New polyurethanes derived from amino acids

Reactive & Functional Polymers 47 (2001) 11–21 www.elsevier.com / locate / react

New polyurethanes derived from amino acids Synthesis and characterization of a,v-diaminooligopeptides by ring-opening polymerization of glutamate N-carboxyanhydrides a ´ ´ Laurent Fontaine a , *, Laurent Menard , Jean-Claude Brosse a , Gerard Sennyey b , b Jean-Pierre Senet a

` , UMR CNRS UCO2 M, Universite´ du Maine, Avenue O. Messiaen, 72089 Le Mans Cedex 9, France LCOM- Chimie des Polymeres b ISOCHEM ( Groupe SNPE), Site du Bouchet, 9, rue Lavoisier, BP 18, 91710 Vert-le-Petit, France Received 17 March 2000; received in revised form 24 August 2000; accepted 5 September 2000

Abstract Strictly aminotelechelic oligopeptides with g-methylglutamate or g-benzylglutamate units were synthesized by oligomerization of the corresponding N-carboxyanhydride initiated by ethylenediamine. The degree of polymerization (DP) was controlled by the ratio of NCA to ethylenediamine concentrations for reaction times of less than 1 h and DP 5 20. The aminotelechelic nature, which greatly depends on the conditions used during oligomerization, was confirmed by amino end-group quantification. Characterization was achieved by 1 H and 13 C NMR and IR spectroscopy.  2001 Elsevier Science B.V. All rights reserved. Keywords: Ring-opening polymerization; N-carboxyanhydrides; Diamino oligopeptides; Amino acids; Polyurethanes; Biomedical applications

1. Introduction Polymeric materials have often been used for biomedical devices and their role in this area has been increasing for the past 2 decades [1]. Biocompatibility is one of the most important features they must fulfil for this kind of application. One method of improving the biocompatibility of polymers for biomedical purposes and restricting the toxicity of compounds released after degradation is to incorporate amino acid segments into their backbone [2]. For this reason oligopeptides and polypeptides with *Corresponding author.

reactive end groups have been known to be promising as polycondensation monomers for the synthesis of new biomedical polymers. Since their discovery by Leuchs at the beginning of the century [3], a-amino acid N-carboxyanhydrides (NCA) have attracted increasing interest as monomers for the synthesis of oligopeptides or polypeptides. Four mechanisms have been described which depend on the NCA and the initiator [4–9]: amine, carbamate, activated monomer and zwitterionic mechanisms. Aminotelechelic oligopeptides are mostly prepared via oligomerization of NCA initiated by a primary diamine which involves an anionic mechanism [4]. Because the primary amine function is more reactive than the growing chain

1381-5148 / 01 / $ – see front matter  2001 Elsevier Science B.V. All rights reserved. PII: S1381-5148( 00 )00057-2

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L. Fontaine et al. / Reactive & Functional Polymers 47 (2001) 11 – 21

extremity, initiation is faster than propagation and therefore the polymerization degree can be predicted by the ratio of the NCA to initiator concentration (at 100% conversion), particularly for a ratio below 100. The use of difunctional oligopeptides obtained by NCA oligomerization initiated by a diamine as polycondensation monomers has been reported in previous literature. Uchida et al. [10] reported the use of a g-methylglutamate oligopeptide as a polycondensation monomer with 4,49-diphenylmethanediisocyanate for the synthesis of new polyureas. Masar and Cefelin [11] carried out phenylalanine–NCA and leucine–NCA polymerizations initiated by a diamine in order to prepare polycondensates with diisocyanates. Segmented polyurethanes with g-benzylaspartate oligopeptide segment for water permeable membranes have been obtained by Yokohama et al. [12]. These oligopeptides have been synthesized by NCA oligomerization initiated by hexamethylenediamine. The aim of this study was the synthesis of aminotelechelic oligopeptides with gmethylglutamate or g-benzylglutamate segments with a controlled degree of polymerization, useful as potential monomers for the synthesis of new biomedical polymers. g-Benzylglutamate–NCA and g-methylglutamate–NCA were

selected as starting materials because the corresponding oligopeptides are soluble in many polar organic solvents [13,14], have good biocompatibility [2] and can be converted by the removal of benzyl or methyl ester groups.

