Polymers from Agricultural Coproducts - American Chemical Society

Ç H 2. Ο. BOC-NHCHÇJNHCHCNH(CH2 )6 NHCCHNHCÇ:HCONH-BOC. Ο. Ο. Ο. Ç H 2. BOC-Tyr-Leu-H-Leu-Tyr-BOC. 4 Ο. C (CH2 )4 C-. 1. NaOH / H 2 0. 2...
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Synthesis of Functionalized Targeted Polyamides P. M . Mungara and Κ. E. Gonsalves Polymer Science Program, Institute of Materials Science, U-136 & Department of Chemistry, University of Connecticut, Storrs, CT 06269

A synthetic approach to polyamides containing the tyrosine-leucine linkage is presented. The diphenyl phosphoryl azide, (DPPA), coupling technique was utilized to synthesize the monomers. Solution polymerization of monomer I, tyrosylleucyliminohexamethylene­ -iminoleucyltyrosine, with adipoyl and sebacoyl chlorides gave polymers with intrinsic viscosities of 0.18 dL/g and 0.13 dL/g respectively in 90% formic acid. Interfacial polymerization of the monomer yielded a fibrous crosslinked material which was found to be a poly(amide-ester) by IR spectroscopic analysis. In another reaction, monomer II, β-alanyltyrosylleucyl-β-alanine, was polymerized using DPPA and triethylamine to give a polyamide with an intrinsic viscosity of 0.07 dL/g in 90% formic acid. A synthetic route to monodispersed alanine peptides is also presented. Polyamides containing the naturally occurring α-L-amino acid linkages belong to a class of potentially biodegradable polymers whose applications are numerous, especially in agriculture and the biomedical field (7). In the latter, polypeptides have had considerable success as controlled drug delivery systems, degradable sutures and artificial skin substitute^-3). In this paper, the synthetic approach to polyamides containing oc-L-amino acids is presented. The first part deals with the synthetic method utilizing diphenyl phosphoryl azide, (DPPA), to incorporate the dipeptide, tyrosine-leucine(Tyr-Leu) in polyamides. The tyrosine-leucine bond is targeted for degradation via enzymes such as chymotrypsin, thermolysin, subtilisin and aspergillopeptidase A. In the second part, a rapid method for making monodispersed polypeptide is presented. Here, Fmoc-Ala-Cl and Benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate, (BOP), coupling techniques were combined to synthesize alanine peptides of upto six units, which are targeted for use in controlled drug delivery systems.

