VOL. 14, 73-81 (1975)
BIOPOLYMERS
Synthesis and Conformational Properties of Polypeptides Containing Long Aliphatic Side Chains. Poly (N'-Stearyl-L-Lysine) and Poly(N'-Pelargonyl-L-Lysine) VALERY SHIBAEV,* A'IANLIO PALUMBO, and EVARISTO PEGGIOW, Institute of Organic Chemistry, University of Padova, 35100 Padua, Italy
Synopsis Poly(N'-stearyl-Llysine) and poly(Nf-pelargonyl-clysine) were synthesized both by polymerization of N'-pelargonyl and N'-stearyl-clysine NCA and by acylation of poly(L1ysine) with pelargonyl and stearyl chloride. This second route has proven to be very useful, since completely acylated polymers are obtained in almost quantitative yield, whereas the usual scheme of preparation of c protected poly(c1ysine) cannot easily be applied due to solubility problems. Poly(N'pelargony1 and stearyl-clysine) are soluble in alcohols containing linear aliphatic chains such as n-butanol and n-octanol and in mixtures of these alcohols with hydrocarbons such as n-hexane and n-heptane. Both polymers show an a-helical conformation in the above solvents, which can be disrupted upon addition of sulfuric acid. Also in the solid state, poly(N'-stearyl-clysine) and poly(Ne-pelargonyl-clysine)show X-ray diffraction patterns typical of order structure.
INTRODUCTION Branched polymers containing long aliphatic side chains are of special interest especially from a structural point of view. Polymers of this kind crystallize quite readily, leading to a structure in which the side chains are packed in a hexagonal lattice typical of the "gas-crystalline state.'-4" This structure is quite independent of the configuration of the main chain and it is largely determined by the strong interactions amongst the long aliphatic side chains. The structure and the physicomechanical properties of some of these branched polymers in the solid state have been extensively investigated.'-6 Thc main feature of these polymeric systems is the presence of a high degree of order. Structural studies have also been carried out in solution, where a structuring effect toward low-molecularweight compounds has been o b ~ e r v e d . ~ I n the present paper we report the results of structural investigations in solution and in the solid state on polypeptides with long aliphatic side
* On leave of absence from the Moscow State University, Dept. of Chemistry, Moscow B-234-U.S.S.R. 73
@ 1975 by John Wiley & Sons, Inc.
74
SHIBAEV, PALUMBO, AND I'E:CGION
chains such as poly (N'-stcaryl-L-lysinc.) and poly(N'-peIargonyl-L-lysine). I n these cases, through the optical rotatory properties of the peptide backbone, it is possiblr t o observe the mutual effect of the packing of the side chains on the conformation of the main chain.
EXPERIMENTAL Poly-L-Lysine Poly-L-lysine hydrochloride was prepared according to Fasman ct a1.8 by polymerization of t h r corresponding N-carboxyanhydride. After exhaustive dialysis against 0.01 N hydrochloric acid and lyophilization, the sample had an intrinsic viscosity of 1.0 dl/g in 1 N hydrochloric acid. W-Stearyl-L-Lysine Copper I1 Complex L-lysinc copper I1 complex (11.2 g)s n-as dissolvcld in 100 ml of distilled water. The pH of the ice-cooled solution \\as adjusted to 9-10 with 2 N sodium hydroxidc. Stcxaryl chloridc (50 g), disso1vc.d in 50 ml (.thy1 rther, n-as then added dropwisc to thc vigorously stirred solution, whose pH was maintained brtwecn 9 and 10 by thc addition of 2 N sodium hydroxide. A blue solid precipitatc.d, 1% hich 11as recovered by filtration and washed several times with water, mc.thano1, and nar m ethyl ether, in order to rrmove all the stearic acid formed by hydrolysis of the stearyl chloride. Anal. Calcd. for C&SBP\TIOBCU:C, 64.8; H, 10.68; K, 6.31. Found: C, 65.0; H, 10.52; IT,5.95. W-Stearyl-L-Lysine Tricthylentetraaminc disulphate (TETD) (3.3 g) was dissolved in 250 ml 2 N sodium hydroxidc. The solution mas brought to boiling and 7 g N'stearyl-L-lysinc copper 11' complex m r c rapidly addcd I\ ith stirring. Only a small portion of the complex dissolved and the reaction took place mainly at the solid-liquid interface. Thcl solution was filtered, and the clear solution neutralized with glacial acetic acid, and cooled in an ice bath. A white solid n a s obtained, which was washed ivith warm water, methanol, and warm ethyl ether. Thc precipitate was also insoluble in most common organic solvents (tetrahydrofuranc, dimethyl formamide, dioxane, dimethyl sulfoxide) ; mp 230-231°C; yicld 8%. Anal. Calcd. for C24H4&203: C, 69.83; H, 11.72; N, 6.78. Found: C, 68.32; H, 11.00; N, 5.S4.