2. Results and discussion

2.1. Synthesis of oligopeptides g-Methylglutamate and g-benzylglutamate oligopeptides were prepared by oligomerization of the corresponding N-carboxyanhydrides (NCA) initiated by ethylenediamine (ED) in dimethylformamide (DMF) as solvent (Scheme 1). DMF is a solvent for both the initial NCA and the oligopeptides. According to the anionic mechanism of this oligomerization, for 100% conversion, the degree of polymerization is given by the ratio of [NCA] to [ethylenediamine] ([NCA] / [ED]). Some authors [4,15,16] have noticed the possibility of modification of the oligopeptide extremities which depends on the reaction conditions and could alter the number of amino end-groups. This modification consists in a cyclization which provides a pyrrolidone ring by amino end-group nucleophilic attack onto the

Scheme 1. Oligomerization of N-carboxyanhydride

L. Fontaine et al. / Reactive & Functional Polymers 47 (2001) 11 – 21

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Scheme 2. Formation of pyrrolidone ring.

ester group of the last amino acid segment, releasing the corresponding alcohol (Scheme 2). By varying the time and the temperature used during the oligomerization reactions, it was possible to predict their effects on the modification of the end-groups (Tables 1 and 2). The results summarized in Tables 1 and 2 show that the presence of g-methylglutamate segments instead of g-benzylglutamate segments leads to an increase of the yields. This variation originates from the presence of benzyl

groups which enhance the solubility of the corresponding oligopeptides in low polarity solvents and therefore limit the oligopeptide precipitation. Since the precipitation of the oligopeptides also depends on their size, the reactions leading to a low degree of polymerization give low yields. It can also be noticed that the yield is neither altered by the temperature nor by the reaction time, but the characterization of the oligopeptides by NMR spectra and amino end-groups

Table 1 Oligomerization of g-methylglutamate–NCA Sample

M1 M2 M3 M4 M5 M6 a b

Oligomerization a

Amino end-group concentration [NH 2 ]310 4

DP

t (h)

T (8C)

Yield (%)

Calculated

HClO 4

DNFB

Ninhydrin

Theor.b

HClO 4

NMR

5 1 5 90 1 0.75

40 40 40 40 20 20

80 70 73 72 80 51

6.84 13.41 13.41 13.41 13.41 21.76

4.15 10.10 6.73 0 11.01 16.75

4.59 9.95 2.16 0 13.53 14.83

4.92 9.96 6.52 0 9.06 13.41

20 10 10 10 10 6

33 13.5 20.3 – 12.3 7.9

19.8 10.6 10.6 9.6 11.5 7.3

[NCA] 0 50.5 mol l 21 . Theoretical degree of polymerization: DPtheor. 5[NCA] 0 / [ED].

Table 2 Oligomerization of g-benzylglutamate–NCA Sample

B1 B2 B3 B4 B5 B6 B7

Oligomerization a

Amino end-group concentration [NH 2 ]310 4

DP

t (h)

T (8C)

Yield (%)

Calculated

HClO 4

DNFB

Theor.b

HClO 4

NMR

5 1 5 67 1 1 0.75

40 40 40 40 20 20 20

68 52 50 52 66 50 54

4.49 8.88 8.88 8.88 8.88 14.54 14.54

2.03 5.71 3.95 0 8.23 12.73 12.01

2.24 –c –c 0 6.03 8.10 9.84

20 10 10 10 10 6 6

44.7 15.7 22.8 – 10.5 6.3 7.5

24.2 10.4 10.8 10.1 10.8 6.9 7.3

[NCA] 0 50.5 mol l 21 . Theoretical degree of polymerization: DPtheor. 5[NCA] 0 / [ED]. c Not determined. a

b

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L. Fontaine et al. / Reactive & Functional Polymers 47 (2001) 11 – 21

quantification demonstrates the influence of these parameters on the oligopeptide end-groups structure.

