Tyrosine-Bearing Polyphosphazenes - Biomacromolecules (ACS

31P NMR chemical shifts are reported in ppm relative to 85% H3PO4 at 0 ppm. ... For polymer 12a, a TA Instruments Q10 DSC apparatus was used. Elementa...
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Biomacromolecules 2003, 4, 1646-1653

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Tyrosine-Bearing Polyphosphazenes Harry R. Allcock,* Anurima Singh, Archel M. A. Ambrosio, and Walter R. Laredo Department of Chemistry, The Pennsylvania State University, University Park, Pennsylvania 16802 Received April 2, 2003; Revised Manuscript Received June 25, 2003

Tyrosine-functionalized polyphosphazenes were synthesized, and their hydrolytic stability, pH-sensitive behavior, and hydrogel-forming capabilities were investigated. The physical and chemical properties of the polymers varied with the type of linkage between the tyrosine unit and phosphazene backbone. Poly[(ethyl glycinat-N-yl)(ethyl tyrosinat-N-yl)phophazenes] (linkage via the amino group of tyrosine) were found to be hydrolytically erodible. The rate of hydrolysis was dependent on the ratio of the two side groups, the slowest rate being associated with the highest concentration of tyrosine. The hydrolysis products were identified as phosphates, tyrosine, glycine, ammonia, and ethanol derived from the ester group. The hydrolytically stable phenolic-linked tyrosine derivatives were prepared from N-t-BOC-L-tyrosine methyl ester and alkoxy-based cosubstituents. Polyphosphazenes with both propoxy and phenolic-linked tyrosine side groups showed a pH-sensitive solubility behavior, which was dependent on the ratio and nature of the two side groups. The polymer was soluble in aqueous media below pH 3 and above pH 4. From pH 3-4, the polymer was insoluble. Replacement of propoxy by trifluoroethoxy units yielded a polymer that was insoluble in aqueous media at all pH values. Replacement of propoxy by methoxyethoxyethoxy groups gave a polymer that was soluble at all pH values. Exposure of both the propoxy and methoxyethoxyethoxy polymers to calcium ions in aqueous media caused gel formation due to ionic cross-linking through the carboxylate groups. Introduction There is an ongoing search for polymeric materials that can be used in biomedical applications. Materials that incorporate R-amino acids have generated considerable interest because they offer certain advantages over conventional polymer systems. They are structurally diverse, relatively nontoxic, and tissue-compatible, and the degradation products may be nontoxic and easily metabolized by living tissues.1 Synthetically derived poly(R-amino acids) have been investigated for applications in degradable sutures, artificial skin, membranes for artificial kidney, wound dressing, tissue engineering, and controlled drug delivery.1-5 However, these materials have some undesirable properties, which include thermal degradation on melting, insolubility in common solvents, and a high cost of production. For these reasons, other methods have been examined for the incorporation of amino acids into polymers to form new materials that might have improved material properties and overcome the undesirable physicomechanical characteristics associated with some conventional poly(amino acids). This has led to the development of two classes of amino acid-derived organic polymers. The first class includes pseudo-poly(amino acids), which are synthetic polymers composed of R-amino acids linked by nonpeptide bonds such as ester, carbonate, or urethane linkages.1 Examples of pseudo-poly(amino acids) have been developed by Kohn et al.6,7 on the basis of tyrosine-derived polycarbonates. These materials show promise as temporary scaffolds for tissue regeneration and as drug-delivery polymers. The second class consists of copolymers that contain amino acids and non-

amino acids within the same polymer backbone. A widely investigated example of this class includes copolymers formed between poly(ethylene glycol) (PEG) and amino acids.8,9 The PEG component of the polymer increases the solubility of the poly(amino acids) in water. In recent years, certain polyphosphazenes have also shown promise as prospective materials for various biomedical applications.10-13 Polyphosphazenes possess a backbone of alternating phosphorus and nitrogen atoms with two organic side groups attached to each phosphorus atom. These polymers are synthesized by the reactions of poly(dichlorophosphazene) with organic nucleophiles, such as alkoxides, aryloxides, or amines. Amino acid ester functionalized polyphosphazenes are biodegradable and have been shown to possess tunable degradation rates for targeted applications.14 One such polymer, poly[bis(ethylglycinat-N-yl)phosphazene] (1, R ) H), readily degrades in aqueous media to form inorganic phosphates, ammonia, glycine, and ethanol, all of which can be metabolized or excreted by the body.15-17 Hydrolysis of the phosphazene backbone is affected by many factors, and a rough correlation exists between hydrolytic instability and side group leaving ability. Amine-linked side groups with relatively low pKa values (∼7-9) render the backbone more susceptible to hydrolytic attack than side groups with higher pKa values (>10). In addition, for the R-amino acids, the relative rates of hydrolysis are also affected by the bulkiness of R groups in the general formula, H2NCH(R)COOR′. The relative rates of hydrolysis of four amino acid-containing polyphosphazenes are shown in structure 1.