0097-6156/94/0575-0160$08.00/0 © 1994 American Chemical Society

11. MUNGARA AND GONSALVES

Functionalized Targeted Polyamides 161

Experimental Materials. The amino acid derivatives and the Castro's reagent, benzotriazolyloxytris(dimethylamino)phosphonium hexafluorophosphate, (BOP), were obtained from Advanced ChemTech Inc., Louisville, K Y , and used without further purification. DPPA, obtained from Aldrich Chemical Company, was purified by distilling under reduced pressure. Adipoyl and sebacoyl chlorides were distilled under reduced pressure while hexamethylenediamine was purified by vacuum sublimation. Triethylamine was dried over CaH2 and distilled at atmospheric pressure. Anhydrous DMF was obtained by drying over BaO and distilling under reduced pressure. Measurements. The intrinsic viscosities of the polymers were measured in a 0.5 g/dL solution of formic acid at 25 °C. The infrared spectra of the compounds ( KBr pellet, or film cast from CHCI3) were recorded on a Nicolet 60SX FTIR spectrometer, while the *H NMR spectra were obtained on an IBM AF-270 NMR spectrometer (270 MHz), with CF3COOD, CDCI3 or DMSO-d6 as the solvents . The molecular weights of the monomers and the alanine peptides were determined using fast atom bombardment (FAB) mass spectrometry on a Kratos MS50RF high resolution magnetic sector mass spectrometer. Results and Discussion Tyr-Leu Polymers. Scheme I gives the general outline of synthesizing adipoyl or sebacoyl polymers containing tyrosylleucyUminohexamethyleneiminoleucyltyrosine (Tyr-Leu-H-Leu-Tyr) unit. Tert-butyloxycarbonyl-L-tyrosine (BOC-TyrOH) was coupled with L-leucine methyl ester.HCl (H-Leu-OMe.HCl) using DPPA and triethylamine in DMF (4-7). The methyl ester protecting group was deblocked using aqueous NaOH. The incorporation of hexamethylenediamine unit was achieved by reacting 1 equivalent of the amino protected dipeptide, BOC-Tyr-LeuOH with 0.5 equivalent of the diamine using DPPA. The resulting peptide, BOCTyr-Leu-NH(CH2)6NH-Leu-Tyr-BOC was reacted with anhydrous trifluoroacetic acid (TFA) at room temperature to give monomer I. The 1H NMR of the monomer in deuterated trifluoroacetic acid (CF3COOD) showed peaks at δ (ppm); 0.81 (m, 12H, (CH3)2, Leu), 1.35-1.58 (m, 14H, CHCH2, Leu, CH2, diamine), 3.1-3.2 (m, 8H, CH2NH, diamine, CH2, Tyr), 4.5-4.6 (t, 4 H , CH-N, Tyr, Leu) and 6.8-7.0 (d, 8H, aromatic H, Tyr). The IR spectrum (KBr pellet) showed a broad peak between 3500-3200 cm"! (O-H stretching ) peaks at 3298 cm" (N-H stretching), 3080 cm" (C-H stretching, aromatic), 2930 and 2858 cm (C-H stretching aliphatic), 1652 cm" (amide I) and 1521 cm' (amide Π). Solution polymerization was achieved by reacting a chloroform solution of monomer I and triethylamine with the chloroform solutions of either adipoyl or sebacoyl chlorides to give the corresponding polymers (8). The adipoyl polymer (PTLA, 0.52 g, yield, 52.2% ) had an intrinsic viscosity, [η], of 0.18 dL/g while that of sebacoyl (PTLS, 0.25 g yield, 37%) had an intrinsic viscosity of 0.13 dL/g in 90% formic acid. The *H NMR of the adipoyl polymer in CF3COOD had peaks at 5(ppm); 0.83 (d, 12H, (CH3)2, Leu), 1.3-1.5 (m, 18H, CHCH2, Leu, CH2, diamine, adipoyl), 2.3 (m, 4H, CH2CO, adipoyl), 2.96-3.30 (m, 8H, CH2 , Tyr, CH2N, diamine), 4.6-4.8 (m, 4H, CH-N, Tyr, Leu), and 6.8-7.0 (d, 8H, aromatic, Tyr). The IR spectrum (KBr pellet) showed peaks between 3500-3200 cm" (O-H, stretching), 3317 cm" (N-H, 1