2.2. Characterization of oligopeptides 2.2.1. Amino end-group quantification Determination of the amino end-group content was performed spectrophotometrically using ninhydrin reagent and 2,4-dinitrofluorobenzene (DNFB), and by potentiometric titration with perchloric acid in acetic acid solution. The determination of amino end-groups by the ninhydrin reagent is often used in the quantitative determination of proteins, amino acids or peptides. This method is based on the measurement of absorbance at 570 nm of the purple complex (Ruheman complex) formed by reaction of amino groups with ninhydrin reagent (Scheme 3) [17–19]. Amino end-group content of g-methylgluta-

mate oligopeptides were determined with a standard curve using g-methylglutamic acid as a model compound. This system obeys the Beer– Lambert law for concentrations below 4 ? 10 25 mol l 21 (Fig. 1). Because of the instability of the complex formed, the colorimetric assays of standard solutions and oligopeptides were performed under exactly the same conditions. Two other quantification methods were useful for both types of oligopeptides, i.e. containing g-methylglutamate or g-benzylglutamate segments. The modification of primary amino groups with DNFB followed by colorimetric determination (Scheme 4) was previously reported for the amino group determination in polyamides [20] and amino acid fragments in proteins [21,22]. The reaction of the oligopeptides with DNFB was conducted in two steps. In the first step a mixture of DNFB, oligopeptide and benzyl alcohol were mixed at 508C for 5 h. In the

Scheme 3. Reaction of amino end group with ninhydrin.

Fig. 1. Standard curves from g-methylglutamic acid determination using ninhydrin reagent.

L. Fontaine et al. / Reactive & Functional Polymers 47 (2001) 11 – 21

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Scheme 4. Rection of oligopeptides with DNFB.

second step, the oligopeptide was reacted with DNFB in sodium hydrogenocarbonate solution as an acid scavenger. The best reaction conditions were determined by varying reaction time with DNFB. The NMR spectra of the modified oligopeptides allowed the determination of the reaction time which provided the best yield (i.e. 90 h). The molar extinction coefficient of N-DNP glutamic acid (e 5 17971 cm 2 mol 21 ) was used in the calculations. Potentiometric titration by a perchloric acid– acetic acid solution proved to be a very convenient method to determine amino end-group concentration [23]. Standard deviation of the method (1.5%) was estimated by titration of pure 1,6-hexamethylenediamine. A mixture of acetic acid and benzyl alcohol (50:50) was required to dissolve oligopeptides with g-methylglutamate units while pure acetic acid was used for oligopeptides with gbenzylglutamate units. Determination of the primary amino endgroup concentration of the prepared oligopeptides was achieved using these methods which demonstrate their aminotelechelic character (see Tables 1 and 2). The different methods did not give exactly the same value for a given oligopeptide. In some cases, the ninhydrin and DNFB methods led to anomalous results which can be ascribed to the incomplete derivatization of oligopeptides by DNFB. Nevertheless, in most cases, con-

sistent results were obtained by the three different methods (i.e. M2 / M1, B1 / B7), but the modification step required by the DNFB method and the optimization of the ninhydrin method were time consuming. Therefore, potentiometric titration method seemed to be the best way in order to obtain accurate, fast and reproducible results. The results obtained by potentiometric titration compared to the calculated (theoretical) concentrations show the influence of the reaction conditions used for the synthesis of oligopeptides on the nature of the end-groups. Amine concentration decreases with reaction time (i.e. B2 / B3 / B4 and M2 / M3 / M4) and with reaction temperature (i.e. B3 / B5 and M3 / M5). No amino end-group could be detected by any of the methods under investigation in the case of oligopeptides M4 and B4 synthesized for 90 h and 67 h at 408C. The alteration of oligopeptides end-groups was influenced by the reaction time and temperature, which in turn caused a decrease in amino end-group concentration. This alteration is related to the cyclization produced by the nucleophilic attack of the terminal amino group on the ester group of the last amino acid segment, as described above, leading to a pyrrolidone ring (Scheme 2). This reaction was confirmed by 1 H NMR analysis (see below). In order to avoid this reaction, the synthesis has to be carried out at 208C for a duration which