W-Stearyl-L-Lysine NCA N'-Stearyl-L-lysine (50 mg) was suspended in 40 ml of anhydrous freshly distilled dioxane. Phosgene was thcn bubbled through the stirred solution for 0.3 hr and the temperature kept at 30-32°C. The suspension became a clear solution, which was filtered and evaporated 271 zlucuo. A pale yellow solid was obtained; thc crud(, matmial i t as thcn rcwystallized from ethyl acetate. The two anhydride bands appear at 1S50 and l h l 0 em-'.
POLY (N'-STEARYL AND PELARGONYL-L-LYSINE)
75
Anal. Calcd. for C25H46N204: C, 68.45; H, 10.56; N, 6.38. Found: C, 68.17; H, 10.93; PIT, 6.23.
Poly (W-Stearyl-L-Lysine) 1 and 2 1) W-stearyl-L-lysine NCA (80 mg) was dissolved in 40 ml freshly distilled dry dioxane. Tricthylaminc was added as initiator (1 :30). The solution was allowed to stand 2 days at room temperature with stirring. T hr 1850- and 1810-cm-' bands of the NCA slowly decreased during polymerization until they completely disappeared in the solution. A whitc precipitate was obtained, which was isolated by filtration and washed with dioxane several times. The product was purified by dissolving it in n-octanol and precipitating with mcthanol; mp 210°C. Anal. Calcd. for C24H4,N202: C, 73.04; H, 11.75; N , 7.09. Found: C, 70.47; H, 11.25; N, 7.02. 2) Poly-L-lysine hydrochloride (0.2 g) was dissolved in 200 ml distilled matcr. The pH was adjusted to 9 with 2 N sodium hydroxidr, and the solution cooled to 4-6°C with an ice bath. Stcaryl chloride (0.7 g) in 30 ml ethyl ether was addcd dropwise with stirring, while keeping the p H a t a value of 8.5-9 with 2 N sodium hydroxide. A white precipitate was obtained, which was washed scveral timm with water, methanol, and ethyl ether. The yield was about 80%; mp 212°C. The product was purified by pouring the solution of n-octanol into methanol. Anal. Calcd. for C24H&202: C, 73.04; H, 11.73; N , 7.09. Found: C, 70.25; H , 11.73; N, 7.00.
Poly (W-Pelargonyl-L-Lysine) Poly-L-lysine hydrochloride (200 mg) was dissolved in 200 ml distilled water. The p H was adjustrd to 8.5-9 with 2 N sodium hydroxide and the solution cooled to 4-6°C with an ice bath. Pelargonyl chloride (0.7 g) in 30 ml ethyl ether was then added dropwise with stirring while keeping the pH value around 9 with 2 N sodium hydroxide. A white precipitate was recovered by filtration and washrd several times with water, mcthanol, and ethyl ether. The yield was 90%. The product was purified by pouring thc solution of n-butanol into mrthanol. Anal. Calcd. for CI5H2sN2O2:C, 67.1; H, 10.4; K, 10.4. Found: C, 65.98; H, 10.25; N, 10.18.
MEASUREMENTS Infrared measurement were performed with a Beckman ir 11 spectrophotometer in the solid state (KBr pellets) in the range of 4000-600 cm-l. Circular dichroism spectra were measured with a Cary 60 recording spectropolarimeter in the region 250-200 nm. X-ray powder diffraction patterns were recorded with a Siemens SRS apparatus.