10.1021/bm030027l CCC: $25.00 © 2003 American Chemical Society Published on Web 08/23/2003

Tyrosine-Bearing Polyphosphazenes

Biomacromolecules, Vol. 4, No. 6, 2003 1647 Scheme 1. Amino Acid-Bearing Polyphosphazenes at Different pH Values

In this study, we have synthesized a group of polymers based on L-tyrosine-substituted polyphosphazenes. These polymers are an addition to the broad series of R-amino acidsubstituted polyphosphazenes that have been studied in our program and elsewhere.10-17 The development of tyrosinefunctionalized polyphosphazenes is of special interest because, unlike the previously studied poly[(amino acid ester)phosphazenes], tyrosine derivatives can be linked to the phosphazene backbone via the amino (2) or the phenolic group (3). The amino-linked tyrosine derivative has the

potential to yield biodegradable polymers with possible applications in both drug delivery and tissue engineering. The attachment of tyrosine to the phosphazene backbone via the phenolic group could impart pH-sensitive solution or gel properties to this system. The presence of both acidic and basic ionizable groups should yield a material that undergoes one or more phase transitions in aqueous media over a wide range of pH values. Moreover, the pendent carboxylate groups can be utilized to form ionic hydrogels in the presence of di- or trivalent cations. Ionic hydrogels are interesting materials because their equilibrium degree of swelling is affected by changes in pH or ionic strength.20,21 This behavior makes them suitable for use in membranes and microcapsules and in biocompatible materials. In a previous study, it was shown that ionic hydrogels are formed by the Ca2+ cross-linking of aqueous solutions of sodium poly[bis(carboxylatophenoxy)phosphazene], [NP(OC6H4COONa)2]n. The resultant hydrogels are useful for biological microencapsulation and complexation to other metal cations.18,19 Results and Discussion Synthesis of Model Cyclic Trimers (6 and 7). Smallmolecule model reactions were carried out initially with the use of hexachlorocyclotriphosphazene (4) as a starting material and with linkage attempted through the amino function of the amino acid reagent as shown in Scheme 2. The synthesis of 6 was attempted by the reaction of 4 with an excess of L-tyrosine ethyl ester with moderate heating.

Scheme 2. Synthesis of Tyrosine-Bearing Cyclic Trimers

However, full replacement of the chlorine atoms was not achieved, as indicated by 31P NMR spectroscopy. The lack of complete substitution is attributed to steric limitations, because it is known that amino acid derivatives with smaller R groups replace all of the chlorine atoms of 4.15 Linkage via the aryloxy functional unit, 7a, was then attempted by the reaction of the sodium salt of L-tyrosine ethyl ester with 4. Complete halogen replacement occurred readily as confirmed by positive ion FAB mass spectroscopy (MH+ 1385) and by 31P NMR spectroscopy (δ ) 9.7 ppm, singlet). The ethyl ester groups of 7a were hydrolyzed with the use of potassium tert-butoxide to yield deprotected 7b. Cyclic trimer 7b showed pH-dependent solubility behavior in aqueous solution, which indicated the presence of free amino and carboxylic acid groups. In basic aqueous media, 7b was soluble. At approximately pH 4, the trimer precipitated from solution. Below pH 2, 7b redissolved. These results indicate the presence of a zwitterionic form of the trimer (Scheme 1). Moreover, when a basic solution of 7b at pH 10 was added to a solution of CaCl2, a precipitate formed immediately. The resultant solid is a cross-linked material formed by ionic linkages in the presence of multivalent cations.19

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Scheme 3. Synthesis of Polyphosphazenes with Tyrosine and Glycine Units

N-Linked Tyrosine Polymers: Synthesis of Poly[(ethyl glycinat-N-yl)(ethyl tyrosinat-N-yl)phosphazenes] (9a-f). Although the synthesis of fully N-linked tyrosine-substituted high polymer 2 was attempted, complete chlorine replacement did not occur. This parallels the behavior of the small molecule cyclic trimer system and is probably due to steric effects or low reactivity of the nucleophile or both. For this reason, the synthesis of mixed-substituent polymers with a smaller cosubstituent than tyrosine was attempted. An initial treatment of poly(dichlorophosphazene) (8) in THF with an excess of L-tyrosine ethyl ester, followed by the addition of excess glycine ethyl ester yielded a fully substituted polymer. The reaction sequence is shown in Scheme 3. A maximum of 83% introduction of the tyrosine units was achieved, the remaining side groups being glycine ethyl ester units (Table 1). Exposure to air and water was minimized during the isolation of the polymers to avoid oxidation of the amino groups or hydrolysis of the macromolecules before characterization. The polymers were vacuum-dried and stored under argon. The mechanical properties of the polymers varied from those of a flexible solid (9a) to brittle materials (9e-f). Although polymers 9b-d were brittle, films fabricated from them were slightly flexible and could be removed from Teflon or glass surfaces without tearing. Unlike previously synthesized amino acid-containing polyphosphazenes, polymers 9a-f were only partially soluble in THF, and the solubilities decreased with an increase in tyrosine content in the order: 9a (most soluble) > 9f (least soluble). The decreased solubilities and brittle properties are attributed to hydrogen bonding imparted by the phenolic group of tyrosine, because structural analogues with phenylalanine-based side groups are flexible materials that are readily soluble.24 However, the polymers were soluble in polar protic solvents such as methanol, ethanol, or 2-propanol (Table 2). Although 31P NMR spectra indicated full chlorine replacement, elemental analysis of the polymer showed the presence of chlorine. This was attributed to hydrogen chloride coordinated either to the backbone or to side group nitrogen

