DesaminotyrosylâTyrosine Alkyl Esters - ACS Symposium Series

Chapter 15

Desaminotyrosyl-Tyrosine Alkyl Esters New Diphenolic Monomers for the Design of Tyrosine-Derived Pseudopoly(amino acids) Joachim Kohn

Downloaded by UNIV OF PITTSBURGH on July 2, 2013 | http://pubs.acs.org Publication Date: August 15, 1991 | doi: 10.1021/bk-1991-0469.ch015

Department of Chemistry, Rutgers University, New Brunswick, NJ 08903

In peptide chemistry, peptide analogs containing nonnatural backbone linkages are classified as "pseudopeptides". In analogy, we used the term "pseudopoly(amino acid)" to denote polymers in which amino acids are linked together by bonds other than conventional peptide bonds. Here we summarize our studies on the identification of desaminotyrosyl-tyrosine alkyl esters as new diphenolic monomers for the design of amino acid derived polyiminocarbonates and polycarbonates. In particular, desaminotyrosyl-tyrosine hexyl ester was found to lead to mechanically strong polymers with good engineering properties. These new pseudopoly(amino acids) appear to be promising materials for a number of biomedical applications.

It is a well established observation that the mechanical strength and toughness of a polymer can be increased by the incorporation of aromatic monomers into the polymer backbone. For example, some of the strongest polymers known today (Kevlar and P E E K ) are both exclusively derived from aromatic monomers. Yet, among the biomaterials, aromatic monomers are very rarely used and high molecular weight poly(L-lactic acid) (a completely aliphatic polymer) is the strongest degradable implant material currently available (7). Since extremely strong, yet degradable polymers would present an important addition to the range of currently available polymeric biomaterials, we attempted to identify aromatic monomers that could be safely used in the design of high strength biomaterials. Unfortunately, the limited biocompatibility of many industrially used aromatic monomers imposes severe restrictions on the use of aromatic backbone structures in degradable polymers intended for medical applications. Diphenols, for instance, which are necessary intermediates in polyurethane, polycarbonate, and polyester chemistry are often too toxic to be considered in the design of a degradable 0097-6156/91/0469-0155$06.00/0 © 1991 American Chemical Society

In Polymeric Drugs and Drug Delivery Systems; Dunn, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.


156

POLYMERIC DRUGS AND DRUG DELIVERY SYSTEMS

biomaterial. In a detailed comparison of relevant toxicological data, we identified Bisphenol A (BPA) as probably one of the least toxic monomers among the industrial diphenols (2). Due to the use of B P A in the fabrication of polycarbonate based food containers, baby bottles and kitchen utensils, the toxicological properties of B P A have been carefully studied (3): B P A has a low level of oral toxicity and was found to be noncarcinogenic in a National Cancer Institute bioassay (4). Most significantly, BPA-derived polyiminocarbonates produced only a relatively mild "foreign body reaction" when implanted subcutaneously in mice, rabbits and rats (2,5). Similar results were recently observed for BPA-containing polyphosphoesters that also showed good tissue compatibility in preliminary implantation studies (6). On the other hand, we found B P A to be highly cytotoxic in vitro using a chick embryo fibroblast tissue culture assay (7). In conjunction with the known irritant properties of B P A (4), our interpretation of these results is that even B P A has only marginal biocompatibility. In spite of the lack of a strong inflammatory tissue response in preliminary, subcutaneous implantation studies, we currently suggest that BPA-containing, degradable polymers may be of only limited value as biomaterials. In an attempt to identify more biocompatible diphenols for the design of degradable biomaterials, we studied derivatives of tyrosine dipeptide as potential monomers. After protection of the amino terminus and the carboxylic acid terminus, the reactivity of tyrosine dipeptide (Figure 1) could be expected to be similar to the reactivity of industrial diphenols. Thus, derivatives of tyrosine dipeptide could be suitable replacements for B P A in the synthesis of a variety of new polymers that had heretofore not been accessible as biomaterials due to the lack of diphenolic monomers with good biocompatibility. In this context, we were particularly interested in the exploration of polyiminocarbonates {2,8) and polycarbonates (5), two structurally related classes of polymers that are best derived from diphenolic monomers. The corresponding BPAderived polymers (Figure 2) had previously been found to be particularly strong materials with excellent engineering properties (2,3). In order to identify tyrosine derivatives that could be used in place of B P A , we initiated a detailed investigation of the structure-property relationships in polyiminocarbonates (9,10) and polycarbonates (11). These studies led to the identification of desaminotyrosyltyrosine hexyl ester (Dat-Tyr-Hex, also abbreviated as DTH) as the first tyrosine derived monomer whose polymerization leads to strong and processible polymers with a wide range of potential biomedical applications. Experimental A l l solvents were of H P L C grade and were used as received, except for tetrahydrofuran (THF) which was freshly distilled from sodium and benzophenone prior to use. M o l e c u l a r Weight. Molecular weights were determined by gel permeation chromatography (GPC) on a chromatographic system consisting of a Perkin-Elmer Model 410 pump, a Waters Model 410 RI detector, and a Perkin-Elmer Model 3600


