Chemical Synthesis and Structural Characterization of Linear and

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Elastin-Based Biopolymers: Chemical Synthesis and Structural Characterization of Linear and Cross-Linked Poly(OrnGlyGlyOrnGly) M. Martino, T. Perri, and A. M. Tamburro* Department of Chemistry, University of Basilicata,Via N. Sauro 85, 85100 Potenza, Italy Received August 10, 2001; Revised Manuscript Received December 15, 2001

Poly(OrnGlyGlyOrnGly) was synthesized by classical procedures in solution. The monomeric sequence -OrnGlyGlyOrnGly- was chosen as a modification of -ValGlyGlyValGly-, typical of elastin, to impart primary amine functionality, susceptible to cross-linking with appropriate bifunctional reagents. Herein we focus on the cross-linking of poly(OrnGlyGlyOrnGly) with glutaraldehyde. The polymers, both linear and cross-linked, were characterized and investigated for their molecular and supramolecular properties. Circular dichroism studies performed on linear poly(OrnGlyGlyOrnGly) revealed a variety of conformations similar to elastin. At a supramolecular level, different kinds of aggregates were found such as the elastin-like twistedrope pattern of filaments and fibrils, together with other specific morphologies, similar to those recently identified in some elastin-mimetic polypeptides. Introduction

Materials and Methods

The domain of polypeptide biomaterials has became an extremely active field of research in recent years. Particular interest has been shown in protein mimetics and their possible utilization as prostheses for the vascular system and thus of importance for cardiovascular surgery. Elastin-based polymers would seem to deserve great attention in view of their potential application as artificial substitutes for arteries and veins. By taking advantage of the unique abundance of repetitive sequences in elastin, many linear sequential polypeptides have been chemically synthesized and investigated for their molecular and supramolecular properties.1-5 These results, taken together, clearly demonstrate the capacity of elastin-based polymers to mimic almost all the physical properties of elastin. This left the problem of obtaining an efficient method of cross-linking. After an initial approach by γ-irradiation,6 it seems that systems based on chemical coupling of functionalized elastin-like sequences7-10 are more efficient. To find the best conditions for efficient crosslinking, we decided to study different sequences comprising side-chain amino groups at different distances from the backbone and different cross-linking reagents with variable distances between the two functional groups. Examples are lysine and ornithine, on one hand, and glutaraldehyde (GTA) and disuccinimidyl glutarate (DSG) on the other. In this paper we report on the classical synthesis of GTA cross-linked poly(OrnGlyGlyOrnGly). The choice of this particular sequence was dictated by previous results on (ValGlyGlyValGly), (ValGlyGlyLeuGly) and (LeuGlyGlyValGly) sequences which demonstrated the presence of structures very similar to those of elastin.4,11-13 The insertion of two basic residues per monomeric unit should hopefully increase the potential for extensive cross-linking.

Synthesis. Amino acids were purchased from Novabiochem AG (Laufelfinger, Switzerland). Glycylglycine ethyl ester hydrochloride was purchased from Sigma Chemical Co (St. Louis, MO). The purity of all synthetic products was ascertained by thin-layer chromatography on butanol-acetic acid-water (3:1:1) or chloroform-methanol-acetic acid (9: 0.8:0.2). Further characterization was obtained by 1H NMR spectra recorded on a Bruker AM-300 spectrometer. The synthesis of peptides and polymers was performed as indicated in Scheme 1. Boc-Orn(Z)GlyGly-OEt. Isobutyl chloroformate (IBCF, 13.4 mmol) was added at -15 °C to a solution of BocOrn(Z)-OH (13.4 mmol) and N-methylmorpholine (NMM, 13.4 mmol) in chloroform (53.6 mL). The temperature was kept at -15 °C for 1 min, then Cl-H2+GlyGly-OEt (13.4 mmol) and NMM (13.4 mmol) were added. The mixture was stirred at room temperature for 24 h, and the organic solution was sequentially washed with 5% sodium bicarbonate, water, 5% citric acid, and water. The solution, dried over sodium sulfate, was evaporated to dryness, and an oily residue was obtained (6.17 g), which did not crystallize (91% yield). Boc-Orn(Z)GlyGly-OH. NaOH (1 N, 13.2 mL) was added to a solution of Boc-Orn(Z)GlyGly-OEt (12 mmol) in acetone (32 mL). The solution was stirred at room temperature for 3 h, then the organic solvent was evaporated under reduced pressure and water was added. The unreacted product was extracted with ethyl acetate, then the aqueous solution was cooled at 0 °C and neutralized with dropwise addition of 1 N HCl (13.2 mL). The obtained crude material was extracted with ethyl acetate and dried over sodium sulfate. The solution was evaporated to dryness, and the solid residue was crystallized from ethyl acetate/petroleum ether to give 4.03 g of tripeptide (70% yield). 1H NMR (DMSO-

