BIOPOLY MERS VOL. 13, 1117-1125 (1974)

Synthesis and Conformational Study of Poly(N6-Carbobenzoxy, N’-Benzyl-L-Ornithine)

and Poly (N’-Benzyl-L-Ornithine)

HIROYUKI YAMAMOTO and TADAO HAYAKAWA, Institute of High Polymer Research, Faculty of Textile Science and Technology,

Shinshu University, Ueda, Japan, and JEN TSI YANG,* Cardiovascular Research Institute and Departm.ent of Biochemistry and Biophysics,

University of California, Sari Francisco, California 94143

Synopsis Poly(N6-carbobenzoxy, N*-benzyl-cornithine) (PCBLO) was prepared by the

standard NCA method. PCBLO was converted into poly(N6-benzyl-cornithine) (PBLO) through decarbobenzoxylation with hydrogen bromide. The monomer N6- benzyl-cornithine was synthesized by reacting Lornithine with benzaldehyde, followed by hydrogenation. The conformation of the two polypeptides was studied by optical rotatory dispersion and circular dichroism. PCBLO forms a right-handed helix in helix-promoting solvents. In mixed solvents of chloroform and dichloroacetic acid (DCA) it undergoes a sharp helix-coil transition at 12% (v/v) DCA at 25OC, as com- pared with 36% for poly(N6-carbobenzoxy-tornithine) (PCLO). Like PCLO, the helix-coil transition is “inverse,” that is, high temperature favors the helical form. PBLO is soluble in water at pH below 7 and has a ‘‘coiled” conformation. In 88% (v/v) 1-propanol above pH (apparent) 9.6 it is completely helical. In 50% 1-propanol the transition pH (apparent) is about 7.4; this compares with a pHt, of about 10 for poly- Lornithine in the same solvent.

INTRODUCTION

The stereochemical influence of side groups on the polypeptide conforma- tion has recently received considerable interest. Such studies can provide information concerning the various factors that affect the conformational stability. One such approach is to study the conformation of a homol- ogous series of polypeptides and determine the influence of chain length of the side groups. For instance, the uncharged PLL,t PLO, and poly- (L-a, y-diaminobutyric acid) show decreasing helical stability with the

* To whom correspondence should be addressed. t Abbreviations used in this work: PLL, poly(L-lysine); PLO, poly(L-ornithine);

PCLL, poly(NC-carbobenzoxy-L-lysine); PCLO, poly(N6-carbobenzoxy-cornithine); PCBLL, poly(N‘-carbobenzoxy, N‘-benzyl-clysine); PCBLO, poly(N’-carbobenzoxy, N*-benzyl-cornithine); PBLL, poly(Ne-benzyl-Llysine); PBLO, poly(N6-benzyl-L- ornithine); DCA, dichloroacetic acid; ORD, optical rotatory dispersion; CD, circular dichroism.

1117

@ 1974 by John Wiley & Sons, Inc.

1118 YAMAMOTO, HAYAKAWA, AND YANG

shortening of the side chain Their carbobenzoxy derivatives, PCLL and PCLO, undergo an “inverse” temperature-induced helix-coil transition in mixed solvents, but poly(NY-carbobenzoxy-L-a, 7-diamino- butyric acid) has a “normal” A second approach is to intro- duce functional groups into the side chain and study the stability of the helical conformation. For instance, PLL containing different substituents on the N’ position has been inve~t igated.~,~ Synthesis and conformational study of PCBLL and PBLL was already r e p ~ r t e d . ~ In this paper we study the helix-coil transition PCBLO and PBLO using ORD and CD. These two polypeptides were synthesized by the aame method as that for PCBLL and PBLL.

EXPERIMENTAL

Materials

PCBLO and PBLO were synthesized according to the following scheme:

Om.HC1- Benzylidene Om. ___) Bz-Om- HC1 I I1 I11

CuCOa. Cu(0H)z COCI, (Et)aN - Z, Bz-Orn d Z, Bz-Orn NCA ___)

CaHsCHO NaBH4

zc1 IV v

HBr Poly(Z, Bz-Om) - Poly(Bz-Om - HBr) +NHCHCOj-,, fNHCHCO+-,

( c: H2)3 ( c: Hz)t I NH.HBr

I NOCO(CH2)HaCs

where Bz = benzyl, Z = carbobenzoxy, and NCA = N-carboxyanhydride. N’-Benzylidene-L-Omithine (11). The I1 crystals were prepared from

I and benzaldehyde and recrystallized from methanol and ether;9 yield, 73%; mp 200°C; [ a ] ~ ~ ~ = -8.4 (c = 1% in water). Calcd. for CEH1602NI: C, 65.4; H, 7.3; N, 12.7. Found: C, 65.5; H, 7.3; N, 12.5.