Table 1. Characterization Data for Polymers 9a-f

polymer

% tyrosinea

% glycinea

Tg (°C)

Td (°C)

9a

8

92

-4.2

205

9b

19

81

5.4

210

9c

27

73

20.3

205

9d

40

60

31.3

216

9e

45

55

51.6

215

9f

83

17

80.5

219

elemental analysis found (calc) C 41.55 (41.15) H 6.36 (6.42) N 15.88 (15.79) Clb 0.38 (0) C 44.65 (44.22) H 6.29 (6.36) N 15.01 (14.51) Clb 0.40 (0) C 46.46 (46.16) H 6.27 (6.33) N 13.85 (13.71) Clb 0.43 (0) C 48.52 (48.89) H 6.36 (6.28) N 12.74 (12.58) Clb 0.41 (0) C 50.14 (49.92) H 6.31 (6.26) N 12.33 (12.19) Clb 0.57 (0) C 55.98 (55.50) H 6.08 (6.15) N 9.98 (9.88) Clb 0.88 (0)

a Determined by 1H NMR spectroscopy. b The residual chlorine was attributed to HCl bound either to the backbone or to side-group N atoms.

atoms rather than to unreacted P-Cl bonds. Thus, triethylamine was added to a solution of the polymer to remove bound HCl. The polymer was then washed quickly with water and immediately dried under vacuum to remove traces of water and triethylamine. Thermal Characterization of Polymers 9a-f. No crystalline transitions were detected in polymers 9a-f by differential scanning calorimetry (DSC) analysis. This was attributed to the lack of symmetry of the polymer repeat units due to the different side units attached to the phosphorus atoms. Although polymer 9f contained 83% tyrosine groups, the 17% glycine ethyl ester units introduced sufficient

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Tyrosine-Bearing Polyphosphazenes Table 2. Solubility of Cyclic Trimers and Polymers in Selected Solvents solvent trimer or polymer

THF

methanol

other alcoholsa

7a 7b 9f 10a 10b 11a 11b 12a 12b

yes no yes no no yes no no no

yes no yes yes no no no yes no

yes no yes yes no no no no no

a

water no yes (pH 4) no no yes (all pH values) no no no yes (pH 4)

Absolute ethanol and 2-propanol.

randomization to prevent ordering at the molecular level. By contrast, previously studied amino-derived, single-substituent polymers have Tm values in the range of 150-250 °C.17 Polymers 9a-f showed complex DSC thermograms at temperatures near 200 °C that suggested thermal decomposition at elevated temperatures. Thermogravimetric analysis (TGA) showed a two-stage thermal decomposition pattern for polymers 9a-f. This was detected beyond the initial onset of decomposition (∼200 °C). The Tg values for polymers 9a-f increased with an increase in tyrosine content. This was attributed to restricted rotation about the P-N bonds due to increased hydrogen bonding. The Tg values are shown in Table 1. Hydrolysis Studies of Polymers 9a-9f. The hydrolytic degradation kinetics of solid polymers 9a-9f were followed by measurements of the percent mass loss of the polymers as a function of time. The method involved die casting of cylindrical pellets at high pressure and subsequent immersion of the pellets in deionized water (1% w/v) during an 8-week period. The polymers were then rinsed with deionized water, dried to constant weight, and weighed. In the hydrolysis studies, all of the polymers underwent a decrease in mass. In general, the higher the tyrosine content, the less hydrolytically sensitive was the polymer. For example, a 20%-25% mass loss was detected in polymer 9a (8% tyrosine) after 30 days, whereas polymer 9f (83% tyrosine) underwent ∼4% mass loss in the same time span. This is consistent with the behavior of other poly[(amino acid ester)phosphazenes], where bulkier groups linked to the R-carbon of the amino acid residue yield materials that are more resistant to hydrolysis.17 The hydrolytic degradation curves for polymers 9a-9f are shown in Figure 1. No gelled products were detected during the hydrolysis studies. However, the initial pale yellow pellets swelled and were progressively bleached during longer exposures to water. The hydrolysis products were identified as phosphates, tyrosine, glycine, ammonia, and ethanol derived from the ester group (see Experimental Section). Several possible mechanisms have been proposed by which poly[(amino acid ester)phosphazenes] might hydrolyze.17,22 In one, water hydrolyzes the ester units of the side groups to form the corresponding polymer-bound amino acid. The phosphorus atoms along the backbone are then susceptible to attack by carboxylic acid units. In a second

Figure 1. Percent mass loss of pellets over time: (9) poly[(92% ethyl glycinat-N-yl)(8% ethyl tyrosinat-N-yl)phosphazene]; (×) poly[(73% ethyl glycinat-N-yl)(27% ethyl tyrosinat-N-yl)phosphazene]; (2) poly[(55% ethyl glycinat-N-yl)(45% ethyl tyrosinat-N-yl)phosphazene]; (b) poly[(17% ethyl glycinat-N-yl)(83% ethyl tyrosinat-N-yl)phosphazene].