15. KOHN

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Desaminotyrosyl— Tyrosine Alkyl Esters

computerized data station. For polymer analysis, two PL-gel GPC columns (300 mm χ 7.7 mm, particle size 5 |im, pore size 10 and 10 A respectively) were placed in series and were operated at a flow rate of 1 mL/min in D M F containing 0.1% (w/v) of LiBr. Molecular weights were calculated relative to polystyrene standards without further corrections. 5

3

Thermal Properties. The glass transition temperature (T ) and the decomposition temperature ( T ) were measured with a DuPont 910 Differential Scanning Calorimeter (DSC) calibrated with indium. The standard heating rate for a l l polymers was 10 °C/min. Thermogravimetric analysis (TGA) was performed on a DuPont 951 Thermogravimetric Analyzer at a heating rate of 20 °C/min. g


d

Polymer Processing. Polymer films were cast in trimethylsilyl coated glass molds from membrane filtered 15% (w/v) methylene chloride or chloroform solutions. Transparent films were obtained which were dried to constant weight in high vacuum. Rectangular strips or round disks were cut from the films. F o r compression molding a Carver laboratory press equipped with thermostated, heated platens was used. Polymers were placed in a stainless steel mold and heated to 40 °C above their glass transition temperature. Then a load of 1-2 tons was applied for 5 min. Mechanical Properties. The mechanical properties of thin, solvent-cast polymer films were measured on an Instron Tensile Tester according to A S T M standard D882-83. In all cases, tensile values were calculated from the average of at least four measurements obtained from four separate specimens per polymer sample. Hydrolytic Degradation Studies. D i s k s (2.3 cm χ 1.1 cm χ 0.05 c m , approximately 150 mg) were cut from solvent-cast films. The disks were incubated at 37 C in phosphate buffer (0.1 M , pH 7.4). The degradation process was followed by recording the weight change of individual disks, by measuring the residual polymer molecular weight after various intervals of exposure to the buffer solution and by FT-IR analysis of partly degraded samples. e

Synthesis of Tyrosine Derivatives. Tyrosine derived monomers were prepared by D C C mediated coupling reactions in THF following standard procedures of peptide chemistry (12). Dat-Tym, Z-Tyr-Tym, Z-Tyr-Tyr-Hex, and Dat-Tyr-Hex (DTH) were purified and characterized according to reference 10. For spectral data of ZTyr-Tyr-Hex, see Kohn and Langer (73). Synthesis of Dicyanates. A l l dicyanates were prepared according to previously published procedures (2). For IR spectral data see Kohn and Langer (2), for elemental analyses, see reference 10. A specific procedure for the preparation of ZTyr-Tyr-Hex-dicyanate has been published (75). Preparation of Polyiminocarbonates. Solution polymerizations of equimolar mixtures of diphenols and dicyanates were carried out in THF using potassium tert-


158


butoxide as the catalyst as described previously (5). Interfacial polymerizations were performed in a two phase system consisting of aqueous NaOH and methylene chloride in the presence of 10% (molar) of tetrabutylammonium bromide as a phase transfer catalyst (#). A l l polyiminocarbonates showed a strong and sharp IR absorption band at 1670 cm" to 1680 c m " , which is characteristic of the iminocarbonate bond.