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L. Fontaine et al. / Reactive & Functional Polymers 47 (2001) 11 – 21

depends on the target polymerization degree. Good results were obtained with samples B5, B6, M5 and M6 which exhibited an amine content close to the calculated (theoretical) one (see Tables 1 and 2). It was also observed that a continuous loss of amino end-group takes place during storage at

Fig. 2. Amino end-group content of g-benzylglutamate oligopeptides stored at 208C.

room temperature. An HPLC trace of B7 sample after 4 months of storage at 208C has shown the presence of benzyl alcohol formed by endgroups cyclisation. In addition, the loss of amino end-group was proved by comparing amino end-group concentration determined by potentiometric titration after 4 months of storage at room temperature (Fig. 2) with the initial values determined immediately after the synthesis. It is therefore necessary to store the prepared oligopeptides at low temperatures (,08C) in order to avoid this side-reaction.

2.2.2. Analysis of oligopeptides by NMR spectroscopy 13 C and 1 H NMR spectra recorded in trifluoroacetic acid (ATF) [24] are consistent with the oligopeptidic structure [25–27] and allowed the calculation of the polymerization degrees. A representative 1 H NMR spectrum of oligopeptide B5 is provided in Fig. 3. Assignments of hydrogen atoms belonging to the asymetric carbon of the amino acid units and belonging to the initiator were done by the analysis of a

Fig. 3. 1 H NMR (ATF solvent) spectrum of oligopeptide B5.

L. Fontaine et al. / Reactive & Functional Polymers 47 (2001) 11 – 21

model compound spectrum, obtained by the reaction of ethylenediamine and acetic anhydride (d CH 2 51.85 ppm and d CH53.05 ppm). The degree of polymerization was calculated by integration of peaks corresponding, respectively to the initiator and to the proton belonging to the asymetric carbon of the amino acid unit, which is not dependent upon the nature of the end-groups (Tables 1 and 2). The results obtained are consistent with the nucleophilic (or anionic-type) mechanism of oligomerization since the degree of polymerization determined by NMR was very close to the theoretical value. Unfortunately, this method could not provide information about the exact nature of the oligopeptide extremities except for oligomers M4 and B4 in which no amino endgroup could be detected. The corresponding NMR spectra showed a signal at d 4.60 ppm attributed to the hydrogen belonging to the asymetric carbon of the pyrrolidone ring. 13 C NMR spectra of M4 and B4 also exhibited three peaks located at 60, 30.5 and 26 ppm and corresponding to the pyrrolidone ring. Another peak with low intensity appeared at 185 ppm which was assigned to a carboxylic acid function caused by the partial hydrolysis of the g-methyl or g-benzyl ester functions. Comparison of the degrees of polymerization determined by NMR spectroscopy with those calculated from amino end-groups concentrations indicates the aminotelechelic nature of oligopeptides B5, B6, B7, M5 and M6 (Tables 1 and 2). The lack of amino end-group of samples M1, M2, M3, B1, B2 and B3 is revealed by the increase of the apparent degree of polymerization calculated from their amino function concentration.

2.2.3. Oligopeptide analysis by infrared spectroscopy The structures of the oligopeptides were also confirmed by FTIR spectra which exhibit the characteristic absorption bands located near 1650 cm 21 (n C=O), 1550 cm 21 (d N–H), 1220 cm 21 (n C–N) and 1740 cm 21 (n COOR).

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Oligopeptides may exist in two different conformations depending on the nature of the initiator used during the oligomerization reaction [4]. In the case of a primary diamine as initiator, a b conformation appears first which evolves to a helicoidal a conformation [28,29]. The evolution of each conformation can be followed by the analysis of the FTIR spectra which exhibit two amide bands for each conformation. a conformation shows the amide band at 1656 cm 21 and 1548 cm 21 and b conformation at 1630 cm 21 and 1536 cm 21 . In many cases, two shouldered bands were found, in which the shoulder represented the minor conformation and depended on the number of amino acid segments (Table 3). Conclusion Aminotelechelic oligopeptides with gmethylglutamate or g-benzylglutamate units were synthesized by the corresponding N-carboxyanhydride oligomerization initiated by ethylenediamine. According to the anionic mechanism of this reaction, the degree of polymerization is controlled by the ratio of NCA to ethylenediamine concentrations. The aminotelechelic nature was confirmed by amino end-group content determination and was found to be greatly influenced by the reaction conditions. The DP and the conformation of the oligopeptides were determined by NMR and IR spectroscopy, respectively. It has been demonstrated that aminotelechelic oligopeptides with DP56 or 10 can be obtained at 208C after 45 or 60 min. Their aminotelechTable 3 Conformation of oligopeptidic chains by FTIR (in solid state) Sample