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Scheme 1. Synthesis of Peptides and Polymers

d6) δ: 11.8 (bs, 1H, OH), 8.11 (m, 2H, NH Gly), 7.35 (A2B2X system, 5H, C6H5CH2O), 7.25 (t, 1H, NHδ Orn), 7.00 (d, 1H, NH Orn), 5.00 (s, 2H, C6H5CH2O), 3.90 (m, 1H, HR Orn), 3.75 (m, 4H, HR Gly), 2.98 (d, 2H, Hδ Orn), 1.60 (m, 2H, Hβ Orn), 1.41 (m, 2H, Hγ Orn), 1.35 (s, 9H, Boc). Boc-Orn(Z)Gly-OEt. Isobutyl chloroformate (16.4 mmol) was added at -15 °C to a solution of Boc-Orn(Z)OH (16.4 mmol) and NMM (16.4 mmol) in chloroform (65.6 mL). The temperature was kept at -15 °C for 1 min, then Cl-H2+Gly-OEt (16.4 mmol) and NMM (16.4 mmol) were added. The mixture was stirred at room temperature for 24 h. The organic solution was sequentially washed with 5% sodium bicarbonate, water, 5% citric acid, and water and dried over sodium sulfate. The solution was evaporated to dryness, and the residue was crystallized from diethyl ether/ petroleum ether to give 6.53 g of protected dipeptide (88% yield). 1H NMR (DMSO-d6) δ: 8.20 (t, 1H, NH Gly), 7.35 (A2B2X system, 5H, C6H5CH2O), 7.23 (t, 1H, NHδ Orn), 6.88 (d, 1H, NH Orn), 5.00 (s, 2H, C6H5CH2O), 4.07 (q, 2H, OCH2CH3), 3.91 (m, 1H, HR Orn), 3.80 (ABX system,