76
SHIBAEV, PALUhIBO, AND PEGGION
RESULTS AND DISCUSSION Synthesis
-
-
The syrithesis of the desired polypeptides is outlined in Schemes I and 11. lysine
+ Cu+'
stearyl chloride
lysine copper complex
N'-stesryl-lysine copper complex
TETD
S'-stearyl-lysine
COCI? dioxane
trletlr\ lamine
V'-stexiyl-ly mc-.V-cai Iio\yanhydride
poly( \ f-itearyl-Irlysme)
SCHEME I stearvl chloride
P o l y - ~ Im > e
poll (,Yf-stear>1-~-1\$me)
SCHKAIE I1
Scheme I involves thc prcparation of thr monomer W-stcaryl- L-lysineN-carboxyanhydride and its subscqucnt polymerization in thc presence of a tertiary aminc. In this proccdurc, the dcblocking step of t h r N'-stearyllysine copper complex is a crucial onr, since the rcaction occurs in an ethcrogencous phase. In fact t h r copper complex is sparingly solublc in thc warm alkalinr solution of triethylcntrtraaminc disulphatc (TETD) and the reaction mainly occurs at tht. liquid-solid interface. The mixture can not bc kept for more than 1 miri in alkalinc conditions, otherwise extensive raccmizatiori occurs. E'or thcsr reasons thc yield of N'-straryl-L-Iysine obtaincd by ncutralization of thr filtrate. is very Ion. (no mow than S-lO%). All t h r remaining steps of Scheme I arr quite rasy and give t h r desired products in almost quantitative yields: thc monomc'r N'-stcaryl Iysine NCA is rasilg solublc in dioxaric and polymerizes in t h r usual way. In Schemc. I1 poly(N'-stcaryl-lysinc.) is prrpared by dircxct acylation of poly-L-lysine a t pH S.5-9. In order to obtain total acylation of the amino groups, a large cxcess of straryl chloridc (dissolwd in acetone) was used. Under these conditions acylation of thc t-amino groups in the sidc chains is essentially complete. Both procedures 1) and 2) wcrc followed t o synthesize poly(W-pclargonyl-L-lysine), using pclargonvl chloridc in t he placc of stearyl chloride. I n this caw thc acyl chloride is a liquid and can therefore be added dircctly to the solution of poly-L-lysinr.
Ir Studies The ir spectra of W-stcaryl-L-1ysinc and of the copper I1 complexes of L-lysinc and N'-stearyl-L-lysinc. in the region 3700-700 em-' are presented in Figure 1. I n the first and third compourids, typical amide bands at 3300, 1640, and 1333 cm-I arc prrscnt, togethcr with bands dut. to the aliphatic chairis at 2S60 and 2930 cm-I (-CH stretching), at 1470 cm-* (-CH2 sym. def.), arid 725 em-' (-CH, rocking). The only rcmarkablc aspcct is the prrscncc of a shoulder at 740 cm-' i n the spectra of N'-strarylL-lgsine copprr I1 complex and of N'-stcaryl-L-lysiric. According to I Fig. 4. CD spectrum of poly(Nf-pelargonyl-clysine) in n-butanol, conc.
=
2.06 mg/ml.
right-handed a! helix with negative bands a t 222 and 208 nm. The relativc intensity of these two bands is a little different from that observed in other peptides; this is probably due t o the solvent effects on the 205-nm The addition of increased amounts of methanol t o the poly(W-steary1-Llysine) in n-octanol does not inducc any conformational change in the polymer
SHIBAISV, PALUMBO, AND PEGGION
80
D Az218nm 0 h=222nm X A1230nm
9
8 7-
0
5
1
I
10
15
20
‘25
3 5 %.+OH
30
Fig. 5. Plot of A E values of poly(M‘-stearyl-i,lysine) vs. solvent composition in noctanol/metlianol mixtures a t 218, 222, and 230 nm.
t
I 0 % HZS4
10
20
Yo
I
I
30
40
50
H2SO.q
Fig. 6. Plots of A, values of poly[i\”-steaiyl-L-lysine) vs. solvent composition in noctanol/sulfuric acid mixtures at 195 and 222 nm.
(Figure 5 ) . Thc amplitude of thc 222-nm band is almost independent of the methanol content of thc solvont mixture until 25% methanol is reached, at which point the polymer prccipitatcs. This shows t h a t t h r packing effect of methanol on thc polymer sidc chains4 docs not influcricc the conformation of the peptidc backbone. Apparently both thc hexagonal packing of the side chain3 and t h r polypcptidc helical backbone can coexist in the same structure.