DP (NMR)

Conformation a

M1 M2–5 M6 B1 B5 B6,7

19.8 9.5–11.6 7.3 24.2 10.8 6.9–7.3

a.b b b a.b a,b b

a

Major conformation.

L. Fontaine et al. / Reactive & Functional Polymers 47 (2001) 11 – 21

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elic properties, their controlled degree of polymerization and their solubility in many organic solvents allow their use as polycondensation monomers for the synthesis of new polymers derived from amino acids [30,31].

3. Experimental

3.3.2. General procedure of oligomerization N-Carboxyanhydride (0.1 mol), V ml (see 21 table below) of DMF ([NCA]50.5 mol l ) were placed into a 500-ml flask equipped with a condenser topped by a bubbler and a mechanical stirrer. V 9 ml of the initiator solution was added quickly by a syringe to the previous homogeneous solution. Immediately after the addition, CO 2 was evolved.

3.1. Methods 1

13

H and C NMR spectra were recorded on Bruker AC 400 (400 MHz) spectrometer in trifluoroacetic acid (ATF) solution excepted otherwise indicated. Tetramethylsilane was used as a reference for 1 H and 13 C NMR. Chemical shifts (d ) are given in ppm. Aromatic protons are denoted H arom . FTIR spectra were recorded on a Fourier transform Perkin-Elmer 1750 spectrometer. Potentiometric titration was performed by pH measurements with a Methrom pH meter in water or in acetic acid with combination electrodes 6.0203.000 and 6.0219.100. UV spectra were recorded with a Unicam UV–vis UV2 spectrometer. HPLC analysis were performed with Waters apparatus equipped with a 510 model pump, U6 K injector, C 18 Radial Pack column and double detection (UV and refractometer) using methanol as eluent.

DP

V9

V

20 10 06

5 10 16

195 190 184

The mixture was stirred for t min (see Tables 1 and 2) and then precipitated in ether (1000 ml) and copiously washed with ether. Oligopeptides were then dried at room temperature under reduced pressure (in the presence of P4 O 10 ) for 24 h.

3.4. Amino end-groups determination

Solvents were purified by the usual methods. Ethylenediamine and dimethylformamide (DMF) were distilled immediately before use. Other materials were commercial products used as received. N-Carboxyanhydrides were provided by Isochem-Snpe.

3.4.1. Potentiometric titration A 0.5-ml volume of a commercial perchloric acid solution (65%) was diluted in a 250-ml volumetric flask with glacial acetic acid. This solution was then titrated by a potassium hydrogenocarbonate solution obtained by mixing 0.408 g of salt with 100 ml of acetic acid ([HClO 4 ]50.02 mol l 21 ); 300 mg of gmethylglutamate (g-benzylglutamate) oligopeptide were dissolved in 10 ml of benzyl alcohol (10 ml of acetic acid and 20 ml of acetic acid). These solutions were then titrated against perchloric acid solution.

3.3. N-Carboxyanhydrides oligomerization

3.4.2. Determination using ninhydrin reagent

3.3.1. Initiator solution A 3-g amount of freshly distilled ethylenediamine was diluted up to 100 ml with dimethylformamide ([ethylenediamine]50.5 mol l 21 ).

3.4.2.1. Ninhydrin solution Sodium propionate (20.18 g), propionic acid (9.3 ml), methylcellosolve (50 ml) and ninhydrin (2 g) were mixed in a 100-ml volumetric flask and water was added to the mark.