mechanism, it has been suggested that water displaces the amino acid esters from the phosphorus atoms to form a hydroxyphosphazene species, which then undergoes chain cleavage to phosphates and ammonia. To test whether acidic conditions would preferentially cleave the ester groups from the amino acid or cleave the amino acid ester from the backbone, polymer 9f was mixed with 80% trifluoroacetic acid and allowed to react for one week. The hydrolysis medium was analyzed intermittently and the results showed that the free amino acid ester was present in greater concentrations in all fractions than was the free amino acid and ethanol. This suggests an attack at the bond that connects the side group to the skeleton. An analogous experiment was carried out with the use of an excess amount of potassium tert-butoxide with trace amounts of water. The results were similar and imply that OH- groups preferentially displace the free amino acid ester molecules. As with the acid hydrolysis mechanism, this would presumably give rise to a hydroxyphosphazene intermediate. Despite these results, the actual mechanism is most likely a combination of both proposed routes and is dependent on the conditions employed. In addition, commercial sources of amino acid esters are known to contain trace amounts of the free amino acids, which may also contribute to the overall degradation process. O-Linked Tyrosine Polymers: Synthesis of Polymers 10-12. Although the synthesis of fully O-linked tyrosinesubstituted polymer 3 was attempted, complete chlorine replacement did not occur. For this reason, two-reagent reactions were carried out with smaller cosubstitutents to yield fully substituted polymers 10a, 11a, and 12a. The mixed substitutent polymers were synthesized by the initial treatment of poly(dichlorophosphazene) (8) with the lesshindered nucleophile, followed by the addition of an excess of sodium salt of N-t-BOC-L-tyrosine methyl ester. The synthetic outline is shown in Scheme 4. The less sterically hindered alkoxy-based side group was introduced first to avoid potential reactions of the alkoxide with the tyrosine ester groups. To obtain free carboxylic and amino groups,

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Scheme 4. Synthesis of Polyphosphazenes with Tyrosine and Alkoxy Units

Scheme 5. Hydrogel Formation of Tyrosine-Bearing Polyphosphazenes in the Presence of Ca2+

deprotection reactions were carried out with the use of potassium-tert-butoxide (to remove the ester group) and trifluoroacetic acid (to remove the tert-butoxycarbonyl group). The first of three alkoxy-based side groups used was the methoxyethoxyethoxy (MEE) unit. Single-substituent polymers with this side group are readily soluble in water and polar organic solvents and possess interesting solution and biomedical properties.23 Copolymer analogues have shown promise in drug-delivery systems.20 The alkyl ether side group also provides sites that can be cross-linked by γ-irradiation.20 Polymer 10b, having 55% MEE and 45% tyrosine, was synthesized. The pH-sensitive solubility behavior of this polymer was examined over the pH range of 2-12. Although cyclic trimer 7b showed pH-dependent solubility behavior, the corresponding high polymer, 10b, did not. This was attributed to the highly hydrophilic alkyl ether side groups, which enhance the water solubility of the polymer. Thus, the hydrophilic properties of the alkyl ether side groups, coupled with the presence of only 45% tyrosine groups, presumably dominate the solution behavior and contribute to solubility in aqueous media over a wide range of pH values. The solubility behavior of polymer 10b in selected solvents is summarized in Table 2. The polymer precipitated from aqueous solution when CaCl2 was added. This demonstrated that polyphosphazene-based ionic hydrogels can be obtained with the use of tyrosine side units. The proposed structure is shown in Scheme 5.

To prepare a polymer with a more amphiphilic character, a mixed-substitutent polyphosphazene of structure 11a was synthesized. The trifluoroethoxy cosubstituent was chosen for its hydrophobicity. Polymer 11b, having trifluoroethoxy and tyrosine side groups in a ratio of 1:1, was synthesized according to Scheme 4. This polymer was insoluble over the entire range of pH values tested (2-12). This insolubility was attributed to the combination of hydrophobic trifluoroethoxy side units and amphiphilic tyrosinyl groups, which appears to prevent dissolution in either hydrophilic or hydrophobic solvents. Polymer 12a was then synthesized to incorporate the lesshydrophobic propoxy side groups. A two-step synthetic strategy was adopted for the preparation of this polymer (Scheme 4). The first step was a macromolecular substitution in which poly(dichlorophosphazene) was allowed to react with the sodium salt of propanol followed by the sodium salt of N-t-BOC-L-tyrosine methyl ester. This yielded the fully substituted polymer 12a. This polymer was a brittle, brown-colored powder that was soluble in methanol. The methyl ester units were then hydrolyzed by treatment with potassium tert-butoxide followed by acidification with hydrochloric acid to form the carboxylic acid. The partially deprotected carboxylic acid polymer was insoluble in water. Thus the polymer was neutralized with sodium bicarbonate to obtain the sodium salt derivative, which was water-soluble. Trifluoroacetic acid was employed for the removal of the

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Tyrosine-Bearing Polyphosphazenes Table 3. Characterization Data for Poly[(60% L-tyrosinyl) (40% propyl)phosphazene] polymer 12b

b

31P

NMR, ppm

MWa

PDI

Tg (°C)b

Td (°C)c

-7.6 -12.5 -18.3

1.3

4.4

19.2

250

a Determined by GPC (×105), using poly(ethylene oxide) standards. Determined by DSC analysis. c Determined by TGA analysis.