POLY(iV'-STEARYL AND PELARGONYL-L-LYSINE)
81
Essentially identical results have been obtained also with poly(N'pelargonyl-L-lysine) . Denaturation of the a-helical structure of poly(N'-stc.aryl-L-lysirie) can be achieved only by addition of concentrated sulfuric acid t,o the polymer solution in n,-octanol. The acid causes a sharp helix-coil transition, which is half-complete a t ~ 2 2 % H2S04in the solvent mixture (Icigure 6).
X-Ray Diffraction Studies X-ray diffraction studies carried out on crude and purified poly(N'stearyl-L-lysine) showed that the material purified by redissolution in noctanol and precipitation with ct.hyl ether is much more crystalline than the crude polymer. There are intcnsc rcflcctions a t 2.42 and a t 1.84 A, which are typical of a hexagonal packing of the aliphatic side chain^.^ Reflections typical of the a-helical conformation \wrc not rcadilg distinguishable. X-ray measuremcmts on oricnted fibcrs arc' undrrn-ay to determine whether the tu-helical form occurs in prccipitatnd polynicm.
References 1. Shibaev, V. P., Pet,rukhin, B. S., Zubov, In. A., Plate', N. A. & Kargin, V. A. (1968) Vysocomolekul. Soedin A10, 216. 2. Plate', N. A,, Shibaev, V. P., Petrukhin, B. S., & Kargin, V. A. (1968) J. Polym. Sci. pt. C 23, 37. 3. Shibaev, V. P., Petrukhin, B. S., Plate', N. A. & Korgin, V. A. (1970) Vysocomolekul. Soedin A12, 140. 4. Plate', N. A. & Shibaev, V. P. (1971) Vysocomolekzcl. Soedin A13,410. 5 . Shibaev, V. P., Plate', N. A. & Korgin, V. A. (1964) Proc. I I I Ezcr. Conf. Electron Microscopy, Vol. A, p. 415. 6. Kargin, V. A., Khalikov, I). Kh., Shibaev, V. P., Plate', N. A. & Lemanovskaya, A. F. (1970) Dokl. A k a d . Nauk SSSR 190,376. 7. Shibaev, V. P., Talrose, 11. V., Petrukhin, B. S. & Plate', N. A. (1971) Vysoconiolekzcl. Soedin A13, 4. 8. Fasman, G. I)., Idelson, AT. & Blout, E. It. (1961) J. A m e r Chem. Soc. 83,709. 9. Keller, A. & Sanderman, J. (1954) J. I'olyna. Sci. 13,511. 10. Chapman, 11. (1957) J . Chem. Soc. (1,onrlon) 4489. 11. Peggion, E., Verdini, A. S., Cosani, A. & Scoffone, E. (1969) Macromolecules 2, 170. 12. Quadrifoglio, F. & Urry W. B. (1968) J. A m e r . Chcm. Soc. 90,2755. 13. Epand, R. F. & Sheraga, H. A. (1968) Biopolymers 6,1383. 14. Dickerson, It. E. (1964) in The Proteins, Neurath, Ed. Vol. 11, p. 679.
Received July 7, 1972 Accepted September 15, 1974
BIOPOLYMERS
Synthesis and Conformational Properties of Polypeptides Containing Long Aliphatic Side Chains. Poly (N'-Stearyl-L-Lysine) and Poly(N'-Pelargonyl-L-Lysine) VALERY SHIBAEV,* A'IANLIO PALUMBO, and EVARISTO PEGGIOW, Institute of Organic Chemistry, University of Padova, 35100 Padua, Italy
Synopsis Poly(N'-stearyl-Llysine) and poly(Nf-pelargonyl-clysine) were synthesized both by polymerization of N'-pelargonyl and N'-stearyl-clysine NCA and by acylation of poly(L1ysine) with pelargonyl and stearyl chloride. This second route has proven to be very useful, since completely acylated polymers are obtained in almost quantitative yield, whereas the usual scheme of preparation of c protected poly(c1ysine) cannot easily be applied due to solubility problems. Poly(N'pelargony1 and stearyl-clysine) are soluble in alcohols containing linear aliphatic chains such as n-butanol and n-octanol and in mixtures of these alcohols with hydrocarbons such as n-hexane and n-heptane. Both polymers show an a-helical conformation in the above solvents, which can be disrupted upon addition of sulfuric acid. Also in the solid state, poly(N'-stearyl-clysine) and poly(Ne-pelargonyl-clysine)show X-ray diffraction patterns typical of order structure.