N’-Benzyl-L-Ornithine. HCl (111). The compound I1 was hydrogenated by sodium bor~hydride.~ The oily product of I11 was dissolved in ethanol and neutrdized with pyridine. The white precipitcite was recrystdlized from water; yield, 47%; mp 229°C; [ a ] ~ ~ ~ = 7.0 (c = 1% in water). Anal. Calcd. for C12H19N202C1: C, 55.7; H, 7.4; N, 10.8. Found: C, 55.7; H,7.3; N, 10.9.

N6-Carbobenzoxy, N6-Benzyl-L-Omithine (IV). The copper complex of I11 was carbobenzoxylated and the oily product of IV was crystallized with yield, 84%; mp 211-213°C; [ a ] ~ ~ ~ = 14.3 (C = 1% in acetic acid). Anal. Calcd for CzoH2404N2: C , 67.4; H, 6.8; N, 7.9. Found: C, 67.5; H,6.8; N,7.9.

Anal.

HELIX-COIL TRANSITION OF POLYORNITHINES 1119

N6-Carbobenzoxy, N'-BenzyI-L-Ornithine NCA (V). The KCA was prepared by the standard procedure using phosgene for 30 min a t 50°C and obtained as a syrup; yield (theoretical), chloride free.

High-molecular-weight PCBLO was prepared by polymerizing a 10% solution of V in dry dioxane with triethylamine as the initiator. The molar ratio of the NCA to the initiator ( A / I ) was 100: 1. The poly- mer was precipitated with water, filtered, and dried; yield, 92%. Anal. Calcd. for CzoHzz03Nz: C, 71.0; H, 6.6; N, 8.3. Found: C, 70.9; H, 6.6; N, 8.3. The intrinsic viscosity of PCBLO was 0.43 dl/g in DCA at 25°C. The degree of polymerization was estimated to be about 300, as- suming that the log [~I-log M plot for PCLL was applicable.5

The polymer was prepared by decarbobenzoxylation of PCBLO in 3:4 (v/v) acetic acid-dioxane with hydrogen bromide (25%) in glacial acetic acid; yield (theoretical). PBLO.HBr was dissolved in water and reprecipitated with 1 : 1 ethanol-ether, centrifuged, and dried; yield, 75%. Anal. Calcd. for C12H170N2Br: C, 50.5; H, 6.0; N, 9.8. Found: C,50.9; H,6.1; N, 10.2.

Methods

PCBLO.

PBLO . HBr.

ORD and CD. ORD was measured with a Cary 60 spectropolarimeter and CD with a Jasco J-10 spectropolarimeter.'O For measurements below 0°C a heat exchanger containing Dry Ice and ethanol, a Haake thermo- regulator, and a pump circulating ethanol were used to maintain constant temperature. Specially designed cells with heavy walls were used for these low-temperature studies.

Elementary Analyses. All analyses were carried out with a Yanagimoto CHN recorder.

RESULTS AND DISCUSSION

Poly(N6-carbobenzoxy, Nd-benzyl-L-Ornithine)

ORD for PCBLO in helix-promoting solvents showed a 233-nm minimum and the corresponding CD a double minimum a t 220 and 208 nm, which are characteristic of a right-handed helix. Table I lists the

Conformation.

TABLE I CD and ORD of Poly(N6-Carbobenzoxy, Nb-Benzyl-L-Ornithine), and

Poly!N'-(=srboben&oxy, N'-Renzyl-~-I~ysine)

Polypeptides Solvent [m'1233 [ e ~ ~ ~ ~ - ~ ~ PCBLO Dioxane - 12400 - 28200 - 35700

- Chloroform - 12500 - 34000 2-chloroethanol - 12800 - 28300 - 37200

PCBLLa Dioxane - 10300 - 27600 -25100 - Chloroform - 9500 - 27300

Zchloroethanol - 9500 - 29000 - 28200

a Data taken from Ref. 9.

1120 YAMAMOTO, HAYAKAWA, AND YANG

numerical values at these extrema, together with those of PCBLL,S which has one more methylene group in the side chain. PCBLO appears to have larger magnitudes a t these extrema than PCBLL. Another difference is that the ratio of the two negative CD bands is [O]20s = 0.8 for PCBLO and [O 122$ [O ]207 = 1.0-1.1 for PCBLL.

Figure 1 shows the ORD in the visible region for PCBLO and also PCBLL for comparison. The data can be fitted with the Moffitt equation, and the bo 60 determined largely reflects the extent of the helical form. Although the two curves in chloroform appear to be very different, their bo indicates the presence of the helical conformation: -555 and -430 deg cm2 dmol-’ for PCBLO and PCBLL, respectively (ao = -130 for PCBLO and +180 for PCBLL). In DCA, the bo is +35 (PCBLO) and +45 (PCBLL) deg cm2 dmol-’, respectively, suggesting the presence of the “coiled” conformation.