BOC protecting group. Characterization data for polymer 12b are discussed in Table 3. Unlike polymers 10b and 11b, polymer 12b showed a pHdependent solubility behavior in aqueous media. The ability of polymer 12b to form hydrogels following the addition of divalent cations was also investigated. The addition of a CaCl2 solution to the aqueous polymer solution at pH 7.4 caused precipitation of the polymer via the formation of ionic cross-links. The proposed cross-linked structure is similar to the cross-linked structure shown for polymer 10b in Scheme 5. pH Studies. Polymer 12b showed pH-dependent solubility behavior in aqueous media that indicated the presence of free amino and carboxylic acid groups. Polymer 12b was soluble in basic aqueous media. At approximately pH 4, the polymer precipitated from solution. Below pH 2, 12b redissolved. This pH-dependent solubility behavior is similar to that observed for trimer 7b. Comparison of this polymer with the corresponding species with oligoethyleneoxy or trifluoroethoxy cosubstituent groups suggests that the propoxy units are a main factor that tip the balance to forming an amphiphilic polymer. The pHresponsive solubility behavior is controlled by the extent of ionization of the pendent carboxylic and amine functionalities. Within the tyrosinyl groups a relatively high degree of hydrogen bonding is present at low pH values (3-4) as a result of the presence of R-COOH units. This, in turn, decreases the solubility of the polymer, which corresponds to the behavior of other polyphosphazenes with pendent carboxylic acid groups. Although the pKa value of R-COOH in free tyrosine is 2.20,26 the insolubility at pH 4 indicates that enough protonated acid groups are present to override the expected water solubility from the influence of the quaternized amine groups. In a separate study, the effect of temperature and side group ratio on the pH-sensitive solubility behavior of polymer 12b was investigated. Within the temperature range of 2537.2 °C, polymer 12b showed a similar dependence of solubility on the pH of the media, as discussed above. However, with a change in the ratio of the two side groups, the solution properties of the polymer changed. A decrease in the amount of tyrosine as a cosubstituent yielded polymers that were no longer soluble in water. Instead, they formed hydrogels that showed a pH-dependent swelling behavior. A polymer with 55% tyrosine and 45% propoxy groups existed in a contracted state between pH 3-4. Below pH 3 and above pH 4, the polymer underwent considerable swelling. At pH 7.4, the percentage of water absorption corresponded to 410%.

Conclusions A class of tyrosine-functionalized polyphosphazenes has been developed. Polyphosphazenes that bear tyrosine groups linked via the amino group to the skeletal phosphorus atoms together with glycine ethyl ester cosubstituents are hydrolytically erodible. The rates of hydrolysis depend on the ratio of the two side groups, the slowest rate being associated with the highest concentration of tyrosine. The phenolic-linked tyrosine derivatives were prepared from N-t-BOC-L-tyrosine methyl ester and alkoxy-based cosubstituents. These polymers are resistant to hydrolysis. Mixed-substituent tyrosine/ oligoethyleneoxy polyphosphazenes were soluble in aqueous media from pH 2 to 12. This polymer formed hydrogels in the presence of divalent cations. Based on previous work, radiation cross-linking of this polymer should yield hydrogels that are acutely responsive to pH changes.21 A polyphosphazene with propoxy and tyrosinyl cosubstituents showed pH-sensitive solubility behavior. This behavior depended on the ratio of tyrosine and propoxy side groups. Polymers with more than 55% L-tyrosine attached to the phosphazene backbone were soluble in aqueous media at pH 2 and were also soluble from pH 5-12. At pH 3-4, the polymers were insoluble. This is the first polyphosphazene to show a complex room-temperature pH-dependent solubility behavior at various pH intervals between pH 2 and 12. This system also offers the possibility to form hydrogels via ionic crosslinks formed in the presence of divalent ions. The ability of these materials to form hydrogels following the addition of divalent cations may be useful in applications such as microencapsulation and drug delivery. Furthermore, the pH-sensitive behavior of these polymers could perhaps be utilized for partitioning of proteins or small biomolecules at specific pH values.27 The tyrosine-functionalized polyphosphazene system offers the opportunity to incorporate properties such as bioerosion or pH-sensitive behavior into one material by structural variations at the molecular level. Experimental Section Reagents and Equipment. The synthesis reactions were carried out under an atmosphere of dry argon using standard Schlenk line techniques. Hexachlorocyclotriphosphazene (Ethyl Corp. and PCS) was obtained from a trimer-tetramer mixture by sublimation (30 °C/0.2 mmHg). Poly(dichlorophosphazene) was prepared by the ring-opening polymerization of hexachlorotriphosphazene in an evacuated Pyrex tube at 250 °C. Tetrahydrofuran (THF), toluene, and triethylamine were distilled from sodium benzophenone ketyl under a dry argon atmosphere. 1-Propanol (Aldrich) was distilled over calcium hydride. All other solvents were obtained from Aldrich and were used without further purification. L-Tyrosine ethyl ester, glycine ethyl ester hydrochloride, N-t-BOC-L-tyrosine methyl ester (all from Sigma), sodium hydride (60% dispersion in mineral oil, Fluka), sodium (Aldrich), potassium tert-butoxide (Aldrich), trifluoroacetic acid (99% Aldrich), boron tribromide (1 M solution in methylene chloride, Aldrich), and buffer solutions (Aldrich) were used as received. 2,2,2-Trifluoroethanol