INTRODUCTION Branched polymers containing long aliphatic side chains are of special interest especially from a structural point of view. Polymers of this kind crystallize quite readily, leading to a structure in which the side chains are packed in a hexagonal lattice typical of the "gas-crystalline state.'-4" This structure is quite independent of the configuration of the main chain and it is largely determined by the strong interactions amongst the long aliphatic side chains. The structure and the physicomechanical properties of some of these branched polymers in the solid state have been extensively investigated.'-6 Thc main feature of these polymeric systems is the presence of a high degree of order. Structural studies have also been carried out in solution, where a structuring effect toward low-molecularweight compounds has been o b ~ e r v e d . ~ I n the present paper we report the results of structural investigations in solution and in the solid state on polypeptides with long aliphatic side
* On leave of absence from the Moscow State University, Dept. of Chemistry, Moscow B-234-U.S.S.R. 73
@ 1975 by John Wiley & Sons, Inc.
74
SHIBAEV, PALUMBO, AND I'E:CGION
chains such as poly (N'-stcaryl-L-lysinc.) and poly(N'-peIargonyl-L-lysine). I n these cases, through the optical rotatory properties of the peptide backbone, it is possiblr t o observe the mutual effect of the packing of the side chains on the conformation of the main chain.
EXPERIMENTAL Poly-L-Lysine Poly-L-lysine hydrochloride was prepared according to Fasman ct a1.8 by polymerization of t h r corresponding N-carboxyanhydride. After exhaustive dialysis against 0.01 N hydrochloric acid and lyophilization, the sample had an intrinsic viscosity of 1.0 dl/g in 1 N hydrochloric acid. W-Stearyl-L-Lysine Copper I1 Complex L-lysinc copper I1 complex (11.2 g)s n-as dissolvcld in 100 ml of distilled water. The pH of the ice-cooled solution \\as adjusted to 9-10 with 2 N sodium hydroxidc. Stcxaryl chloridc (50 g), disso1vc.d in 50 ml (.thy1 rther, n-as then added dropwisc to thc vigorously stirred solution, whose pH was maintained brtwecn 9 and 10 by thc addition of 2 N sodium hydroxide. A blue solid precipitatc.d, 1% hich 11as recovered by filtration and washed several times with water, mc.thano1, and nar m ethyl ether, in order to rrmove all the stearic acid formed by hydrolysis of the stearyl chloride. Anal. Calcd. for C&SBP\TIOBCU:C, 64.8; H, 10.68; K, 6.31. Found: C, 65.0; H, 10.52; IT,5.95. W-Stearyl-L-Lysine Tricthylentetraaminc disulphate (TETD) (3.3 g) was dissolved in 250 ml 2 N sodium hydroxidc. The solution mas brought to boiling and 7 g N'stearyl-L-lysinc copper 11' complex m r c rapidly addcd I\ ith stirring. Only a small portion of the complex dissolved and the reaction took place mainly at the solid-liquid interface. Thcl solution was filtered, and the clear solution neutralized with glacial acetic acid, and cooled in an ice bath. A white solid n a s obtained, which was washed ivith warm water, methanol, and warm ethyl ether. Thc precipitate was also insoluble in most common organic solvents (tetrahydrofuranc, dimethyl formamide, dioxane, dimethyl sulfoxide) ; mp 230-231°C; yicld 8%. Anal. Calcd. for C24H4&203: C, 69.83; H, 11.72; N, 6.78. Found: C, 68.32; H, 11.00; N, 5.S4.
W-Stearyl-L-Lysine NCA N'-Stearyl-L-lysine (50 mg) was suspended in 40 ml of anhydrous freshly distilled dioxane. Phosgene was thcn bubbled through the stirred solution for 0.3 hr and the temperature kept at 30-32°C. The suspension became a clear solution, which was filtered and evaporated 271 zlucuo. A pale yellow solid was obtained; thc crud(, matmial i t as thcn rcwystallized from ethyl acetate. The two anhydride bands appear at 1S50 and l h l 0 em-'.