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2H, HR Gly), 2.80 (d, 2H, Hδ Orn), 1.51 (m, 2H, Hβ Orn), 1.41(m, 2H, Hγ Orn), 1.39 (s, 9H, Boc), 1.19 (t, 3H, OCH2CH3). TFA- H2+Orn(Z)Gly-OEt. Boc-Orn(Z)Gly-OEt (14.5 mmol) was dissolved in dichloromethane (32.7 mL), then trifluoro acetic acid (TFA, 32.7 mL) was added at 0 °C. After being stirred at 0 °C for 30 min, the solution was kept at room temperature for 2 h. The solvent was evaporated to dryness, giving 6.07 g of an uncrystallizable oil, which was used in the following step (90% yield). 1H NMR (DMSOd6) δ: 9.00 (t, 1H, NH Gly), 8.30 (bs, 3H, NH Gly), 7.437.30 (m, 6H, C6H5CH2O and NHδ Orn), 5.05 (s, 2H, C6H5CH2O), 4.10 (q, 2H, OCH2CH3), 4.05-3.80 (m, 3H, HR Orn and HR Gly), 3.02 (d, 2H, Hδ Orn), 1.73 (m, 2H, Hβ Orn), 1.52 (m, 2H, Hγ Orn), 1.20 (t, 3H, OCH2CH3). Boc-Orn(Z)GlyGlyOrn(Z)Gly-OEt. IBCF (8.35 mmol) was added at -15 °C to a solution of Boc-Orn(Z)GlyGlyOH (8.35 mmol) and NMM (8.35 mmol) in chloroform (33.4 mL). The temperature was kept at -15 °C for 1 min, and TFA-H2+Orn(Z)Gly-OEt (8.35 mmol) and NMM (8.35 mmol) were added. The solution was stirred at room temperature for 24 h. The obtained solid product was filtered, washed with water and diethyl ether, and finally crystallized from ethanol/diethyl ether to give 5.75 g of pentapeptide (85% yield). 1H NMR (DMSO-d6) δ: 8.48 (t, 1H, NH Gly), 8.11 (t, 1H, NH Gly), 8.01-8.00 (m, 2H NH Gly and NH Orn), 7.40-7.20 (m, 12H, A2B2X system, C6H5CH2O and NHδ Orn), 6.90 (d, NH Orn), 5.00 (s, 2H, C6H5CH2O), 4.28 (m, 1H, HR Orn), 4.08 (q, 2H, OCH2CH3), 3.90 (m, 1H, HR Orn), 3.85-3.70 (m, 6H, HR Gly), 2.90 (m, 4H, Hδ Orn), 1.65 (m, 4H, Hβ Orn), 1.45 (m, 4H, Hγ Orn), 1.35 (s, 9H, Boc), 1.18 (t, 3H, OCH2CH3). Boc-Orn(Z)GlyGlyOrn(Z)Gly-OH. NaOH (1 N, 7.75 mL) was added to a solution of Boc-Orn(Z)GlyGlyOrn(Z)Gly-OEt (7.08 mmol) in acetone (19 mL). After 3 h at room temperature, the organic solvent was evaporated under reduced pressure and water was added. The unreacted product was extracted with ethyl acetate, then the aqueous solution was cooled at 0 °C and neutralized with dropwise addition of 1 N HCl (7.75 mL). The crude material was extracted with ethyl acetate and dried over sodium sulfate. The organic solution was evaporated to dryness and the obtained residue was crystallized from ethanol/diethyl ether to give 3.80 g of pure solid (68% yield). 1H NMR (DMSOd6) δ: 8.22 (t, 1H, NH Gly), 8.10 (t, 1H, NH Gly), 8.087.98 (m, 2H, NH Gly and NH Orn), 7.35 (A2B2X system, 10H, C6H5CH2O), 7.22 (t, 2H, NHδ Orn), 6.98 (d, 1H NH Orn), 5.00 (s, 4H, C6H5CH2O), 4.30 (m, 1H, HR Orn), 3.90 (m, 1H, HR Orn), 3.73 (m, 6H, HR Gly), 2.99 (m, 4H, Hδ Orn), 1.65 (m, 4H, Hβ Orn), 1.43 (m, 4H, Hγ Orn), 1.37 (s, 9H, Boc). TFA- H2+Orn(Z)GlyGlyOrn(Z)Gly-OH. TFA (11 mL) was added at 0 °C to a solution of Boc-Orn(Z)GlyGlyOrn(Z)Gly-OH (4.80 mmol) in dichloromethane (11 mL). The reaction mixture was kept at 0 °C for 30 min and at room temperature for 2 h. The solution was evaporated to dryness, and the crude material was crystallized from methanol/diethyl ether to give 3.58 of pentapeptide (92% yield). 1H NMR (DMSO-d6) δ: 8.72 (t, 1H, NH Gly), 8.30 (t, 1H, NH Gly),

Elastin-Based Biopolymers

Figure 1. SDS-PAGE gel of linear (lane 3) and GTA-cross-linked (lanes 1 and 2) poly((OrnGlyGlyOrnGly). Lane 4 contains standards having the indicated molar masses in daltons.

8.20 (t, 1H, NH Gly), 8.17-8.02 (m, 2H, NH Orn), 7.407.20 (m, 12H, C6H5CH2O and NHδ Orn), 5.00 (s, 4H, C6H5CH2O), 4.30 (m, 1H, HR Orn), 3.89-3.70 (m, 7H, HR Gly and HR Orn), 3.00 (d, 4H, Hδ Orn), 1.68 (m, 4H, Hβ Orn), 1.45 (m, 4H, Hγ Orn). Poly[Orn(Z)GlyGlyOrn(Z)Gly]. Diphenyl phosphoryl azide (DPPA, 0.62 mmol) and triethylamine (TEA, 1.03 mmol) were added to a solution of TFA-H2+Orn(Z)GlyGlyOrn(Z)Gly-OH (0.41 mmol) in dimethyl sulfoxide (DMSO, 4.35 mL) (ref 14). The reaction mixture was held at room temperature for 48 h, and the resulting crude material was triturated with diethyl ether to give 2.0 g of benzyloxycarbonylated polymer (69% yield). Poly(OrnGlyGlyOrnGly). A 1.18 g portion of 5% Pd/C were suspended in 4.50 mL of 80% acetic acid and left under vigorous stirring and under nitrogen for 30 min. Then a solution of poly[Orn(Z)GlyGlyOrn(Z)Gly] (2.0 g) in 181 mL of the same solvent was added dropwise. The reaction was held at room temperature until the evolution of CO2 had stopped and kept under nitrogen for a further 15 min. The catalyst was filtered and repeatedly washed with 80% acetic acid.15 The solution obtained after filtration was evaporated

Figure 2. MALDI-TOF spectrum of poly(OrnGlyGlyOrnGly).