-1

1

1

1

1

1

POLYMERS FROM AGRICULTURAL COPRODUCTS

Scheme I. Synthesis of Tyr-Leu-NH(CH ) NH-Leu-Tyr polymers 2

BOC-Tyr-OH

+

6

H-Leu-OMe.HCl DPPA / Et N DMF 3

BOC-Tyr-Leu-OMe NaOH/H 0 2

BOC-Tyr-Leu-OH 0.5 eq. H N(CH ) NH DPPA/Et N DMF 2

2

6

2

3

BOC-Tyr-Leu-NH(CH ) NH-Leu-Tyr-BOC 2 6

TFA TFA.H-Tyr-Leu-NH(CH ) NH-Leu-Tyr-H.TFA(Monomer I) 2 6

CHCl /Et N Adipoyl or Sebacoyl Chloride 3

3

Tyr-Leu-NH(CH ) NH-Leu-Tyr-NHCO(CH ) CO -j-jj 2

6

2

n

η = 4, adipoyl, PTLA; η = 8, sebacoyl, PTLS

11. MUNGARA AND GONSALVES

Functionalized Targeted Polyamides 163

1

1

stretching), 3084 cm" (C-H stretching, aromatic), 2933 and 2853 cm" (C-H stretching, aliphatic), 1653 c m (amide I) and 1518 cm" (amide II). Similar results were obtained for the sebacoyl polymer. The interfacial polymerization of monomer I was carried out by reacting the sodium hydroxide solution of the monomer with chloroform solutions of adipoyl or sebacoyl chloride (9). A crosslinked poly(amide-ester) was obtained in each case. This could be attributed to the reaction of both the amino and the hydroxyl groups in the tyrosine unit with the acid chloride in alkaline medium. In order to confirai the reaction due to the hydroxyl group in the tyrosine unit, interfacial polymerization of an alkaline solution of amino protected monomer I and a chloroform solution of adipoyl chloride was carried out, (Scheme II). The IR spectrum of the polyester obtained was then compared with that of crosslinked poly (amide-ester). The two spectra had peaks at 1765 cm~l (ester), 1656 cm'l (amide I) and 1517 c m (amide II) (10). The amide peaks in the polyester are due to the peptide bond originally present in the monomer. The IR spectrum of the polymer made by solution polymerization of monomer I in triethylamine does not show the ester peak at 1765 cm" (Figure 1). The results confirm that the hydroxyl group in tyrosine can react readily in an alkaline medium. The synthesis of poly^-alanyltyrosylleucyl^-alanine), PATLA, is outlined in Scheme III. The amino acids, tert-butyloxycarbonyl^-alanine (BOC^-Ala-OH) and L-tyrosine methyl ester.HCl (H-Tyr-OMe.HCl) were coupled using the DPPA method as explained above. The dipeptide BOC^-Ala-Tyr-OMe was obtained. The methyl ester protecting group was deblocked using aqueous NaOH to give the amino protected dipeptide, BOC-P-Ala-Tyr-OH. In another reaction, tertbutyloxycarbonyl-L-leucine (BOC-Leu-OH) and β-alanine methyl ester.HCl, (βAla-OMe.HCl) were coupled using DPPA to give the dipeptide BOC-Leu-P-AlaOMe. The BOC protecting group was deblocked using TFA to give H-Leu^-AlaOMe which was utilized in segment condensation with BOC-p-Ala-Tyr-OH. The segment condensation using DPPA and triethylamine in DMF afforded the tetrapeptide, BOG^-Ala-Tyr-Leu^-Ala-OMe. The NMR of the tetrapeptide in CF3COOD showed peaks at δ (ppm); 0.81 (d, 6H, (CH3)2, Leu), 1.2-1.5 (m, 3H, CH2CH, Leu), 2.7 (t, 2H , CH2CO, β-Ala ), 2.98 (d , 2H, CH2, Tyr), 3.6 (t, 2H, CH2-N, β-Ala), 4.51-4.70 (t, 2H, CH-N, Tyr, Leu), and 6.8-7.0 (d, 4H, aromatic H, Tyr). Hydrolysis of the methyl ester protecting group of the tetrapeptide using aqueous NaOH followed by the deblocking of the BOC group with TFA afforded the TFA salt of monomer II, β-Α&-ΤνΓ-ίευ-β-Α&. Poly (β-Ala-Tyr-Leu-β-Ala) was obtained by dissolving monomer II in DMF and reacting with DPPA in the presence of triethylamine for 2 days (11). The polymer (PATLA, 0.50 g, yield, 55.4% ) had an intrinsic viscosity, [ η], of 0.07 dL/g in 90% formic acid. The K NMR of the polymer in CF3COOD gave peaks at δ(ρριτι); 0.81 (6H, (CH3)2, Leu), 1.5 (3H, CH2-CH, Leu), 2.7 (4H, CH2-CO, β-Ala), 2.97 (2H, CH2, Tyr), 3.56 (4H CH2-N, β-Ala), 4.50-4.75 (2H, CH-N, Leu, Tyr) and 6.8-7.0 (4H, aromatic H, Tyr). The IR spectrum (KBr pellet), showed peaks between 3500-3200 c m ' (O-H stretching), 3298 cm" (N-H stretching), 3082 cm" (C-H stretching, aromatic) , 2955 and 2858 cm" (C-H stretching, aliphatic), 1670 cm" (amide I) and 1516 cm" (amide II). PATLA-II is the polymer made by repolymerizing -1