POLY (N'-STEARYL AND PELARGONYL-L-LYSINE)
75
Anal. Calcd. for C25H46N204: C, 68.45; H, 10.56; N, 6.38. Found: C, 68.17; H, 10.93; PIT, 6.23.
Poly (W-Stearyl-L-Lysine) 1 and 2 1) W-stearyl-L-lysine NCA (80 mg) was dissolved in 40 ml freshly distilled dry dioxane. Tricthylaminc was added as initiator (1 :30). The solution was allowed to stand 2 days at room temperature with stirring. T hr 1850- and 1810-cm-' bands of the NCA slowly decreased during polymerization until they completely disappeared in the solution. A whitc precipitate was obtained, which was isolated by filtration and washed with dioxane several times. The product was purified by dissolving it in n-octanol and precipitating with mcthanol; mp 210°C. Anal. Calcd. for C24H4,N202: C, 73.04; H, 11.75; N , 7.09. Found: C, 70.47; H, 11.25; N, 7.02. 2) Poly-L-lysine hydrochloride (0.2 g) was dissolved in 200 ml distilled matcr. The pH was adjusted to 9 with 2 N sodium hydroxidr, and the solution cooled to 4-6°C with an ice bath. Stcaryl chloride (0.7 g) in 30 ml ethyl ether was addcd dropwise with stirring, while keeping the p H a t a value of 8.5-9 with 2 N sodium hydroxide. A white precipitate was obtained, which was washed scveral timm with water, methanol, and ethyl ether. The yield was about 80%; mp 212°C. The product was purified by pouring the solution of n-octanol into methanol. Anal. Calcd. for C24H&202: C, 73.04; H, 11.73; N , 7.09. Found: C, 70.25; H , 11.73; N, 7.00.
Poly (W-Pelargonyl-L-Lysine) Poly-L-lysine hydrochloride (200 mg) was dissolved in 200 ml distilled water. The p H was adjustrd to 8.5-9 with 2 N sodium hydroxide and the solution cooled to 4-6°C with an ice bath. Pelargonyl chloride (0.7 g) in 30 ml ethyl ether was then added dropwise with stirring while keeping the pH value around 9 with 2 N sodium hydroxide. A white precipitate was recovered by filtration and washrd several times with water, mcthanol, and ethyl ether. The yield was 90%. The product was purified by pouring thc solution of n-butanol into mrthanol. Anal. Calcd. for CI5H2sN2O2:C, 67.1; H, 10.4; K, 10.4. Found: C, 65.98; H, 10.25; N, 10.18.
MEASUREMENTS Infrared measurement were performed with a Beckman ir 11 spectrophotometer in the solid state (KBr pellets) in the range of 4000-600 cm-l. Circular dichroism spectra were measured with a Cary 60 recording spectropolarimeter in the region 250-200 nm. X-ray powder diffraction patterns were recorded with a Siemens SRS apparatus.
76
SHIBAEV, PALUhIBO, AND PEGGION
RESULTS AND DISCUSSION Synthesis
-
-
The syrithesis of the desired polypeptides is outlined in Schemes I and 11. lysine
+ Cu+'
stearyl chloride
lysine copper complex
N'-stesryl-lysine copper complex
TETD
S'-stearyl-lysine
COCI? dioxane
trletlr\ lamine
V'-stexiyl-ly mc-.V-cai Iio\yanhydride
poly( \ f-itearyl-Irlysme)
SCHEME I stearvl chloride
P o l y - ~ Im > e
poll (,Yf-stear>1-~-1\$me)
SCHKAIE I1
Scheme I involves thc prcparation of thr monomer W-stcaryl- L-lysineN-carboxyanhydride and its subscqucnt polymerization in thc presence of a tertiary aminc. In this proccdurc, the dcblocking step of t h r N'-stearyllysine copper complex is a crucial onr, since the rcaction occurs in an ethcrogencous phase. In fact t h r copper complex is sparingly solublc in thc warm alkalinr solution of triethylcntrtraaminc disulphatc (TETD) and the reaction mainly occurs at tht. liquid-solid interface. The mixture can not bc kept for more than 1 miri in alkalinc conditions, otherwise extensive raccmizatiori occurs. E'or thcsr reasons thc yield of N'-straryl-L-Iysine obtaincd by ncutralization of thr filtrate. is very Ion. (no mow than S-lO%). All t h r remaining steps of Scheme I arr quite rasy and give t h r desired products in almost quantitative yields: thc monomc'r N'-stcaryl Iysine NCA is rasilg solublc in dioxaric and polymerizes in t h r usual way. In Schemc. I1 poly(N'-stcaryl-lysinc.) is prrpared by dircxct acylation of poly-L-lysine a t pH S.5-9. In order to obtain total acylation of the amino groups, a large cxcess of straryl chloridc (dissolwd in acetone) was used. Under these conditions acylation of thc t-amino groups in the sidc chains is essentially complete. Both procedures 1) and 2) wcrc followed t o synthesize poly(W-pclargonyl-L-lysine), using pclargonvl chloridc in t he placc of stearyl chloride. I n this caw thc acyl chloride is a liquid and can therefore be added dircctly to the solution of poly-L-lysinr.