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under vacuum, and water was added. After lyophilization, the obtained crude product was crystallized from methanol/ diethyl ether to give 200 mg of de-benzyloxycarbonylated polymer (30% yield). Its molecular weight was estimated by SDS-PAGE to be comprised of between 6000 and 14 000 Da (Figure 1). A further characterization by matrix-assisted laser desorption ionization (MALDI) mass spectrometry has assessed a distribution of molecular weights centered around 2000 Da (Figure 2). As a matter of fact, the MALDI-TOF technique underestimates the molecular weight distribution of polydisperse polymers, as our case is.16,17 Chemical Cross-Linking of Poly(OrnGlyGlyOrnGly) with GTA. Poly(OrnGlyGlyOrnGly) (47.5 mg, 0.119 mmol) was dissolved in 0.1 M phosphate buffer, pH 7.0. A 1.43 mL portion of a 2.5% GTA solution was dropwise added to the polymer solution (GTA-to-NH2 ratio 1.5), and the reaction mixture was kept under gentle stirring at room temperature for 24 h.18,19 Then NaBH3CN (750 mg) was added at 0 °C, in large excess (about 50 times) with respect to the number of -NH2 groups per pentameric unit.20 After 5 h at room temperature, the reaction was stopped and dialyzed for 20 h against water, using 3500 Da molecular weight cutoff dialysis membranes. The solution was lyophilized to give 13 mg of a solid product. Its molecular weight was estimated by SDS-PAGE to be in the range 30000120000 Da (Figure 1). Estimation of Residual Amino Residues. To estimate the content of residual amino groups and the efficiency of the cross-linking reaction between GTA and poly(OrnGlyGlyOrnGly), the resulting cross-linked product was treated with a 0.2 M ethanolic ninhydrin solution.21 This method was based on the formation of a blue colored compound by reaction of ninhydrin and the ornithine -NH2 groups, which can be spectroscopically detected at 570 nm with high sensitivity. The total amine content of a non-cross-linked sample of poly(OrnGlyGlyOrnGly) was also determined as a benchmark for the analysis of the cross-linked sample. To

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this purpose, several solutions of poly(OrnGlyGlyOrnGly) in 0.2 M citrate buffer, pH 5.0, were prepared, having concentrations ranging from 2.0 to 6.0 mM. A 2 mg portion of GTA cross-linked poly(OrnGlyGlyOrnGly) was dissolved in 1 mL of citrate buffer, and serial dilutions of this solution afforded the samples for the analysis. A 1.0 mL portion of ninhydrin solution was added to 0.1 mL of either linear or cross-linked samples and to a blank containing 0.1 mL of citrate buffer instead of the polymer solution. Each sample was neutralized with 1 N NaOH (0.010 mL) and heated at 70 °C for 20 min in a water bath. Three milliliters of diluent solution, containing equal volumes of water and ethanol, were added to each tube, and the contents were mixed. Readings were taken starting 15 min after the samples had been removed from the water bath. Two distinct measurements were conducted for each concentration, and the average absorbances values ( SD (standard deviation) were plotted vs millimolar concentration of -NH2 groups per pentameric unit (data not shown). Statistical significance was tested by the Student’s t-test and a p value < 0.05 was considered statistically significant. The data points are well described by a linear fit (equation: y ) 0.078x; R2 ) 0.993). The residual amine content of the cross-linked polymer was estimated as about 8% of the theoretical value for non-crosslinked poly(OrnGlyGlyOrnGly), indicating that more than 90% of the ornithine residues, originally present in poly(OrnGlyGlyOrnGly), reacted with GTA. MALDI Mass Spectra. MALDI mass spectra were recorded on a PerSeptive Biosystem Voyager DE mass spectrometer operating in the positive-ion mode with the delayed extraction. Mass calibration was performed using insulin and matrix molecular ions at m/z 5734.59 and 379.93 Da, respectively. The MALDI matrix was prepared by dissolving 10 mg of R-cyano-4-hydroxycinnamic acid in 1 mL of acetonitrile/0.1% TFA/ethyl alcohol (30:30:30 v/v/ v). Typically, 1 µL of analyte was applied to metallic sample plate and 1 µL of matrix was then added. CD Spectra. CD spectra were recorded on a Jasco J-600 dichrograph. Cylindrical cells with optical path of 0.1 cm were used. Sample concentrations were 0.1 mg mL-1. The data are expressed in terms of [θ]M, the molar ellipticity (per pentameric unit), in units of deg cm2 dmol-1. Estimation of Secondary Structures from CD Spectra. The estimation of the relative content of secondary structures for poly(OrnGlyGlyOrnGly) was performed using a modified version by Alix and co-workers22 of the program CONTIN, by Provencher.23 Laser Scanning Microscopy (LSM). Poly(OrnGlyGlyOrnGly) was put on a glass for microscopy and analyzed under 488 and 514 nm laser beams of a confocal laser scanning microscope (ZEISS LSM 3) with the filter set LP 520. Environmental Scanning Electron Microscopy (ESEM). Linear and GTA-cross-linked poly(OrnGlyGlyOrnGly) were put on a stub with a biadhesive film of graphite to ensure sample adhesion and apparatus conductivity. The analysis was performed with the environmental scanning electron microscope PHILIPS ESEM XL 30, endowed with a

