluoromethyl toluene signal at -63.2 and the signal due to CF₃ (F-4 in Figure 3) at -81.4 ppm. The total moles of repeat units was determined by comparison ofthe relative signal intensities ofthe 1H signal due to trifluorotoluene (5 hydrogens) and the methine protons (1 hydrogen) H-2, H-10 and H-16 whose signals were not resolved. This information was then used to determine the mole- % of NF3HNA repeat units in the product. The identical strategy was used to determine the mole-% of TF3HHxA, HpF3HOA, and TDF3HUDA repeat units in polymers formed from mixtures of NA with TFHxA, HpFOA, and TDFUDA, respectively, and the results are presented in Tables I and II. The ¹⁹F and 1H chemical shifts of TF3HHxA, HpF3HOA and NF3HNA repeat units are listed in Table m. The strategy used to assign 1H and ^I9F peaks for TF3HHxA and HpF3HOA followed exactly as was described above for NF3HNA. It should be mentioned that no evidence was found from ^!H and ¹⁹F NMR analyses for the formation of fluorinated repeat units that differed in chain length from that used as the carbon source in fermentations As seen in Table m, fluorines Fal of TF3HHxA repeat units show two signals at -66.79 and -66.88 ppm. Table IH.

1H and ¹F NMR chemical shifts of fluorinated repeat units incoφorated into PHAs.

TF3HHxA HpF3H0A NF3HNA

Chemical shifts in Η NMR analysis (ppm)

TF3HHxA HpF3HOA NF3HNA

CH₂ (Hal) 2.63 CH₂(Ha2) 2.68 CH₂ (Ha3) 2.59 CH(HB1) 5.18 CH(Hbl) 5.18 CH(Hbl) 5.18 CH₂(Hcl) 1.88,1.93 CH₂(Hc2) 1.82,1.97 CH₂(Hc3) 1.85,1.92 CH₂(Hdl) 2.15 CH₂(Hd2) 2.14 CH₂ (Hd3) 2.08

Chemical shifts in ¹⁹F NMR analysis (ppm)

TF3HHxA HpF3H0A NF3HNA

CF₃(Fal) -66.79,-66.88 CF₃(Fa2) -81.2 CF₃(Fa3) -81.4

CF₂ (Fb2) -128.2 CF₂ (Fb3) -126.5 CF₂(Fc2) -116.0 CF₂ (Fc3) -124.8 CF₂(Fd3) -115.1 In a few cases, the mole-% of fluorinated side groups in products was also determined by elemental analysis (see Tables I and T). The estimations were made using the following assumptions: 1) fluorinated repeat unit structures in products have the identical chain length as the fluoro-carbon source used; 2) the ratio of HN to HH units is 62:29 (based on GC analysis). The values of mole-% fluorinated repeat units from elemental analysis were found to be in excellent agreement with those from NMR measurements (see Tables I and II).

Relationship Between Fluoroalkanoate Structure and Fluoro- PHA Formation. The mole-% values of fluorinated PHA repeat units formed by P. oleovorans and P. putida as a function ofthe fluorinated cosubstrates and second stage cultivation time are presented in Tables I and LT, respectively. Inspection of Tables I and π shows that cultivation of both P. oleovorans and P. putida on 1 : 1 mixtures of fluorinated adds with NA resulted in substantial incoφoration of fluorinated repeat units. The highest level of incoφoration observed was 17.3 mole-% for a 3-day cultivation of P. putida with NA/NFNA. When this cultivation was carried for 2 days, as opposed to 3 days, the PHA volumetric yield was significantly higher (0.16 relative to 0.01 g/L) but the mole-% of NF3HNA repeat units was lower (9.8 mole-% relative to 17.3 mole-%). P. putida showed comparatively lower incoφoration of shorter chain length fluorinated substrates. The maximum mole-% of fluorinated side chains formed by P. putida using NA/TFHxA and NA/HpHO A was 3.3. Unfortunately, due to low PHA yields, compositional analyses of PHAs formed by P. putida from NA/TFHxA at culture times of ≥ 2 days was not obtained. Interestingly, the use ofthe longer chain fluoroacid TDFUDA for polymer formation by both organisms resulted in relatively high PHA yields but little to no incoφoration of fluoroalkanoate repeat units (see Tables I and IT). It may be that the rigid and long (6 carbons) fluorocarbon chain length of TDFUDA does not allow its metabolism to activated monomer and subsequent polymerization. Fatty acid β-oxidation of TDFUDA would produce a substrate of relatively shorter chain length but which now lacks a hydrocarbon spacer between the pendent fluorocarbon substituent and the 3 -position ofthe chain. Thus, further metabolism of this substrate to the 3-hydroxyl-thiocoenzyme A activated monomer and subsequent polymerization would likely not be possible.