3.2. Materials

L. Fontaine et al. / Reactive & Functional Polymers 47 (2001) 11 – 21

This solution was kept at low temperature in the dark.

3.4.2.2. Standard solutions The standard solutions (0.5310 24 ,C ,33 10 24 mol l 21 ) were obtained by dissolution of an initial solution prepared by dissolving 0.133 g of g-methylglutamic acid in 100 ml of water. A 0.9-ml volume of each standard solution was introduced into a 25-ml tube equipped with a screwtop and a magnetic stirrer; 0.1 ml of isopropanol, 1 ml of pyridine–water (10:90, v / v) solution and 2 ml of the ninhydrin solution were added. 3.4.2.3. Oligopeptide solutions To 1310 23 mol of oligopeptide in a 25-ml tube equipped with a screwtop and a magnetic stirrer, 1 ml of an isopropanol–water (10:90) solution, 1 ml of a pyridine–water solution (10:90) solution and 2 ml of the ninhydrin solution were added. 3.4.2.4. General procedure The tubes containing the previous solutions

1

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3.4.3. Determination using dinitrofluorobenzene 3.4.3.1. Modification reaction An 11-ml volume of benzyl alcohol, 0.83 24 10 mol of oligopeptide and 0.2 ml of dinitrofluorobenzene were placed into a 50-ml round-bottom flask. This mixture was stirred for 5 h at 508C. After cooling, 0.9 ml of sodium hydrogenocarbonate solution (5%) was added and then the mixture was stirred for 90 h at room temperature in the dark. The modified oligopeptide was then precipitated in 300 ml of ether, filtered and washed by 50 ml of water– ethanol (50:50) solution, 50 ml of water, 50 ml of water–ethanol (50:50) and 100 ml of ethyl acetate (g-methylglutamate) or 100 ml of ether (g-benzylglutamate). The oligopeptides were then extracted with ethyl acetate or diethyl ether in a Soxlhet for 70 h to remove residual DNFB. 3.4.3.2. General procedure The concentration of the chromophor group was determined from 1 mg of each oligopeptide in 10 ml of 1,1,1,3,3,3-hexafluoro-2-propanol by spectrophotometry (e : 17971 cm 2 mol 21 ) at 348 nm.

H NMR (C 2 HCl 3 ):

were immersed into an oil bath at 1008C for 30 min. The tubes were then removed from the bath and 20 ml of a solution of ethanol–water (50:50) were added. The mixtures were stirred at room temperature for 20 min and then poured into a 100-ml volumetric flask and water added to the mark. A blank run was run without oligopeptide. Absorbance was read at 570 nm.

* Oligopeptides with g-methylglutamate units: d H 1 : 3.40 (s); d H 2 : 4.58; d H 3 : 1.95–2.05; d H 4 : 2.50; d H 5 : 3.60 (s); d H 6 : 6.84 (s); d H 7 : 8.18 (d); d H 8 : 9.05 (s). *Oligopeptides with g-benzylglutamate units: d H 1 : 3.54 (s); d H 2 : 4.78; d H 3 : 2.10–2.33; d H 4 : 2.60; d H 5 : 5.24 (s); d H 6 : 6.90 (s); d H 7 : 8.25 (d); d H 8 : 9.15 (s); d H arom. : 7.40.

L. Fontaine et al. / Reactive & Functional Polymers 47 (2001) 11 – 21

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g-Methylglutamate oligopeptides:

3.4.4. NMR analysis of oligopeptides 3.4.4.1. Acetylation of ethylenediamine Ethylenediamine (0.017 mol) and 6 ml of chloroform were placed into a round-bottom flask equipped with a condenser, a dropping funnel and a magnetic stirrer. 0.039 mol of acetic anhydride in 10 ml of chloroform was then added dropwise. After stirring at room temperature for 2 h, the solvent was evaporated under reduced pressure. After recrystallization in chloroform, the product was dried under reduced pressure at 408C for 24 h. Yield: 40%, F 51708C. IR (in KBr disk): n (C=O, amide I): 1630 cm 21 ; d (N–H, amide II): 1531 cm 21 ; n (C–N, amide III): 1620 cm 21 . 1 H NMR (C 2 HCl 3 , d in ppm): 1.85 (s, CH 3 ); 3.05 (s, CH 2 ); 6,2 (br, NH). g-Benzylglutamate oligopeptides:

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C NMR (C 2 HCl 3 , d in ppm): 22.54 (CH 3 ); 38.16 (CH 2 ); 169.27 (CO).