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lanthanum hexaboride (LaB6) filament, at different voltages and different pressures of the chamber. Transmission electron microscopy (TEM). The synthetic polypentapeptide poly(OrnGlyGlyOrnGly) was suspended either in “HPLC grade” water (method A) or in phosphate buffer, pH 7.00 (method B), to a final concentration of 20 mg/mL. Specimen preparation was afforded in the following way: a drop of sample suspension was deposited on a thin carbon film coated grid, and it was left for 4 h at room temperature in a humidified Petri dish. With regard to GTAcross-linked poly(OrnGlyGlyOrnGly), the specimen preparations were performed according to three different procedures: (i) A carbon-coated grid was put on a drop of sample suspension for 2-3 min. (ii) A drop of sample suspension was deposited on a thin carbon film coated grid, and it was left for 4 h at room temperature in a humidified Petri dish. (iii) A drop of sample suspension was deposited on a thin carbon film coated grid, and it was left for 10 h at room temperature in a humidified Petri dish. In every case the grids were rinsed with 20 drops of water and then stained with 10 drops of a 2% uranyl acetate solution, pH ) 3.5. Specimens were examined on a Zeiss EM 10 electron microscope at an accelerating voltage of 60 kV. Results and Discussion Poly(OrnGlyGlyOrnGly). CD Studies. In Figure 3 are reported the CD spectra of poly(OrnGlyGlyOrnGly), at different temperatures, at pH 7, and in TFE. With an increase in temperature from 25 to 70 °C, the curves recorded in aqueous solution display a significant decrease in the intensity of the shorter wavelength band, usually attributed to structureless polypeptide chains and a corresponding increase of the intensity of the shoulder around 220 nm. This result, indicative of an increase of intramolecular order, may be ascribed to an inverse temperature transition, such as that occurring in elastin and in many of its natural and synthetic derivatives.5 Taken together, the observed CD patterns could be tentatively attributed to an equilibrium mixture of different conformations, such as β-sheets, β-turns, unordered (undefined) conformations, and very low amounts of R-helix. The estimation of secondary structure from CD spectra, performed by applying the modified version of the program CONTIN,22,23 has shown that no significant change in the relative percentages are revealed by varying the temperature, resulting in almost constant percentages of β-sheets (40%), β-turns (20%), unordered (30%), and R-helix (10%). Obviously, this kind of calculation of secondary structure is unable to reveal subtle conformational changes in terms of standard ordered secondary structures. In addition, we recorded a spectrum in TFE in order to get further insight into the propensity of the polymer to adopt ordered structures. This is a well-established methodology, according to the literature.24 As expected, the polymer adopts a more ordered conformation, as denoted by the considerable reduction of the intensity of the negative band at shorter wavelengths, suggesting the presence of folded structures.2



Figure 3. CD spectra in phosphate buffer, at pH 7, of poly(OrnGlyGlyOrnGly) at 0 °C (- - -), 25 °C (4), 38 °C (0), and 70 °C (s). The spectrum of poly(OrnGlyGlyOrnGly) in TFE at 25 °C (O) is also reported. Sample concentrations are 0.1 mg/mL.