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(Aldrich) and di(ethylene glycol) methyl ester (Aldrich) were distilled before use. All 31P NMR (145 MHz), 1H NMR (360 MHz), and 13C NMR (90.27 MHz) data were obtained with use of a Bruker 360 MHz spectrometer. 31P NMR chemical shifts are reported in ppm relative to 85% H3PO4 at 0 ppm. Gel permeation chromatography was carried out with use of a Hewlett-Packard HP-1090 liquid chromatograph fitted with an HP-1037A refractive index detector and a Polymer Laboratories gel 10-µm column. Calibration of the columns was accomplished by means of a narrow molecular weight poly(ethylene oxide) standard. Glass-transition temperatures for polymers 9a-9f were determined with use of a PerkinElmer DSC-7 apparatus with TAS-7 software with a heating rate of 10 °C/min under a nitrogen atmosphere. For polymer 12a, a TA Instruments Q10 DSC apparatus was used. Elemental analyses were obtained by Galbraith Laboratories. Synthesis of Cyclic Trimer 6. A solution of hexachlorocyclotriphosphazene (4) (1.0 g, 2.9 mmol) in dry THF (100 mL) was added to a solution of L-tyrosine ethyl ester (10.8 g, 51.7 mmol) in THF (50 mL). The reaction was allowed to proceed at room temperature for 48 h, followed by warming for 5 h at 35 °C. Full replacement of the chlorine atoms by the tyrosine ethyl ester groups was not achieved as indicated by 31P NMR spectroscopy. Synthesis of Cyclic Trimer 7a. L-Tyrosine ethyl ester (18.0 g, 86.2 mmol) was allowed to react with an excess of sodium (3.0 g, 129 mmol) in dry THF (200 mL). After 48 h, unreacted sodium was removed from the reaction mixture, and a solution of 4 (2.0 g, 5.8 mmol) was added to the icecooled tyrosine ethyl ester reaction mixture. After 12 h, the reaction mixture was allowed to warm to room temperature. After 3 days, the mixture was filtered, concentrated, and precipitated into hexanes. The trimer 7a was further purified by dialysis against THF (5 days). 31P NMR (CDCl3): δ 9.73 ppm. 1H NMR (CDCl3): δ 7.00 (2H), 6.69 (2H), 4.14 (2H), 3.85 (1H), 3.75 (1H), 2.99 (1H), 2.82 (1H), and 1.22 (3H). Positive FAB mass spectrometry showed an m + 2 peak at 1385, which corresponds to the species formed by protonation of the carbonyl and amino groups. Synthesis of Cyclic Trimer 7b. Trimer 7a (0.6 g, 0.434 mmol) was dissolved in THF (100 mL). A solution of potassium tert-butoxide (5.8 g, 52.1 mmol) in THF, was added dropwise to the polymer solution, along with a catalytic amount of water. After 48 h, THF was decanted from the reaction mixture to leave an adhesive material. This residue was dissolved in water, and the resultant solution was acidified with concentrated hydrochloric acid. Precipitation occurred at approximately pH 4. The mixture was filtered and the residue was washed several times with water. The product was dried under vacuum. 31P NMR (NaOD/ D2O) δ 9.61 ppm. Synthesis of Polymers 9a-9f. The synthesis of these polymers was carried out in a similar manner. The procedure for polymer 9f is given as a typical example. Poly(dichlorophosphazene) (8) (3.00 g, 25.9 mmol) in THF (300 mL) was added dropwise to L-tyrosine ethyl ester (32.5 g, 155 mmol) in THF (200 mL). The reaction was carried out at room temperature for 3 days. The mixture was then heated to 35 °C for 5 h to maximize substitution. Glycine ethyl ester

Allcock et al. Table 4. Reaction Conditions for Synthesis of Polymers 10 and 11

[NPCl2]n polymer (mmol)

NaOR (mmol) R

R′c

solvent (reaction t-BuO-K+ TFA time, days) (mmol) (mL)

10

17.2

17.2a 26

THF (3)

24.4

20

11

17.2

17.2b 34.4 THF (1)

24.4

20

a

31P

NMR (ppm)

-7.5 -13.1 -18.2 -9.1 -12.6 -15.3

R ) methoxyethoxyethanol. b R ) trifluoroethanol. c R′ ) N-t-BOCmethyl ester.