1

-1

1

l

1

1

1

1

1

1

POLYMERS FROM AGRICULTURAL COPRODUCTS

164

Scheme Π. Synthesis of peptide polyester by interfacial method OH

Œ(CH ) 3

CH(CH )

2

3

2

Ç2 ÇH ÇH Ο BOC-NHCHÇJNHCHCNH(CH ) NHCCHNHCÇ:HCONH-BOC H

2

2

2

Ο

6

Ο

Ο

ÇH

2

BOC-Tyr-Leu-H-Leu-Tyr-BOC

1. NaOH / H 0 2. Adipoyl chloride/CHCl 2

4

3

Ο C (CH ) C2

4

ÇH(CH ) 3

ÇH

Œ(CH ) 3

2

CH

2

ι

2

1

2

Ο

II

B O C - N H C H C N H C H C N H ( C H ) N H C C H N H C ÇHCONH-BOC 2

0

0

peptide polyester (PTLE)

6

Ο

ÇH

2

3000

2000

Wavenumbers (cm— 1 )

2500

1500

1000

Figure 1. Infrared spectra of polymers: (a) poly(amide-ester), cross-linked, (b) peptide polyester, PTLE, (c) polyamide, PTLA.

3500

POLYMERS FROM AGRICULTURAL COPRODUCTS

166

Scheme ΙΠ. Synthesis of P-Ala-Tyr-Leu^-Ala polymer BOC-p-Ala-OH + H-Tyr-OMe.HCl DPPA/Et N DMF 3

BOC- Leu-OH + Η-β-Ala-OMe.HCl DPPA/Et N DMF 3

BOC-Leu-p-Ala-OMe

BOC-p-Ala-Tyr-OMe NaOH/H 0

TFA

2

BOC-p-Ala-Tyr-OH

TFA.H-Leu^-Ala-OMe DPPA/Et N DMF 3

BOC-p-Ala-Tyr-Leu-p-Ala-OMe 1. NaOH/H 0 2. TFA 2

TFA .Η-β- Ala-Tyr-Leu-p-Ala-OH (Monomer Π) DPPA/Et N DMF, 2 days 3

—|- p-Ala-Tyr-Leu-p-Alaj^-

(PATL)