Figure 5. ESEM micrograph of linear poly(OrnGlyGlyOrnGly) demonstrating a twisted fibrous morphology. Bar represents 20 µm. Figure 4. LSM image of linear poly(OrnGlyGlyOrnGly). Notice the typical fibrous elastin-like organization. Bar represents 63.2 µm.

Microscopy Studies. Extensive studies of microscopy (LSM, ESEM, TEM) were performed on linear poly(OrnGlyGlyOrnGly). The emerging supramolecular organization is characterized by an ubiquitous fibrous motif, which strongly resembles that frequently occurring in native elastin and many elastin-derived peptides. Figure 4, obtained by laser scanning microscopy (LSM), shows a typical elastin-like fibrous organization, with fibers having diameters ranging between 19 and 4 µm. A fiber with a diameter of 16 µm is shown in the ESEM micrograph, Figure 5, confirming the data obtained by LSM. Of particular interest is the smallest one, having a diameter of 2.8 µm. This is a clear example of structural hierarchical organization where the greater diameter fiber is, in its turn, constituted by small fibrils, as is typical of elastin.4,25-29 A TEM image (Figure 6a), obtained by procedure A (see Methods section), shows fibrils which are interwound to give bundles exhibiting the twisted rope pattern, according to the characteristic supramolecular or-

ganization of elastin. The diameter size distribution of fibrils (mean, 4.7 nm; standard deviation, 2.4 nm; Figure 6b) comprises diameters from 1 to 12 nm, suggesting a complex lateral alignment of elemental filaments. The pattern does not depend on the pH of the initial solution, as confirmed by the absence of any significant morphological changes in the presence of phosphate buffer (method B). The scale invariant organization of the sample in fibers and fibrils, occurring at increasing resolution (i.e., going from LSM to ESEM and TEM), confirms the fractal hierarchy of supramolecular structures, in agreement with that found for elastin. GTA-Cross-Linked Poly(OrnGlyGlyOrnGly). Microscopy Studies. A variety of different forms have been revealed by ESEM studies performed on the cross-linked polymer. A new morphological pattern emerges from Figure 7. The fibers, in this case short and fragmented, strongly resemble the triangle- or spindle-shaped ones recently identified in some elastin-mimetic polypeptides.8 Of particular interest is the morphology shown in Figure 8, exhibiting a sort of fibrillar network, somewhat reminiscent

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Figure 8. ESEM micrograph of GTA-cross-linked poly(OrnGlyGlyOrnGly) showing a sort of extended network. Bar represents 20 µm.

Figure 6. (a) TEM image of linear poly(OrnGlyGlyOrnGly) suspended in water. Notice twisted fibrils interwound to give bundles. Bar represents 50 nm. (b) Diameter size distribution of linear poly(OrnGlyGlyOrnGly).

Figure 7. ESEM micrograph of GTA-cross-linked poly(OrnGlyGlyOrnGly). Notice the triangle- and spindle-shaped morphologies of fibers. Bar represents 20 µm.

of a membrane structure, that is very similar to those already displayed by R-elastin samples.12 This supramolecular organization, although not so frequent as the fibrous structures, appears to be an intrinsic feature of the cross-linked polymer because, when using the ESEM technique, we are dealing with essentially unperturbed samples, observed under the same experimental conditions. Figure 9a shows the supramo-

Figure 9. (a) TEM micrograph of GTA-cross-linked poly(OrnGlyGlyOrnGly) suspended in water: highly aligned fibrils, parallel oriented with respect to the axes of fibrils. Bar represents 150 nm. (b) Diameter size distribution of GTA-cross-linked poly(OrnGlyGlyOrnGly).

lecular aggregation of GTA-cross-linked poly(OrnGlyGlyOrnGly) as seen by TEM in unbuffered aqueous solution. In brief, many aspects of the structure are very similar to those of mature elastin.25-29 In particular, a filamentous suborganization of the fibrils (diameter distribution ranging