3.4.4.2. g -Methylglutamate oligopeptides 1 H NMR (ATF, d in ppm): Oligopeptide M5: d H 1 : 3.65; d H 2 : 4.82; d H 29 : 4.63; d H 3,39 : 2.21– 2.38; d H 4,49 : 2.69–2.86; d H 5,59 : 3.90. Oligopeptide M4: d H 1 : 3.65; d H 2 : 4.80; d H 29? : 4.70; d H 3,39 : 2.20–2.37; d H 4,49 : 2.70– 2.90; d H 5 : 3.90. 13 C NMR (ATF, d in ppm): Oligopeptide M5: d C 1 : 40.4; d C 2 : 54.8; d C 29 : 55.4; d C 3 : 28.2; d C 39 : 27.2; d C 4,49 : 30.9; d C 5,59 : 171; d C 6,69 : 53.9; d C 7 : 178.2; d C 79 : 175 Oligopeptide M4: d C 1 : 41; d C 2 : 55; d C 29 : 59.6; d C 3 : 28.1; d C 39? : 25.8; d C 4 : 30.4; d C 49 : 30.4; d C 5 : 171; d C 59 : 176.3; d C 6 : 53.8; d C 7,79 : 178.2.

L. Fontaine et al. / Reactive & Functional Polymers 47 (2001) 11 – 21 1

H NMR (ATF, d in ppm): Oligopeptide B5: d H 1 : 3.56; d H 2 : 4.75; d H 29 : 4.50; d H 3,39 : 2.17– 2.32; d H 4,49 : 2.59–2.66; d H 5,59 : 5.24; d H arom. : 7.40. Oligopeptide B4: d H 1 : 3.55; d H 2 : 4.75; d H 29? : 4.60; d H 3,39 : 2.17–2.32; d H 4,49 : 2.60– 2.75; d H 5 : 5.30.; d H arom. : 7.40. 13 C NMR (ATF, d in ppm): Oligopeptide B5: d C 1 : 41; d C 2 : 56; d C 29 : 57; d C 3 : 28.1; d C 39 : 26.1; d C 4,49 : 31.3; d C 5,59 : 175; d C 6,69 : 69.9; d C 7,79 : 137.4; d C 8,89 : 129.8; d C 9,99 : 129.4; d C 10,109 : 129.9; d C 11 : 177.2; d C 119 : 172. Oligopeptide B4: d C 1 : 41; d C 2 : 56; d C 29? : 60; d C 3 : 28.1; d C 39? : 26.1; d C 4 : 31.3; d C 49 : 30; d C 5 : 175; d C 59 : 176; d C 6 : 69.9; d C 7 : 137.4; d C 8 : 129.8; d C 9 : 129.4; d C 10 : 129.9; d C 11,119 : 177.2.

3.4.4.3. FTIR analysis The assignments of oligopeptides M5 and B5 are given: Oligopeptide M5: n (N–H): 3297 cm 21 ; n (C=O ester): 1739 cm 21 ; n (C=O amide I): 1634 cm 21 ; d (N–H amide II): 1531 cm 21 ; n (C–N amide III): 1260 cm 21 . Oligopeptide B5: n (N–H): 3290 cm 21 ; n (C–H arom.): 3065 cm 21 ; n (C=O ester): 1734 cm 21 ; n (C=O amide I): 1628–1652 cm 21 ; d (N–H amide III): 1520–1525 cm 21 ; d (C=C): 1500, 1455 cm 21 ; n (C–N amide III): 1260 cm 21 ; n (C–O ester): 1166 cm 21 ; d (C–H arom.): 739 cm 21 . References [1] J.B. Park, Biomaterials Science and Engineering, Plenum Press, New York, 1984.

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