Ir Studies The ir spectra of W-stcaryl-L-1ysinc and of the copper I1 complexes of L-lysinc and N'-stearyl-L-lysinc. in the region 3700-700 em-' are presented in Figure 1. I n the first and third compourids, typical amide bands at 3300, 1640, and 1333 cm-I arc prrscnt, togethcr with bands dut. to the aliphatic chairis at 2S60 and 2930 cm-I (-CH stretching), at 1470 cm-* (-CH2 sym. def.), arid 725 em-' (-CH, rocking). The only rcmarkablc aspcct is the prrscncc of a shoulder at 740 cm-' i n the spectra of N'-strarylL-lgsine copprr I1 complex and of N'-stcaryl-L-lysiric. According to I Fig. 4. CD spectrum of poly(Nf-pelargonyl-clysine) in n-butanol, conc.
=
2.06 mg/ml.
right-handed a! helix with negative bands a t 222 and 208 nm. The relativc intensity of these two bands is a little different from that observed in other peptides; this is probably due t o the solvent effects on the 205-nm The addition of increased amounts of methanol t o the poly(W-steary1-Llysine) in n-octanol does not inducc any conformational change in the polymer
SHIBAISV, PALUMBO, AND PEGGION
80
D Az218nm 0 h=222nm X A1230nm
9
8 7-
0
5
1
I
10
15
20
‘25
3 5 %.+OH
30
Fig. 5. Plot of A E values of poly(M‘-stearyl-i,lysine) vs. solvent composition in noctanol/metlianol mixtures a t 218, 222, and 230 nm.
t
I 0 % HZS4
10
20
Yo
I
I
30
40
50
H2SO.q
Fig. 6. Plots of A, values of poly[i\”-steaiyl-L-lysine) vs. solvent composition in noctanol/sulfuric acid mixtures at 195 and 222 nm.
(Figure 5 ) . Thc amplitude of thc 222-nm band is almost independent of the methanol content of thc solvont mixture until 25% methanol is reached, at which point the polymer prccipitatcs. This shows t h a t t h r packing effect of methanol on thc polymer sidc chains4 docs not influcricc the conformation of the peptidc backbone. Apparently both thc hexagonal packing of the side chain3 and t h r polypcptidc helical backbone can coexist in the same structure.
POLY(iV'-STEARYL AND PELARGONYL-L-LYSINE)
81
Essentially identical results have been obtained also with poly(N'pelargonyl-L-lysine) . Denaturation of the a-helical structure of poly(N'-stc.aryl-L-lysirie) can be achieved only by addition of concentrated sulfuric acid t,o the polymer solution in n,-octanol. The acid causes a sharp helix-coil transition, which is half-complete a t ~ 2 2 % H2S04in the solvent mixture (Icigure 6).
X-Ray Diffraction Studies X-ray diffraction studies carried out on crude and purified poly(N'stearyl-L-lysine) showed that the material purified by redissolution in noctanol and precipitation with ct.hyl ether is much more crystalline than the crude polymer. There are intcnsc rcflcctions a t 2.42 and a t 1.84 A, which are typical of a hexagonal packing of the aliphatic side chain^.^ Reflections typical of the a-helical conformation \wrc not rcadilg distinguishable. X-ray measuremcmts on oricnted fibcrs arc' undrrn-ay to determine whether the tu-helical form occurs in prccipitatnd polynicm.
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Received July 7, 1972 Accepted September 15, 1974