Conclusions

Figure 10. (a) TEM micrograph of GTA-cross-linked poly(OrnGlyGlyOrnGly) suspended in phosphate buffer, pH 7.0. Interconnected fibrils giving rise to a branched network. Bar represents 150 nm. (b) Diameter size distribution of GTA-cross-linked poly(OrnGlyGlyOrnGly), suspended in phosphate buffer, pH 7.0.

from 8 to 27 nm; mean, 17.1 nm; standard deviation, 3.9 nm; Figure 9b) can be seen. Furthermore, it is worthwhile to note that the framework reported is characterized by aligned, highly oriented fibrils, running parallel to the direction of the fibril axes. On the other hand, when the starting solution was buffered at pH 7.0, the supramolecular structure shown in Figure 10a was observed. Moreover, a deposition of random-oriented fibrils (diameter size distribution ranging from 10 to 26 nm; mean, 19.3 nm; standard deviation, 4.2 nm; Figure 10b) was observed, giving rise to a network very similar to that exhibited by another crosslinked elastin-like polymer, that is cross-linked poly(LysGlyGlyValGly).30 At present we have no reasonable explanation for the influence of the starting solution on the arrangement of the fibrils. However, it is to be stressed that in both cases the cross-linked polymer shows diameter size distributions shifted to values significantly higher than those found in the linear polymer.

The results obtained at molecular level on poly(OrnGlyGlyOrnGly) revealed the presence of different conformations, essentially β-sheets, β-turns, and unordered ones, and almost certainly in equilibrium. This finding is in agreement with current ideas about the conformation(s) of elastin and many of its derivatives. The source of entropic elasticity of elastin actually arises from a wide conformational flexibility, which characterizes the relaxed state. Furthermore, the observed inverse temperature transition, a process quite peculiar to tropoelastin, R-elastin, and elastin-based polypeptides,5 further corroborates the physical-chemical similarity between our polymers and the parent protein. At a supramolecular level, both the synthetic linear and cross-linked polymers adopt a variety of morphologies, some of them being very similar to those exhibited by elastin. In particular, the twisted-rope organization of filaments and fibrils, occurring at different scales, represents an example of structural hierarchy, ubiquitously found in elastin and in many elastinlike polypeptides. This finding clearly demonstrates the supramolecular fractality of the polymer, a feature considered crucial for the elastic function of elastin.4,25-29 Moreover, it should be stressed that fibrils shown by glutharaldehydecross-linked polymer are characterized by a clear tendency to assume a lateral alignment with preferred elongation direction, parallel to the fibril axes. On the contrary, a branched network appears when the cross-linked sample is observed in the buffered aqueous solution, at pH 7.0. Both morphologies seem to be features peculiar to the cross-linked polymer. In addition, other morphologies, recently found in some cross-linked elastin-mimetic polymers,9 were observed, giving further confirmation, also for poly(OrnGlyGlyOrnGly), of the complex pattern currently considered typical of elastin. On this basis, it appears that polypeptides comprising sequences of the type X-Gly-Gly-X-Gly (X ) Orn or Lys) are able to undergo efficient cross-linking by bifunctional reagents such as glutaraldehyde. Furthermore, the obtained cross-linked polymers mimic almost entirely the physical-chemical and supramolecular properties of elastin. They could display elastic properties similar to those of elastin. Furthermore, as the polypeptide sequences are essentially those of elastin, the polymers should also be biocompatible and stable toward the attack by proteases, such as elastases. Therefore, one can hope that elastin-based biomaterials could soon find wide applications in cardiovascular surgery as artificial substitutes for arteries and veins. Nomenclature Boc: t-butoxycarbonyl CD: circular dichroism DMSO: dimethyl sulfoxide DPPA: diphenylphosphoryl azide ESEM: environmental scanning electron microscopy Gly: glycine GTA: glutaraldehyde HPLC: high-pressure liquid chromatography IBCF: isobutyl chloroformate LSM: laser scanning microscopy

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Leu: leucine Lys: lysine NMM: N-methylmorpholine Orn: ornithine SDS-PAGE: sodium dodecyl sulfate polyacrilamide gel electrophoresis TEA: triethylamine TEM: transmission electron microscopy TFA: trifluoroacetic acid Val: valine Z: benzyloxycarbonyl