For short cultivation times where PHA yields were relatively high, the results obtained using P. oleovorans and NA HpFOA were of interest (see Table I). Specifically, after only a 1-day second stage cultivation period, the PHA formed (0.3 g L) had 6.4 mole-% of fluoroalkanoate side groups. This indicates that conversion of HpFOA to HpF3HOA repeat units occurs on a relatively rapid time scale when compared to the results using the other fluoroalkanoates for both organisms (see Tables I and II). The yields of PHAs by P. oleovorans using NA NFNA were almost identical to that using NA HpFO A while the mole-% of incoφorated fluorinated side groups with the former was slightly lower (see Table I). Interestingly, n-octanoic acid (OA) and NA were preferred carbon source chain lengths for PHA production by P. oleovorans. Also, the selectivity of . oleovorans for the production of 3 -hydroxyoctanoate and 3 -hydroxynonanoate repeat units from OA and NA, respectively, was almost identical. Thus, in this work using P. oleovorans, the trend for the dependence ofthe fluoroalkanoate chain length on its incorporation into PHA and PHA yield was remarkably similar to that observed using n-alkanoic acids as sole carbon sources. In a number of cases above the mole-% incoφoration of fluorinated side chains increased as the PHA yield decreased with extended cultivation times. Two factors which may contribute to such behavior are: (1) the use of NA as the primary carbon source for PHA production so that the fluorinated carbon source will be utilized only after NA reaches relatively low media concentrations; and (2) preferential degradation and cellular recycling ofthe PHA product fraction that contains n-alkanoate hydrocarbon side groups when available carbon becomes insufficient to maintain cellular functions.

Molecular Weights of PHAs Produced from Multi-Fluorinated Alkanoates. The molecular wdghts of PHAs isolated from P. oleovorans and P. putida grown on NA/fluorinated alkanoates were measured by GPC and the results are presented in Tables I and π. In all cases, even with fluorinated side group contents up to 17.3 mole-%, high molecular weight products were formed. M„ values of PHAs produced by P. oleovorans containing up to 6.4 mole-% fluorinated side chains ranged from 102,300 g/mole to 169,300 g/mole. PHAs produced by P. putida containing up to 17.3 mole-% fluorinated repeat units had M_n values that ranged from 67,000 g/mole to 114,000 g/mole.

Thermal Properties of PHAs Produced from Mult Fluorinated Alkanoates. Sdected solution predpitated fluoro-PHAs were characterized by DSC to determine whether the iricoφoration of fluorinated side groups influenced product thermal transitions. DSC thermograms for first heating scans were used to determine T_m and ΔH_f values (see Table TV and Figure 4). Second heating scans after rapid quenching of samples from the melt were also recorded to determine T_g values (see Table IV and Figure 5).

Table IV.

DSC measurements^* of PHAs formed with and without NF3HNA repeat units.

a) The samples used in this study were from solution precipitation. b) Determined by ^XH and ¹⁹F NMR spectroscopy. c) The midpoint glass transition temperature measured during the second heating scan after samples were rapidly quenched from the melt. d) Measured during the first heating scan where the value reported is the peak melting temperature for the largest endotherm transition. e) Measured during the first heating scan where the value reported is the cumulative heat of fusion taken over the entire melting range. f) Cultivation period for the second or polymer producing stage ofthe fermentation.

