Chapter 12
Downloaded by UNIV OF MICHIGAN ANN ARBOR on October 22, 2014 | http://pubs.acs.org Publication Date: July 18, 1991 | doi: 10.1021/bk-1991-0467.ch012
Peptide Graft Copolymers from Soluble Aminodeoxycellulose Acetate William H . Daly and Soo Lee Macromolecular Studies Group, Louisiana State University, Baton Rouge, L A 70803
The synthesis of 6-amino-6-deoxycellulose acetate, 1, by conversion of partially hydrolyzed cellulose triacetate to 6-azido-6deoxycellulose acetate followed by selective reduction of the azide with 1,3-propane- dithiol is described. Utilizing 1 as a macroinitiator for γ-benzyl-α,L-glutamate N-carboxyanhydride (BLG-NCA) leads to the formation of soluble peptide cellulose conjugates. We are engaged in the synthesis of a chitin composite analog based upon cellulose derivatives. Completely biodegradable graft copolymers of cellulose and polypeptides could serve as drug delivery systems or as calcium binding matices for prosthetic devices. As we reported earlier,(l) the utilization of insoluble 0-(3aminopropyl)cellulose as a macroinitiator did not produce soluble graft copolymers with predictable properties. The heterogeneous conditions limited the extent of grafting and prevented accurate control of the graft length. The resultant graft copolymers remained insoluble and thus were difficult to characterize. These difficulties reinforced the need for an easily characterizable soluble aminocellulosic, which could be employed as a macroinitiator of graft copolymerization to produce a completely biodegradable polymeric delivery system. In this paper we report the synthesis of soluble 6-amino-6-deoxycellulose acetate, 1, and its utilization as a macroinitiator for the polymerization of Y-benzyl-ct,L-glutamate N carboxyanhydride (BLG-NCA) to produce the desired cellulose polypeptide graft copolymers, 6. Replacement of one hydroxyl group with an amino function, i.e production of aminodeoxyanhydroglucose repeat units, has received considerable attention. The two most common approaches to introduce the aminodeoxy function have been either a reduction of azidodeoxycellulose, which can be prepared from halodeoxyor tosylated cellulose, or reduction of the oxime derived from selectively oxidized cellulosics.(2) Horton and Clode Q ) first reported the preparation of 6-azido-6deoxycellulose acetate with relatively low D.S.(0.25) by tosylating the residual hydroxyls of commercial cellulose acetate, followed by nucleophilic displacement with sodium azide. A regiospecific preparation of 6-azidodeoxycellulose prepared
0097-6156/91/0467-0189$06.00/0 © 1991 American Chemical Society
In Water-Soluble Polymers; Shalaby, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
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by a blocking-unblocking scheme involving tritylation at C-6, substitution of hydroxyl groups at C-2 and C-3 with acetate, detritylation, tosylation, and nucleophilic substitution of tosylhydroxyl group with sodium azide led to higher DS derivatives, but migration of the acetate groups to the 6-position during the deblocking step prevented absolute regiospecificity.(4) Direct chlorination of cellulose with methanesulfonyl chloride in Ν,Ν-dimethylformamide was quite stereospecific; subsequent displacement of the chloride afforded 6-azido-6deoxycellose with D.S.'s ranging up to 0.67.(5). Photolysis of the azidocelluloses followed by mild hydrolysis converted the derivatives into the corresponding 6aldehydocellulose. Horton did not convert either of these derivatives into amino celluloses but either derivative is a good precusor for such a conversion. Reduction of 6-azidodeoxycellulose with L i A l H ^ was reported by Usov et al.(6), and Teshirogi et al.(7-10). The azido derivatives were prepared by the same procedure as described above, but to mitigate the problems associated with the acetate migration, phenyl isocyanate was used to protect the 2,3-hydroxyls. Teshirogi reported that the phenylcarbamoyl groups were retained, but in fact we have found, as reported by Usov, that the reduction effects the cleavage of the carbamate linkage as well as the azide. Tritylation of cellulose followed by tosylation yielded 2(3)-0-tosyl-6-0-tritylcellulose which afforded the corresponding 2(3)-aminodeoxy cellulose upon tosyl displacement, reduction with L i A l H ^ , and detritvlation.(ll) 6-Amino-6-deoxycellulose was also prepared by the oxidation of 2,3diphenylcarbamoylcellulose to the corresponding 6-carboxaldehyde derivatives with dicyclohexylcarbodiimde-pyridine trifluoroacetate in dimethyl sulfoxide, oximation, and reduction the oxime with LiAlH^Q2). Oxidation of a homogeneous cellulose solution in DMSO-paraformaldehyde with acetic anhydride leads to 3-oxocellulose, in contrast, oxidation of a 6-substituted cellulose, such as 6-O-acetylcellulose, occurred mainly at C-2 under the same conditions.Ql) The ketocelluloses were utilized extensively to generate aminodeoxy derivatives; oximation followed by catalytic hydrogenation(14), diborane(14), sodium borohydrideQS) or LiALH^Qo) reduction led to the expected products. Both Usov's group(17) and Yalpani and Hall(18.19) have reported a process for 2(-3)-aminodeoxycellulose with various D.S.'s (0.45-0.55) by reaction of oxocellulose with ammonium acetate and sodium cyanoborohydride [NaB(CN)H^] in methanol. Synthesis of aminodeoxycellulose by the techniques described above led to either insoluble derivatives or derivatives which were soluble in polar solvents only. Further, the procedures tended to be accompanied by extensive chain degradation so the resultant derivatives would not be useful macroinitiators. We elected to use a more direct approach based upon acetate blocking groups, because the resultant aminodeoxycellulose derivatives must remain soluble in non-polar organic solvents to serve as macroinitiators for N C A grafting. 