Acknowledgment. MURST and LAMI grants supported this work. Thanks are due to Dr. A. De Stradis (Centre for Microscopy, University of Basilicata) for making microscopy measurements. References and Notes (1) Debelle, L.; Tamburro, A. M. Int. J. Biochem. Cell Biol. 1999, 31, 261 and references therein. (2) Tamburro, A. M.; Guantieri, V.; Pandolfo, L.; Scopa, A. Biopolymers 1990, 29, 855. (3) Guantieri, V.; Grando, S.; Pandolfo, L.; Tamburro, A. M. Biopolymers 1990, 29, 845. (4) Castiglione Morelli, M. A.; De Biasi, M.; De Stradis, A.; Tamburro, A. M. J. Biomol. Struct. Dyn. 1993, 11, 181. (5) Urry, D. W. J. Protein Chem. 1988, 7 (1), 1 and references therein. (6) Urry, D. W.; Luan, C. H.; Harris, C. M.; Parker, T. M.; McGrath, K.; Kaplan, D. Protein- Based Materials; Birkhauser: Boston, MA, 1997. (7) McMillan, R. A.; Conticello, V. P. Macromolecules 2000, 33, 4809. (8) Huang, L.; McMillan, R. A.; Apkarian, R. P.; Pourdeyhimi, B.; Conticello, V. P.; Chaikof, E. L. Macromolecules 2000, 33, 2989. (9) Welsh, E. R.; Tirrell, D. Biomacromolecules 2000, 1, 23. (10) McMillan, R. A.; Caran, K. L.; Apkarian, R. P.; Conticello, V. P. Macromolecules 1999, 32, 9067.

Martino et al. (11) Martino, M.; Coviello, A.; Tamburro, A. M. Int. J. Biol. Macromol. 2000, 27, 59. (12) Tamburro, A. M.; De Stradis, A.; D’Alessio, L. J. Biomol. Struct. Dyn. 1995, 12, 1161. (13) Tamburro, A. M.; Daga Gordini, D.; Guantieri, V.; De Stradis, A. Properties and Chemistry of Biomolecular Systems; Kluwer Publishers: Dordrecht, 1994; p 389. (14) Nishi, N.; Hagiwara, K.; Tokura, S. J. Peptide Protein Res. 1987, 30, 275. (15) Sivanandaiah, K. M.; Suresh Babu, V. V.; Shankaramma, S. C. Int. J. Peptide Protein Res. 1994, 44, 24. (16) Chen, H.; Guo, B. Anal. Chem. 1997, 69, 4399. (17) Kassis, C. E.; Desimone, J. M.; Linton, R. W.; Remsen, E. E.; Lange, G. W.; Friedman, R. M. Rapid Commun. Mass Spectrom. 1997, 11 (10), 1134. (18) Cheung, D. T.; Nimni, M. E. Connect. Tissue Res. 1982, 10, 201. (19) Reichlin, M. Methods Enzymol. 1980, 70, 159. (20) Borch, R. F.; Bernstein, M. D.; Dupont Durst, H. J. Am. Chem. Soc. 1971, 93, 2897. (21) Spies, R. J. Methods Enzymol. 1957, 3, 468. (22) Alix, A. J. P. Personal communication. (23) Provencher, S. W. Comput. Phys. Commun. 1982, 27, 213-227, 229-242. (24) Reiersen, H.; Rees, A. R. Protein Eng. 2000, 13, 739. (25) Tamburro, A. M.; Guantieri, V.; Daga-Gordini, D. J. Biomol. Struct. Dyn. 1992, 10, 441. (26) Gotte, L.; Mammi, M.; Pezzin, G. Connect. Tissue Res. 1972, 1, 61. (27) Gotte, L.; Giro, M. G.; Volpin, D.; Horne, R. W. J. Ultrastruct. Res. 1974, 46, 23. (28) Bressan, G. M.; Pasquali-Ronchetti, I.; Fornieri, C.; Mattioli, F.; Castellani, I.; Volpin, D. J. Ultrastruct. Mol. Struct. Res. 1984, 94, 209. (29) Serafini-Fracassini, A.; Field, J. M.; Hinnie, J. J. Ultrastruct. Res. 1978, 65 (2), 190. (30) Martino, M.; Tamburro, A. M. Biopolymers 2001, 59, 29. *To whom correspondence may be addressed. E-mail: [email protected].

BM010129G

Chemical Synthesis and Structural Characterization of Linear and

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