Inspection of Table TV shows that incorporation of fluorinated side groups in PHAs lead to large increases in ΔHf. Considering products of P. oleovorans which contained 0 and 8.8 mole-% NF3HNA repeat units, the ΔH_f values increased from 3.2 to 5.9 calories/gram. Comparison ofthe first heating scans for these two products (thermograms a and c, respectively, Figure 4) shows that the sample containing 8.8 mole-% NF3HNA has a relatively broader melting transition and a slightly increased T_B (see Table TV). Similarly, fluoro-PHAs formed by P. putida that contain 0 and 12.4 mole-% NF3HNA had ΔH_f (T_m ) values of 3.2 calories/gram (49.3°C) and 8.0 calories/gram (50.5°C). Further inspection of thermogram d in Figure 4 for the 12.4 mole-% NF3HNA sample shows that unlike PHAs with 0 mole-% NF3HNA, substantial sample melting was still observed at temperatures of 55-80°C. Also, unlike the 0 mole-% PHA samples, the 12.4 mole-% product showed melting transitions at 60 °C and 68°C during the second heating scan (thermogram d, Figure 5) which suggests that NF3HNA repeat units accelerates crystallization during the cooling cycle. Using the midpoint value ofthe T, step transition there was no substantial change in the lowest temperature T, as a function of NF3HNA content (values ranged from - 41.7°C to -41.2°C, see Table TV). Another endotherm transition during second heating scans was observed at temperatures between -28°C and -25°C for all ofthe PHA samples studied (see Figure 5). The step-like character ofthe transition suggests that it is an additional T_g. Furthermore, this transition has an assodated enthalpy of relaxation that increases in magnitude with increased NF3HNA content. It may be that the copolymers of HN and HH formed in this work from NA by the two stage cultivation method deviate significantly from random copolymers. Furthermore, a densification ofthe amoφhous phase with increased NF3HNA content would explain the apparent increase in the enthalpy of relaxation. In the results presented above, possible changes in the ratio of HN, HH, and HV caused by the use of NFNA could also be important in altering the thermal transitions ofthe products formed. To study this further, a number of samples formed by P. oleovorans and P. putida having variable NF3HNA contents were degraded by acid catalyzed methanolysis to their corresponding methyl esters and analyzed by GC. The results of this work clearly showed that the products formed had almost identical molar ratios of n-alkanoate repeat units. Therefore, it was concluded that the observed changes in thermal properties in this work can indeed by attributed to the incoφoration of NF3HNA repeat units. Surface Properties of PHAs Containing Multi-Fluorinated Side

Chain Substituents. To investigate the surface properties of microbially synthesized PHAs containing fluorocarbon side chain substituents, surface contact angles were measured using the direct tangent water-drop method. In Figure 6, the surface contact angle is shown as a function ofthe NF3HNA mole-%. There was no prominent effects of fluorine incoφoration on the surface contaα angle up to about 2 mole-%. Upon increasing the NF3HNA content from 2 mole-% to 3.3 mole-%, the surface contact angle increased from 87° to 92°. Further increase in the NF3HNA content up to 17.3 mole-% led to only a modest increase in the surface contaα angle to 94°. It is interesting to compare the maximum surface contact angle ofthe fluoro-PHAs to other commercial polymers. High surface contaα angles of about 108 were reported for the commercial fluoroplastic polytetrafluoroethylene while other common plastics such as polystyrene, polyvinylchloride, polyethyleneterephthalate, and polymethylmethacrylate were reported to have surface contaα angles of 91, 87, 81 and 80, respectively. Thus, although some ofthe polymers ofthe present invention have surface contaα angles that are less than that for polytetrafluoroethylene, as the amount of fluorine- containing monomeric units increases, the surface contaα angle increases (and the surface energy decreases), and thereby the release chararteristics increase, approaching that of polytetrafluoroethylene. Furthermore, the surface of lowest energy ever found is that comprised of closest packed CF₃ groups. The replacement of a single fluorine atom by a hydrogen atom in a terminal CF₃ group more than doubles the surface energy. See, Advances in Chemistry Series 43: Contact Angle. Wettabilitv. and Adhesion, page 21 (1964). Thus, the higher the mole-% fluorine in the PHAs ofthe present invention, the lower the surface energy, and the higher their release charaαeristics. The complete disclosure of all patents, patent documents, and publications dted herein are incoφorated by reference. The foregoing detailed description and examples have been given for clarity of understanding only. No unnecessary limitations are to be understood therefrom. The invention is not limited to the exaα details shown and described, for variations obvious to one skilled in the art will be included within the invention defined by the claims.