L-tyrosine

was prepared by treatment of glycine ethyl ester hydrochloride (3.62 g, 25.9 mmol) in boiling dry toluene (200 mL) in the presence of triethylamine (3.60 mL) as a HCl acceptor. After 10 h, the reaction mixture was filtered and the filtrate was added to the solution containing the partially substituted polymer. This reaction was allowed to proceed at room temperature for 3 days. The mixture was then filtered and the residue was dissolved in and dialyzed against methanol for 4 days. Attempts were made to remove the hydrogen chloride as Et3N‚HCl, by dissolving the polymer in methanol and adding triethylamine. This resulted in the precipitation of the polymer from the methanolic solution. The polymer was then washed quickly with water to remove traces of Et3N‚HCl and was then immediately dried under vacuum. The resultant polymer was partially soluble in THF and fully soluble in DMSO and dimethyl acetamide. A brittle, yellowcolored polymer was obtained. Table 1 discusses the characterization data for polymers 9a-f. Synthesis of Polymers 10-12. These reactions were carried out in a similar manner. The procedure for the synthesis of polymer 12 is given as a typical example. Details for the synthesis of polymers 10 and 11 are listed in Table 4. Poly(dichlorophosphazene) (8) (2.00 g, 17.3 mmol) was dissolved in 400 mL of THF. In a separate reaction vessel, 1-propanol (1.26 g, 20.9 mmol) was added to a suspension of sodium hydride (0.76 g, 18.98 mmol, 60% dispersion in mineral oil) in 50 mL of THF and was allowed to react for 24 h. Sodium propoxide solution was then added slowly to the polymer solution via an addition funnel. The reaction was allowed to proceed at room temperature for 24 h. N-tBOC-L-tyrosine methyl ester (7.86 g, 26.6 mmol) was then added to a suspension of sodium hydride (0.97 g, 24.15 mmol, 60% dispersion in mineral oil) in 50 mL of THF and allowed to react for 24 h, at room temperature. After the reaction was complete, this solution was added dropwise via an addition funnel to the reaction mixture that contained the partially substituted polymer. The reaction solution was then heated at reflux for 48 h. The completion of the reaction was checked by 31P NMR spectroscopy. The polymer was purified by precipitation from heptane (2×), and by dialysis against methanol for 72 h. 31P NMR (D2O), ppm: δ -7.6, -12.5, -18.3. 1H NMR, ppm: δ 0.6 (3H), 1.3 (2H), 1.4 (9H), 3.0 (2H), 3.5 (2H), 3.7 (3H), 4.1 (1H), 7.01 (4H). 13 C NMR, ppm: δ 180 (1C), 158 (1C), 152 (1C), 135 (2C), 132 (2C), 122 (1C), 81 (1C), 71 (1C), 59 (1C), 53 (1C), 39 (1C), 29 (3C), 25 (1C), 11 (1C). IR, cm-1: 3400-3200 (NH), 1738 (CdO), 1687 (NHCdO), 1393, 1366 (tert-butyl), 1214 (PdN/P-O), 1168 (OCH3).

Tyrosine-Bearing Polyphosphazenes

Synthesis of Polymer 12b. Polymer 12a (1.0 g, 2.5 mmol) was allowed to swell in 50 mL of THF. Potassium tertbutoxide (3.928 g, 35 mmol) was dissolved in 50 mL of THF. The solution was cooled to 0 °C, and a catalytic amount of water was added. The resultant solution was added slowly to the polymer suspension in THF, via an addition funnel. This reaction was allowed to proceed at room temperature for 24 h. Cold water was added to the mixture, and the polymer was precipitated by the addition of concentrated hydrochloric acid. The polymer was then dialyzed against deionized water for 48 h. The solvent was evaporated, and the polymer was dried under vacuum. The dried polymer was then treated with a 50% trifluoroacetic acid/methylene chloride solution (20 mL), and the reaction was allowed to proceed for 8 h. Excess acid was neutralized by the addition of a saturated solution of sodium bicarbonate. The polymer was then dialyzed against deionized water for 2 days, after which time the solvent was evaporated to yield polymer 12b. The polymer was further dried under vacuum. 31P NMR (D2O), ppm: δ -7.6, -12.5, -18.3. 1H NMR, ppm: δ 0.6 (3H), 1.3 (2H), 3.0 (2H), 3.5 (2H), 4.1 (1H), 7.01 (4H). 13C NMR, ppm: δ 174 (1C), 151 (1C), 132 (2C), 131 (2C), 121 (1C), 70 (1C), 56 (1C), 36 (1C), 24 (1C), 10 (1C). IR, cm-1: 3500-2600 (NH2, COOH), 1724 (CdO), 1591, 1401 (COO-), 1218 (PdN/P-O). Hydrolysis Studies of Polymers 9a-9f. A stainless steel casting die was filled with approximately 200 mg of polymer and then heated above the glass-transition temperature of the polymer. The polymer was compressed at high pressure to form 1/4 in. × 1/4 in. cylindrical pellets. Analysis of the pellets by 31P NMR spectroscopy indicated no change in the phosphorus peak nor the presence of additional peaks as a result of side reactions. Deionized water (20 mL) was then added to each sample of polymer (concentration 1% w/v). During the hydrolysis experiments, the swollen and partially degraded polymers were dried and weighed. Detection of Hydrolysis Products of Polymers 9a-9f. The presence of tyrosine, glycine, and ammonia was detected qualitatively with the use of ninhydrin. Ninhydrin reacts to form an intensely colored purple anion (λmax ) 570 nm). Phosphates were detected semiquantitatively by the addition of zirconyl chloride or silver nitrate to the hydrolysis media and the precipitation of zirconyl phosphate or silver phosphate. Ethanol was detected by 1H NMR spectroscopy. Ionic Cross-Linking Reactions with Polymers 10b and 12b. Polymers 10b and 12b were dissolved in 0.2 mL of deionized water. To each of the polymer solutions was added an aqueous solution of CaCl2 (0.006 mmol). The solutions were stirred for 1 min to produce the cross-linked gels.