Table I. Yields, T and GPC Data of the Tyr-Leu Polymers /Oligomers m

Sample Yield, % M M M /M T (°C) w

n

w

m

a

n

PTLA 52.2 14200 7200 1.97 230

not determined

PTLS 37 20800 8100 2.57 160

PTLE 44.4 3500 2500 1.40 131

PATLA 55.4 5300 2900 1.83 a_

PATLA-II 55.3 6500 5700 2.89 138

11. MUNGARA AND GONSALVES

167 Functionalized Targeted Polyamides

PATLA under similar condition as before. As shown in Table I, there was some improvement in the molecular weight although the polydispersity also increased. The result from Table I indicate medium to low yields of the polymers. This could be attributed mainly to the insolubility of the polymers in the solvent medium used in the case of solution polymerization or insufficient stirring speed in the interfacial method. Besides, the bulky nature of the monomers cannot be discounted as contributing to steric hindrance to the reactions resulting into low yields and low molecular weights. Alanine Peptides. The synthesis scheme utilizing Fmoc-Ala-Cl is given in Scheme IV and is essentially that of Carpino et al. (72). Fmoc-Ala-Cl was synthesized by refluxing Fmoc-Ala-OH (6.0 g) with SOC12 (12 mL) using CH2CI2 (30mL) as the solvent. The amino acid chloride was purified by washing several times in CH2CI2 and evaporating off the solvent under reduced pressure to ensure complete removal of the SOC12. Recrystallization of the product from hexane-CH2Cl2 gave FmocAla-Cl (5.90 g, yield, 92.8% , m.p. 112- 114 ο C) which was used in acylation without further purification. A methylene chloride solution of Fmoc-Ala-Cl was interfacially coupled with H-Ala-OtBu.HCl in 5% Na2C03. The reaction mixture was easily purified to afford the dipeptide, Fmoc-Ala-Ala-OtBu (4.0 g, 91.3 % m.p. 161-162 op. The *H NMR of the compound in CDCI3 showed peaks at δ (ppm); 1.2 (d, 6H, CH-CH3), 1.4 (s, 9H, C(CH3)3), 4.2-4.4 (m, 5H, CH-CH3 and CH-CH2O), 5.3 (d, 1H, NH), 6.3 ( d, 1H, NH), and 7.3-7.8 (m, 8H, aryl). IR spectrum (film, solvent; CHCI3) gave peaks at 3286 cm" (N-H stretching ), 3065 cm-1 (aryl C-H stretching), 2963 cm"l (aliphatic C-H stretching), 1739 cirri (c=0, CH2-0-C=0), and 1670 cm"l (C=0, amide). The Fmoc protecting group of the dipeptide was deblocked using Et2NH in acetonitrile and the resulting carboxyl protected dipeptide was acylated with FmocAla-Cl to give the tripeptide, Fmoc-Ala-Ala-Ala-OtBu (2.0 g yield 78.4% m.p. 189190 O Q . NMR, δ (pmm), CDCI3; 1.2 (d, 9H, CH-CH3), 1.5 (s, 9H, C(CH3)3 ), 4.2-4.4 (m, 6H, N-CH-CO and CH-CH2-O), 5.41 (broad s, 1H, NH), 6.6 (broad s, 2H, NH), 7.3-7.8 (m, aryl). IR (film, solvent; CDCI3, cm"l) ; 3286 (N-H stretching), 3050 (aryl C-H stretching) 2946 (aliphatic C-H stretching), 1722 (CO stretching, CH2-OC=0 ), 1020 (C=0 stretching, amide). The peptides obtained by the rapid Fmoc-Ala-Cl acylation technique were utilized in a segment condensation reaction using BOP coupling reagent and diisopropylethylamine, DIEA (13,14). Scheme V gives the outline of the synthetic process leading to the tetrapeptide, Fmoc-Ala-Ala-Ala-Ala-OtBu. Here the Fmoc group was deblocked using Et2NH to give the dipeptide, H-Ala-Ala-OtBu. On the other hand, TFA was used to deblock the tertiary butyl group to give Fmoc-AlaAla-OH. The two dipeptides were then coupled at room temperature using BOP and DIEA in DMF to afford the tetrapeptide, Fmoc-Ala-Ala-Ala-Ala-OtBu (1.3 g, yield, 89.6%, decompose at 190 °C). The *H NMR of this peptide showed peaks at δ (ppm), (DMSO-d6); 1.2 (d, 12H, CH-CH3), 1.4 (s, 9H, C(CH3)3), 4.1-4.3 (m, 7H, N-CH-C=0 and CH-CH2O), 7.3-8.3 (m, 12H, N-H and aryl ). IR (KBr pellet, cm" ); 3295 (N-H stretching), 3050 (aryl C-H stretching), 2963 and 2912 (aliphatic C-H stretching), 1722 (s, C=0 stretching, CH2-0-C=0), 1620 (C=0, amide). Similar coupling technique as 1