1

1

Preparation of Polycarbonates. About 30 mmoles of a tyrosine derived diphenolic monomer was placed into a 250 mL flask. After addition of 75 mL of dry methylene chloride and 11 mL of anhydrous pyridine, a pale yellow solution was obtained. At room temperature, a molar excess of phosgene (as a 1.94 M solution in toluene) was added slowly to the vigorously stirred solution over a period of about 90 minutes. The molecular weight of the polymeric product was monitored by G P C and the addition of phosgene was terminated after the maximum molecular weight had been obtained. Stirring was continued for an additional 120 minutes. Thereafter, the reaction mixture was diluted with 500 mL of methylene chloride, transferred into a separatory funnel and extensively extracted with 0.2 Ν aqueous H C L . The organic phase (containing the polymer) was then dried over magnesium sulfate and concentrated to 150 m L . The polymer was precipitated by slowly adding the concentrated solution into 750 mL of hexane. Yield: about 85%; intrinsic viscosity (chloroform, 30 C): 0.6 to 1.0 dL/g, depending on monomer purity. e

Results and Discussion In an attempt to identify new, biocompatible diphenols for the synthesis of polyiminocarbonates and polycarbonates, we considered derivatives of tyrosine dipeptide as potential monomers. Our experimental rationale was based on the assumption that a diphenol derived from natural amino acids may be less toxic than many of the industrial diphenols. After protection of the amino and carboxylic acid groups, we expected the dipeptide to be chemically equivalent to conventional diphenols. In preliminary studies (14) this hypothesis was confirmed by the successful preparation of poly(Z-Tyr-Tyr-Et iminocarbonate) from the protected tyrosine dipeptide Z-Tyr-Tyr-Et (Figure 3). Unfortunately, poly(Z-Tyr-Tyr-Et iminocarbonate) was an insoluble, nonprocessible material for which no practical applications could be identified. This result illustrated the difficulty of balancing the requirement for biocompatibility with the need to obtain a material with suitable "engineering" properties. In order to identify tyrosine derivatives that would lead to polymers that are processible, mechanically strong, and also biocompatible, we initiated a detailed investigation of the structure-property relationships in polyiminocarbonates and polycarbonates. Since the amino and carboxylic acid groups of tyrosine dipeptide (the Ν and C termini) provide convenient attachment points, selected pendent chains can be used to modify the overall properties of the polymers. This is an important structural feature of tyrosine dipeptide derived polymers.



15. KOHN

Desaminotyrosyl— Tyrosine Alkyl Esters

159

protected tyrosine dipeptide

Figure 1. Molecular structures of Bisphenol A and fully protected tyrosine dipeptide. The amino and carboxylic acid groups of the dipeptide are rendered unreactive by protecting groups (schematically represented by X and Y). This leaves the phenolic hydroxyl groups as the only reactive sites of the molecule.

poly(BPA-carbonate)

poly(BPA-iminocarbonate)

Figure 2. Molecular structures of poly(Bisphenol A carbonate) and poly(Bisphenol A iminocarbonate).

NH [ -CH—C —NH—CH—CH. 2

1

I 1

NH

c=o

1

c=o I 1 0 1 CH

Y = ethyl: Y = hexyl: Y = palmityl:

1 ι

Ο1 1

Y

2

poly(Z-Tyr-Tyr-Et Iminocarbonate) poly(Z-Tyr-Tyr- Hex iminocarbonate) poly(Z-Tyr-Tyr-Pal iminocarbonate)

Figure 3. A homologous series of three tyrosine dipeptide derivatives gave rise to three new polyiminocarbonates that differed only in the length of the alkyl chain attached to the carboxylic acid group of the dipeptide.