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Acknowledgment. This work was supported in part through the Ethicon division of Johnson and Johnson. References and Notes (1) Nathan, A.; Kohn, J. In Biomedical Polymers, Designed-to-Degrade Systems, Shalaby, S. W., Ed.; Hanser Publishers: New York, 1994; p 117. (2) Kohn, J. In Biodegradable Polymers in Drug DeliVery Systems; Chasin, M., Langer, R., Eds.; Marcel Dekker: New York, 1990; p 195. (3) Huang, S. J.; Ho, L.-H. Macromol. Symp. 1999, 144, 7. (4) Walton, A. G. In Biomedical Polymers, Polymeric Materials and Pharmeceuticals for Biomedical Use; Goldberg, E. P., Nakajima, A., Eds.; Academic Press: New York, 1980; p 53. (5) Stupp, S. I.; Ciegler, G. W. J. Biomed. Mater. Res. 1992, 26, 169. (6) Ertel, S. I.; Kohn, J. J. Biomed. Mater. Res. 1994, 28, 919. (7) Hooper, K. A.; Macon, N. D.; Kohn, J. J. Biomed. Mater. Res. 1998, 41, 443. (8) Yokoyama, M.; Miyauchi, M.; Yamada, N.; Okano, T.; Sakurai, Y. Cancer Res. 1990, 40, 1693. (9) Yokoyama, M.; Inoue, S.; Katako, K.; Okano, T.; Sakurai, Y. Makromol. Chem. 1989, 190, 2041. (10) Allcock, H. R. In Chemistry and Applications of Polyphosphazenes; Wiley-Interscience: Hoboken, NJ, 2003; p 504. (11) Allcock, H. R. In Biodegradable Polymers as Drug DeliVery Systems; Chasin, M., Langer, R., Eds.; Marcel Dekker: New York, 1990; p 163. (12) Langone, F.; Lora, S.; Veronese, F. M.; Caliceti, P.; Parnigotto, P. P.; Valenti, F.; Palma, G. Biomaterials 1995, 16, 347. (13) Schacht, E.; Vandorpe, J.; Dejardin, S.; Lemmouchi, Y.; Seymour, L. Biotechnol. Bioeng. 1996, 52, 102. (14) Laurencin, C. A.; Norman, M. E.; Elgendy, H. M.; El-Amin, S. F.; Allcock, H. R.; Pucher, S. R.; Ambrosio, A. A. J. Biomed. Mater. Res. 1993, 27, 963. (15) Allcock, H. R.; Fuller, T. J.; Mack, D. P.; Matsumura, K.; Smeltz, K. M. Macromolecules 1977, 10, 824. (16) Allcock, H. R.; Pucher, S. R.; Scopelianos, A. G. Biomaterials 1994, 15, 563. (17) Allcock, H. R.; Pucher, S. R.; Scopelianos, A. G. Macromolecules 1994, 27, 1071. (18) Allcock, H. R.; Kwon, S. Macromolecules 1989, 22, 75. (19) Cohen, S.; Bano, M. C.; Visscher, K. B.; Chow, M.; Allcock, H. R.; Langer, R. J. Am. Chem. Soc. 1990, 112, 7832. (20) Ambrosio, A. M. A. Ph.D. Thesis, The Pennsylvania State University, University Park, PA 1996. (21) Allcock, H. R.; Ambrosio, A. M. A. Biomaterials 1996, 17, 2295. (22) Allcock, H. R.; Fuller, T. J.; Matsumura, K. Inorg. Chem. 1982, 21, 515. (23) Allcock, H. R.; Dudley, G. K. Macromolecules 1996, 29, 1313. (24) Poly[bis(ethyl phenylalaninat-N-yl) phosphazene] forms partially flexible films, which increase in flexibility with the introduction of glycine ethyl ester cosubstituents. (25) Precedence exists for rearrangement reactions of ethoxy-linked polyphosphazenes. (26) Voet, D.; Voet, J. G. In Biochemistry; John Wiley & Sons: New York, 1990. (27) Sassi, A. P.; Shaw, A. J.; Prausnitz, J. M. Polymers 1996, 37, 2151.

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