6-Amino-6deoxycellulose acetate,l, proved to the most interesting macroinitiator. Experimental A l l reagents and solvents used in the synthesis were of reagent grade or purified by standard techniques. Partial hydrolysis of cellulose triacetate,2, under acidic conditions afforded the starting cellulose derivative^, with a D.S. of acetylation = 2.45.(20) Nuclear magnetic resonance spectra were obtained with a Bruker W P 200 spectrometer at 298°K unless otherwise indicated. Infrared spectra were
In Water-Soluble Polymers; Shalaby, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
12. DALY & L E E
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Peptide Graft Copolymers
recorded with a Perkin-Elmer 283 spectrometer; films were cast of polymer samples from suitable solvents. Synthesis of 6-O-Tosvlcellulose Acetate.4. Into a solution of 25.0 g of partially hydrolysed cellulose acetate,3, in 400 mL of anhydrous pyridine were added 40.0 g of p-toluenesulfonyl chloride. The mixture was stirred at 25°C for 24 hr before diluting with 400 mL of acetone. The diluted solution was poured into 5 L of methanol, and the precipitate was recovered by filteration, washed and dried in vacuo at 50°C for 48 h to yield 27.3 g (82.7%) of a light brown derivative^. C Nmr (acetone-d ): 20.7, 20.8, 21.7, (acetate C H ) ; 63.3 (acetylated C-6); Downloaded by UNIV OF MICHIGAN ANN ARBOR on October 22, 2014 | http://pubs.acs.org Publication Date: July 18, 1991 | doi: 10.1021/bk-1991-0467.ch012
1 3
6
3
68.6 (tosylated C-6); 73.4, 76.0 (C-2, C-3, C-5); 77.1 (C-4); 101.2 (acetylyated C - l ) ; 103.8 ( C - l ) ; 128.9, 131.4, 134.0, 147.0 (aromatic C s ) ; 170.0, 170.1, 170.9 (acetate C=0). Synthesis of 6-Azido-6-deoxycellulose Acetate.5. Into a solution of 20.0 g of 4 (D.S. of tosylation = 0.45) in 400 mL of D M F were added 30.0 g of sodium azide. The mixture was stirred for 48 h at 100°C, and then precipitated into 4 L of distilled water. After two reprecipitations (acetone/water), 15.4g (92.6%) of light 13 brown 5 was obtained. C Nmr (acetone-d^): 20.6, (acetate CH^); 51.2 (azidodeoxy C-6); 63.2 (acetylated C-6); 72.8, 73.4 (C-2, C-3, C-5); 77.2 (C-4); 101.2 (acetylated C-l); 103.9 (glucose C-l); 169.7, 170.1, 170.9 (acetate C=0). IR (film from THF): 2130 cm" (N ). Synthesis of 6-Amino-6-deoxycellulose Acetate. 1. To 5.00 g of 5 in 150 mL of THF and 50 mL of methanol were added 4.1 mL of triethylamine and 7.5 mL of 1,3-propanedithiol. The mixture was stirred for 48 h at 60°C under nitrogen, and then precipitated in 500 mL of ethyl ether. The precipitate was filtered, washed with 2 L of hexane, air dried for 3 h at room temperature and then dried for 48 h at room temperature in vacuo. Partially reduced 6-azido-6-deoxycellulose acetate, 1, 4.12 g ( 0.47 meq/g of amino content) was obtained. This product is insoluble in acetone, T H F , and chloroform, but soluble in hot organic solvents, such as 1
3
pyridine, D M F , DMSO, and THF-ethyl alcohol (5:1 v/v).
1 3
C Nmr (pyridine-d , 5
353°K): 20.6, (acetate C H ) ; 41 (aminodeoxy C-6); 51.2 (azidodeoxy C-6); 63.2 3
(acetylated C-6); 73.1, 73.7 (C-2, C-3, C-5); 77.1 (C-4); 101.2 (acetylated C - l ) ; 104.4 (C-l); 169.6, 169.9, 170.5 (acetate C=0). Graft Copolymerization of B L G - N C A with Macroinitiator 1. The designated quantity of 1 was dissolved in hot dried D M F , the solution was cooled to 25°C, and a solution of B L G - N C A in D M F was added. The graft copolymerization remained homogeneous through out the reaction. After stirring for 48 h the copolymer solutions were poured into 100 mL of distilled water. White, fibrous products, 6a-6f, were obtained after freeze drying. The results are summarized in Table I. Results and Discussion In order to synthesize soluble aminodeoxycellulose derivatives, commercially available cellulose triacetate, 2, (D.S. = 2.8) was employed as a starting material. As shown in Scheme 1, initial partial hydrolysis of 2 under acidic conditions at room temperature afforded derivative 3 with a low concentration of free hydroxyl groups located primarily at the C-6 position. Derivative 3 (D.S. of acetylation = 2.45) was readily soluble in most organic solvents including acetone, THF, D M F , pyridine, and DMSO. Treatment of 3 with p-toluenesulfonyl chloride in pyridine
In Water-Soluble Polymers; Shalaby, S., et al.; ACS Symposium Series; American Chemical Society: Washington, DC, 1991.
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WATER-SOLUBLE POLYMERS
Table I. Graft Copolymers of y-Benzyl-ct,L-Glutamate with 6-Amino-6-deoxycellulose Acetate, 1
1
Cpd. (g)
ADCA (g)
6a
NCA (mol/mol)
NCA/ADCA Efficiency
Grafting
DI*
Conform
0.35C
0.15
3.7
38.5%
1.4
6b 6c 6d
0.25 0.15 0.04
0.25 0.35 0.46
8.2 20 89
64.7 92.4 95.0
5.3 19 88
Random & α-Helix α-Helix α-Helix α-Helix
6e 6f
0.35