Solvent-Induced Transition. Figure 2 shows the helix-coil transition of PCBLO in mixed solvents of chloroform-DCA. A sharp transition occurs a t 12% (v/v) DCA, suggesting a high degree of cooperativity. For comparison, the middle point of the transition for PCBLL occurred near 8% DCA.g Both the unbenzylated polypeptides, PCLO and PCLL, showed a helix-coil transition at 36: 64 (v/v) DCA-chloroform at 25°C.4-11 Thus, the introduction of the N-benzyl group to PCLO and PCLL appears to destabilize these polypeptides and requires less DCA to break up the helical conformation.

- 51 [a 1

-101

-15’

I I

A, nm Fig. 1. ORD of poly(N6-carbobenzoxy, N6-benzyl-Irornithine) (PCBLO) and poly-

(Nkarbobenzoxy, N‘-beneyl-~rlysine) (PCBLL) in chloroform a and dichloroacetic acid c at 25OC. Polymer concentration, 0.55-0.60%; light path, 10 mm.

HELIX-COIL TRANSITION OF POLYORNITHINES 1121

Thermal-Induced Transition. We chose a solvent composition in the transition region (Fig. 2 ) and measured [a]350 to follow the thermal-induced transition for both PCBLO and PCRLL (Fig. 3). At 350 nm the helical form of PCBLO is more levorotatory than the coiled form, but the reverse is true for PCBLL (Fig. 1). As in the case of the unbenzylated polypeptide PCLL, raising the temperature of the solution induces an “inverse” transition; that is, high temperature favors the helical form and low temperature the coiled form.

7 40 80

% DCA in CHCI, ( v / v l

Fig. 2. Helix-co transition of poly(N6-carbobenzoxy, N6-benzj - L-ornithine) in PCBLO, 0.30 mixed solvents of chloroform and dichloroacetic acid (DCA) at 25OC.

0.55%; light path, 10 mm.

80

60

0 10 c) c

d 40

20

- 40 0 40

Temp, “C Fig. 3. Thed- induced helix-coil transition of poly(N’-carbobenzoxy, Ns-benzyl-

cornithine) (PCBLO) and poly(N=carbobenzoxy, N*-benzyl-Irlysine) (PCBLL) in mixed solvents of chloroform and dichloroacetic acid (DCA). Polymer concentration, 0.42-0.90%; light path, 10 mm.

1122 YAMAMOTO, HAYAKAWA, AND YANG

Polj(N6-BenzJrl-L-Ornithine) Conformation in Mixed Solvents; pH Effect. Figure 4 shows the CD

for PBLO in mixed solvents of water and 1-propanol (PBLO was more soluble in 1-propanol than in methanol or ethanol). The aqueous solution of PBLO below pH 7 (without alcohol) has a small positive CD band near 217 nm, followed by a negative band below 210 nm. This is typical of a “coiled” conformation. (We only show a portion of the well-known CD of a “coil” to illustrate the different conformations of PBLO in the presence and absence of alcohol.) This polypeptide does not dissolve in water above pH 7; however, it can be solubilized in mixed solvents of water and alcohol. The latter enhances the helical conformation as evidenced by the appear- ance of a CD double minimum at 220 and 208 nm. We chose 8870 1- propanol as the solvent medium in order to cover a wide range of pH (apparent) from 1 to 13. If the percent alcohol is too high, the polypeptide would have been in the helical conformation over the entire range of pH. Such was the case of poly-L-lysine in 95% methanol or 76y0 2 - p r o p a n 0 1 . ~ ~ ~ ~ ~

The absolute value of the rotations for PBLO in 88% 1-propanol in- creases with the apparent pH and approaches a plateau at pH 12-13, where the polypeptide adopts a helical conformation (Fig. 5 ) . At low pH’s, where PBLO is positively charged, the helical form is unstable be- cause of electrostatic repulsion. However, the changes in [m’]233 with respect to pH are broad unlike that of PLL or other ionic polypeptides in water. This must be due to the use of a high percentage of alcohol, since to the titration of a weak polymeric ion,

pH = pKo + log[E/(l - E)] + (0.434/RT)bG/&Z

where KO is the intrinsic dissociation constant of the secondary amine, -RR’NHz+, in our case, and 5 the average degree of dissociation. The

T 0 x - 0 Y

I I I -40‘ *Lo 2x) 240

A, nm

Fig. 4. CD of poly(N6-benzyl-cornithine) a t various pH’s in water (broken line) and 88% (v/v) 1-propanol (solid line) at 25OC. PBLO, 0.10%; light path, 0.1 mm.