Claims

WHAT IS CLAIMED IS:

1. A fluorine-containing pory(β-hydroxyorganoate) ofthe general formula:

wherein: R is an organic group containing 1-30 carbon atoms; m is about 0-7; n is about 0-10; x is at least about 10; and z is about 0-1,000.

2. The fluorine-containing pory(β-hydroxyorganoate) of claim 1 wherein n is about 0-6.

3. The fluorine-containing poly(β-hydroxyorganoate) of claim 1 wherein n is about 2-4.

4. The fluorine-containing poly(β-hydroxyorganoate) of daim 3 wherein p is about 3-7.

5. The fluorine-containing polyφ-hydroxyorganoate) of daim 1 wherein p is about 3-7.

6. The fluorine-containing pory(β-hydroxyorganoate) of claim 1 wherein n is about 2-3 and p is about 4-6.

7. The fluorine-containing polyφ-hydroxyorganoate) of claim 1 wherein x is about 10-1,000

8. The fluorine-containing poly(β-hydroxyorganoate) of claim 1 wherein x is about 30-500.

9. The fluorine-containing polyφ-hydroxyorganoate) of claim 1 wherdn z is about 10-1,000.

10. The fluorine-containing polyφ-riydroxyorganoate) of daim 1 wherein z is about 20-500.

11. A fluorine-containing polyφ-hydroxyorganoate) of the general formula:

wherdn: m is about 0-7; n is about 0-10; R is -(CH₂)pCH₃ and p is about 0-15; x is about 50-500; and z is about 50-100.

12. The fluorine-containing poly(β-hydroxyorganoate) of claim 11 wherein n is about 0-6.

13. The fluorme-containing poly(β-hydroxyorganoate) of claim 11 wherein n is about 2-4.

14. The fluorine-containing poly(β-hydroxyorganoate) of claim 13 wherein p is about 3-7.

15. The fluorine-containing poly(β-hydroxyorganoate) of claim 11 wherein p is about 3-7.

16. The fluorine-containing poly(β-hydroxyorganoate) of claim 11 wherein n is about 2-3 and p is about 4-6.

17. A fluorme-containing pory(β-hydroxyorganoate) coating composition comprising a fluorme-COrttaining pory(β-hydroxyorganoate) ofthe general formula: CF&FMQBύa O R O

-fCHCH₂ — C ] [ O-CHCH₂-C-]—

wherein: R is an organic group containing 1-30 carbon atoms; m is about 0-7; n is about 0-10; x is at least about 10; and z is about 0-1,000.

18. The fluorme-containing poly(β-hydroxyorganoate) coating composition of claim 17 further including a plasticizer.

19. The fluorine-containing polyφ-hydroxyorganoate) coating composition of claim 17 fiirther including a crosslinking agent.

20. A cured fluorine-corrtaining poryφ-hydroxyorganoate) coating comprising a fluorme-containing poly(β-hydroxyorganoate) ofthe general formula:

wherein: R is an organic group corrtaining 1-30 carbon atoms; m is about 0-7; n is about 0-10; x is at least about 10; and z is about 0-1,000.

21. The cured fluorme-containing poty(β-hydroxyorganoate) coating of claim 20 further including a plasticizer.

22. The cured fluorme-containing polyφ-rrydroxyorganoate) coating of claim 20 wherein the fluorme-containing poly(β-hydroxyorganoate) is crosslinked. DRAWINGS MISSING AT FILING DATE