160


The Effect of Pendent Chains on the Physicomechanical Properties of Tyrosine Derived Polymers. In order to investigate the influence of the C-terminus protecting groups on the polymer properties, we prepared the ethyl, hexyl and palmityl esters of N-benzyloxycarbonyl-L-tyrosyl-L-tyrosine (75). These monomers represented a homologous series which made it possible to prepare three new polyiminocarbonates differing only in the length of the alkyl group attached to the C-terminus (Figure 3). The first member of this series, poly(Z-Tyr-Tyr-Et iminocarbonate), was a virtually insoluble polymer that was not processible. Poly(Z-Tyr-Tyr-Hex iminocarbonate), the second member of the series, was soluble in many common organic solvents such as chlorinated hydrocarbons, T H F , D M F , and D M S O . Solvent casting yielded transparent, but brittle films. The increasing length of the pendent alkyl group also reduced the polymer glass transition temperature (T ) by 29 °C (Table I). Poly(Z-Tyr-Tyr-Pal iminocarbonate), the third member of the series, was very similar to poly(Z-Tyr-Tyr-Hex iminocarbonate) in spite of a large increase in the length of the C-terminus protecting group from C$ to C . These observations indicate that the C-terminus protecting group must have a minimum length in order for the tyrosine derived polyiminocarbonate to become soluble in organic solvents. However, increasing the length of the C-terminus alkyl ester chain beyond C did not have any further advantage. We therefore selected the hexyl ester as the standard C-terminus protecting group throughout the remainder of our studies. Currently, a more detailed investigation of the relationship between solubility, T , and mechanical strength on one hand and the length of the ester side chain on the other hand is in progress. In a second series of experiments (9,70), tyrosine derived dipeptides were synthesized in which the amino group and/or the carboxylic acid group of tyrosine were replaced by hydrogen atoms. This was achieved by using the tyrosine derivatives desaminotyrosine (Dat), and tyramine (Tym) instead of tyrosine (Figure 4). Dat occurs naturally in plants and Tym is a human metabolite. Using these building blocks, four structurally related dipeptide derivatives were prepared that carried either no pendent chains at all (Dat-Tym), a N-benzyloxycarbonyl group as the only pendent chain (Z-Tyr-Tym), a hexyl ester group as the only pendent chain (Dat-Tyr-Hex, further abbreviated as D T H ) , or both types of pendent chains simultaneously (Z-Tyr-Tyr-Hex). This series of monomers (Figure 4) made it possible to investigate the contribution of each type of pendent chain separately. Of course, Z-Tyr-Tyr-Hex is identical to the monomer used before (Figure 3). The corresponding polyiminocarbonates (Figure 5) were prepared first, using recently developed polymerization procedures (8). Poly(Dat-Tym iminocarbonate), the polymer carrying no pendent chains at all, was an insoluble material. Thermal processing techniques could not be used due to the low thermal stability of the polymer in the molten state. Thus poly(Dat-Tyr iminocarbonate) was a virtually non-processible material without practical applications. The presence of a stiff benzyloxycarbonyl group in Z-Tyr-Tym did not significantly improve the processibility or solubility of the corresponding polyiminocarbonate: Poly(Z-Tyr-Tym iminocarbonate), like poly(Dat-Tym iminocarbonate), was an insoluble, and nonprocessible material. On the other hand, g

1 6

6

g


In Polymeric Drugs and Drug Delivery Systems; Dunn, R., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991. 149 62 86

Poly(BPA carbonate)[109,000] Poly(Dat-Tyr-Hex carbonate)[262,000] Poly(Z-Tyr-Tyr-Hex carbonate)[400,000]

f

e

d

c

b

a

400

625 335 340

175

486 355 261

158

416 305 232

510 -

171 163 160 163 147

151 145 143 141 130

2

2

22,000 13,900 13,200

16,300

22,000 -

6 3 4

3.5

3.5 -

yes no yes limited* limited* yes

yes yes yes

4.0 7.5

60 100 5

c

Solubility*

weight average molecular weight as determined by GPC relative to polystyrene standards without further correction. polymers were classified as soluble if clear solutions in chlorinated hydrocarbons, DMF, THF, and DMSO were obtained at a concentration of at least 10 mg/mL. no solvent could be identified. limited solubility in DMF/LiBr or DMF/chloroform mixtures only. due to the insoluble nature of the polymer, the molecular weight could not be determined. no glass transition was observed from 0*C up to the onset of thermal decomposition.

Poly(Dat-Tyr-Hex iminocarbonate)[103,000]

69 91 62 60 not observed* 55

e

m

m

Glass Transition Decomposition Tensile Tensile Elongation at peak strength modulus yield break onset Tg (%) (kg/cm ) (kg/cm ) (%) CO

Poly(BPA iminocarbonate)[109,000] Poly(Z-Tyr-Tyr-Et iminocarbonate)[n/a] Poly(Z-Tyr-Tyr-Hex iminocarbonate )[54,000] Poly(Z-Tyr-Tyr-Pal iminocarbonate) [69,000] Poly(Dat-Tym iimnocarbonate)[71,500]

0

Polymer [molecular weight]

Table I. Physicomechanical Properties of Selected Polymers



162


_

Ο

HO-^~^-CH

2

-ÇH -C-NH-CH-CH —^"~"^-OH 2

Η

Η

desamino-tyrosyl-tyramine (Dat-Tym) _

Ο

HOH^~^-CH

2

-ÇH -C-NH-CH-CH —

DesaminotyrosylâTyrosine Alkyl Esters - ACS Symposium Series

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DesaminotyrosylâTyrosine Alkyl Esters - ACS Symposium Series