1

POLYMERS FROM AGRICULTURAL COPRODUCTS

168

Scheme IV. Fmoc-Ala-Cl acylation technique Fmoc-Ala-OH

+

SOCl CH C1 2

2

2

Fmoc-Ala-Cl 5%Na C0 /CH Cl H-Ala-OtBu.HCl 2

w

3

2

2

Fmoc-Ala-Ala-OtBu Et NH / CH CN 2

3

H-Ala-Ala-OtBu Fmoc-Ala-Cl 5%Na C0 /CH Cl 2

3

2

2

Fmoc-Ala-Ala-Ala-OtBu Scheme V. BOP 2 + 2 segment coupling * Fmoc-Ala-Ala-OtBu

Et NH/CH CN 2

3

H-Ala-Ala-OtBu

TFA/CH C1 2

2

Fmoc-Ala-Ala-OH

BOP / DIEA / DMF

Fmoc-Ala-Ala-Ala-Ala-OtBu *The same procedure was followed to make hexa-alanine

11. MUNGARA AND GONSALVES

169 Functionalized Targeted Polyamides

described for the tetrapeptide was used to synthesize Fmoc-Ala6-OtBu, (0.87 g yield, 84.3 %, m.p. 236 °C dec), from trialanine. NMR, δ (ppm), DMSO-d6; 1.2 (d, 18H, CH-CH3), 1.4 (s, 9H, C(CH3)3). A3 (m, 9H, N-CH-CO and CH-CH2-O), 7.3-8.3 (m, 14H, N-H and aryl). IR (KBr pellet, cm- ); 3330 (N-H stretching ), 3050 (aryl C-H stretching) 2967 and 2933 (aliphatic C-H stretching), 1733 (C=0 stretching, CH2-0-C=0 ), 1633 (s, C=0 amide). Table II gives a summary of the yields and the melting points of the alanine peptides synthesized by both Fmoc-Ala-Cl acylation and BOP coupling technique. 1

Table II. Yields and M.p. of the Alanine Peptides Sample

Yield(%)

m.p. (OQ

Fmoc-dialanine-OtBu Fmoc-trial anine-OtBu Fmoc-tetra-alanine-OtBu Fmoc-hexa-alanine-OtBu

91.3 78.4 89.6 84.3

161-162 189-190 190(dec.) 236 (dec.)

Conclusions These studies indicate that the DPPA technique has been successful in synthesizing peptides containing the tyrosine-leucine linkage, a potential target for enzymatic degradation. These peptides can be utilized in both solution and interfacial, polymerization (Schemes I & II). Direct polymerization is also possible using DPPA (Scheme III). From Scheme II, it has been demonstrated that a functionalized polymer can be synthesized e.g., polyamides containing pendant hydroxyl groups or conversely polyesters containing pendant amino groups. Modifications to obtain higher molecular weight polymers are underway. The Fmoc-Ala-Cl and BOP coupling techniques are convenient for synthesizing the alanine peptides at room temperature using very mild conditions. The yields obtained are also high. Due to the encouranging results, we intend to synthesize longer alanine peptides which can serve as model compounds in the study of peptide structures. Literature Cited 1. Kumar, G. S. Biodegradable Polymers, Prospect and Progress; Marcel Dekker, Inc. : New York, 1987. 2. Anderson, J. M.; Gibbons, D. F.; Martin, R. L.; Hiltner, Α.; Woods, R. J. Biomed. Mater. Res. Symp. 1974, 5(1), 197. 3. Hayashi, T.; Iwatsuki, M. Biopolymers 1990, 29, 549. 4. Gonsalves, K. E.; Mungara, P. M. Chem. Mater. 1993, 5, 1242. 5. Shioiri, T.; Ninomiya, K.; Yamada, S.J.Amer. Chem. Soc. 1972, 94, 6203. 6. Yamada, S.; Ikota, N.; Shioiri, T.J.Amer. Chem. Soc. 1975, 97, 7175. 7. Gonsalves, Κ. E.; Mungara, P. M. Polym. Commun. 1994, 35(3), 663. 8. Morgan, P. W.; Kwolek, S. L.J.Polymer Sci. 1964, A2, 185 . 9. Gonsalves, Κ. E.; Chen, X.; Wong, T. K.J.Mater. Chem. 1991,1(4),643.

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10. Gonsalves, Κ. E.; Chen, X.; Cameron, J. A. Macromolecules 1992, 25, 3309. 11. Nishi, N.; Tsunemi, M.; Hayasaka, H.; Nakajima, B.; Tokura, S. Makromol. Chem. 1991, 192 , 1789. 12. Carpino, L.A.; Aalaee, D. S.; Beyermann, M.; Bienert, M.; Niedrich, H. J. Org. Chem. 1990, 55, 721. 13. Ten Kortenaar, P. B. W.; Kruse, J.; Hemminga, Μ. Α.; Tesser, G. I. Int. J. Peptide Protein Res. 1986, 27, 401. 14. Fehrentz, J. Α.; Seyer, R.; Heitz, Α.; Fulcrand, P.; Castro B. ; Corvol, P. Int. J. Peptide Protein Res. 1986, 28, 620. RECEIVED May 24, 1994