HELIX-COIL TRANSITION OF POLYORNITHINES 1123

pH (opporent)

Fig. 5. at 25°C. light path, 1-5 mm.

last term is the electrostatic free encrgy change with respect to charge 2 which for PBLO can be written as 0.86s W, or c(l - a)(W, or W , is a constant and the subscripts h and c refer to the helical and coiled regions of a titration curve).'O If the term W , is large, the change in z i would automatically be small when the pH of the solution is varied. In SS% 1-propanol, which has a low dielectric constant as compared with water, the protonation of the -(CH2)3(C6H5CH2)NH side groups of PBLO is difficult and the term W , is therefore large. Accordingly, the breaking-up of the helix is difficult and the transition from helix (uncharged) to coil (charged) with respect to pH is broad. To test further the effect of solvent, we include in Figure 5 the results obtained in 50,70,80, and S5Y0 1-propanol. PBLO is more easily protonated in these solvents than in SS% 1-propanol; that is, the term W, in these cases is smaller. Thus, a sharp helix-coil transition occurs near pH (apparent) 7.5. On the other hand, in 90% 1- propanol PBLO remains mostly helical even at pH 1.

One of our objectives is to study the effect of introducing a benzyl group to the -NH2 side group of PLO. We chose to compare the pH- induced transition of PBLO and PLO in 50% (v/v) 1-propanol (Fig. 6). Previous results of PBLL and PLL are also included for comparison (PLL did not dissolve in SS% 1-propanol). Both PBLO and PBLL show a sharp helix-coil transition near pH(apparent) 7.3-7.7. This compares with a transition pH above 10 for PLO and PLL in the same mixed solvents. (In the absence of alcohol, PLL is known to be completely helical upon deprotonation of the e-NH, group, but PLO has only about 20% helix even when the polypeptide is completely unionized. 1--3 However, PLO under- goes a pH-induced transition in mixed solvents of alcohol and water; the transition pH(apparent) was about 9.5 in 40% methanol or 50% dioxane3

Effects of pH and alcohol content on [m']233 of poly(N6-benzyl-L-ornithine) Numerals, volume percents of 1-propanol in water. PBLO, 0.24-0.49%;

1124 YAMAMOTO, HAYAKAWA, AND YANG

T 0 X

m N m

E - - Y

I

I 1 I

Fig. 6. pH-induced helix-coil transition of poly(N6-benzyl-cornithine) (PBLO), poly(Nf-benzyl-clysine) (PBLL), poly(Lornithine) (PLO), and poly(L1ysine (PLL) in 1: 1 1-propanol-water (v/v) a t 25°C. Polymer concentration, 0.19-0.40%; light path, 1-5 mm.

or 8.7 in 50% methanol.2) That the bensylated polypeptides, PBLO and PBLL, have a lower transition pH by more than two units than PLO and PLL in the mixed solvents used can be attributed to the lowering of the pK of the -RNH2+ group. It is known that amines become a stronger acid with the introduction of a phenyl group. For instance, the pK of n-amylamine in water was 10.6 and that of N-ethylaniline 5.1 (taken from the Handbook of Chemistry and Physics). Thus, at pH(apparent) 8, for instance, the side groups of PLO and PLL are completely charged as -(CH2) .NH2 +, which make the helical conformation unstable, but their

TABLE I1 Comparison of [m'] 233 of Four Helical Polypeptides

Polypeptide Solvent [ r n ' l ~ Reference

Poly ( N6-benzyl-L 88% 1-propanol, pH 13 - 12300 This work ornithine)

lysine) Poly(N@-benzyl-c 65% 1-propanol, pH 13 - 12000 9

Poly (~ornithine) 50% methanol, pH 12 - 14000 2 43% methanol, pH 12 - 16000 3 50% dioxane, pH 13 - 16000 3

95% methanol, pH 6-12 - 12000 12 Poly(c1ysine) Water, pH 11-12 -12000 to -14700 2, 5, 15, 16

HELIX-COIL TRANSITION OF POLYORNITHINES 1125

benzylated derivatives, PBLO and PBLL, are still mostly uncharged and retain the helical conformation.

It is now known that the magnitude of the Cotton effects of helical homopolypeptides is to some extent dependent upon the nature of the side groups (see, for example, Ref. 14), the state of aggregation and also the solvent medium used. Table I1 lists the [ r n ' l 2 3 3 for the four related poly- peptides in their helical conformation. The numerical values are close to each other but by no means identical (part of the difference must, of course, be due to experimental errors).

Mild heating of helical PLL at pH 12-13 is known to induce a helix-p transition."+'* But partial helices in aqueous solution or helices in methanol-water cannot be converted to the form by heating. This is also the case for PBLO, which does not show such a transition in l-propanol- HzO (pH 13) a t 50-70°C.

This work was aided in part by grants from the U.S. Public Health Service (GM- 10880 and HL-06285).

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Received November 6, 1973 Accepted February 5, 1974