NMRSTUDY
83
O F POLY-y-BENZYL-L-GLUTAMATE
Nuclear Magnetic Resonance Study of Liquid Crystalline Solutions of
Poly-7-benzyl-L-glutamatein Dichloromethane and 1,2-Dichloroethane by B. M. Fung, Michael J. Gerace, and Loretta S. Gerace Department of Chemistry, Tufts University, Medford, Massachusetts 08166
(Received March 84, 1969)
Nuclear magnetic resonance study on the liquid crystalline phase of solutions of poly-ybenzyl-L-glutamate in dichloromethane and 1,2-dichloroethane and their deuterated analogs is reported. The proton second moment of the polypeptide in l12-dichloroethane-d4 was calculated to be 0.11-0.14 GZ. When the sample was spun along an axis perpendicular to the magnetic field, all proton dipole-dipole interactions in l12-dichloroethane were reduced to ‘/4 of the original values. The 35Clspectra were analyzed according to the Redfield relaxation theory and the correlation time rawas found to be 6 X 10-lZt o 8 X 10-l2sec.
In recent years it has been found that certain systems of biological interest, e.g., some lipids and peptides, form liquid crystalline phases under suitable conditions. Poly-y-benzyl-L-glutamate (PBLG) forms a liquid crystal in a number of solvents at room temperaturea2 The liquid crystalline solutions acquire additional ordering in a strong magnetic field such that the helical axes of the peptide are presumably parallel to the magnetic field.334 The magnetic ordering of the solution restricts the motion of the solvent molecules. The dipole-dipole and nuclear quadrupole interactions are then not averaged to zero. The nonzero interactions give rise to fine structures in the nuclear magnetic resonance (nmr) spectra of the solvent.a-6 Proton nmr spectra of CHzClz in PBLG solutions have revealed that the dipole-dipole interaction is concentration d e ~ e n d e n t . ~Quadrupole ~~ splittings for a5Cl and for deuterium in CHzClz and CDzCl2 solutions have been studied;6 however, the concentration dependence of the splittings and the line widths of the 35Clresonance have not been reported. Now we wish to present additional results of nmr study on PBLG solutions in CHZC12, CDzC12, CHZClCHtCl, and CD2C1CDzC1. The line widths of the a5Cl resonance are analyzed by considering the density matrix with quadrupole relaxation.
The high-resolution proton resonance spectra for solvent molecules were recorded with a Varian A-60A spectrometer at 35”. Poly-y-benzyl-L-glutamate (mol wt 200,000) was obtained from the Cyclo Chemical Corporation, Los Angeles, Calif. The same polypeptide (mol wt = 45,000, 99,100, 240,000) was obtained from Miles Laboratories, Inc., Elkhart, Ind. In calculating the mole fraction of the solute, the formula weight (that is, per peptide unit) of the polypeptide was used. The molecular weight of the polypeptide in those solutions was 200,000 except where specified.
Results Proton Second Moment of PBLG. The broadline proton nmr of polycrystalline PBLG consists of a broad peak and a sharper peak centering at the same position. The proton spectra of PBLG in solutions of CDzCICDzCl in the liquid crystalline phase are smoother and have the shape of a “super Lorentzian” curve’ (broad at the base but having a sharper slope at the center). The second moment was calculated from the experimental spectrum with the trapezoid formula, using an IBM 1130 computer.8 The value for solid PBLG is 7.5 G2. The proton second moment for the liquid crystal solutions of PBLG in 1,2-dichloroethane-dr varied from 0.11 to 0.14 G2 when
Experimental Section The 2H, 35Cl,and lH (for the second moment study of the polypeptide) nuclear magnetic resonances were recorded with a Varian Associates Model VF-16 wideline spectrometer a t frequencies of 8.02, 6.67, and 16.00 MHz, respectively. The spectrometer was operated in the absorption mode at 25”. The line positions and line widths were measured directly from the derivative curves. h Varian C1024 time-averaging computer was employed for signal enhancement for the a5Cl spectra. The number of scans for each spectrum ranged from 5 to 70.
(1) “Ordered Fluids and Liquid Crystals,” Advances in Chemistry Series, No. 63,American Chemical Society, Washington, D. C., 1966, p 141. ( 2 ) C. Robinson, Trans. Faraday Soc., 5 2 , 571 (1956); C. Robinson, J. C. Ward, and R. B. Beevers, Discussions Faraday Soc., 2 5 , 29 (1958). (3) 8.Sobajima, J . Phgs. SOC.,Jap., 23, 1070 (1967). (4) M. Panar and W. D. Phillips, J . Amer. Chem. Soc., 90, 3880, (1968). (5) D. Gill, M.P. Klein, and G. Kotowycz, ibid., 90,6870 (1968). (6) E. T. Samulski and A. V. Tobolsky, Mol. Cryst., 7 , 433 (1969). (7) T. J. Flautt and K. D. Lawson, in ref 1, p 26. (8) The program used was a modification of a program written by F . Ktipper, Department of Chemistry, Harvard University. Volume 74,Number 1
January 8, 1970
B, M. FUNGI., M. J. GERACE,AND L. S. GERACE
84
0
100
HI
Figure 1. The solvent proton nmr of PBLG solutions of CHBClCH&l at 60 MHz. Spinning rate: A and B, 0 cps, C, 78 cps; D, 64 cps. Spectrum A, peptide mole fraction = 0.136; spectrum B-D, peptide mole fraction = 0.259. The computed transitions are represented by vertical lines.
the solute mole fraction was changed from 0.09 to 0.18. High-Resolution Proton Nuclear Magnetic Resonance for Dichloromethane and i ,%Dichloroethane. The highresolution proton nmr of CH2C12in PBLG solutions has two peaks due to the dipole-dipole interaction of the solvent.a Spectra of CHnCl2 in solutions of polypeptides with varying degrees of polymerization showed that the dipolar splitting increased slightly with the molecular weight of the polymer. For example, the splitting for a solution of 0.10 mole fraction in PBLG was 79, 89, and 95 Hz for peptides having molecular weights 45,000, 99,100, and 240,000, respectively. For CHzC1CH2C1,the proton nmr spectra are more complicated because of more interactions (Figure 1A and B). The spectra were analyzed by using a modified L A O C N ~ programg with an IBM 360 computer. The results showed that the two geminal dipole-dipole coupling constants are the same and the four vicinal coupling constants are equal. Whereas those constants vary with the concentration of the peptide (Figure 2), the indirect spin-spin coupling = 6.0 H,, J,,, unimportant) remain constants (.Izrto unchanged. Spinning of the samples along an axis perpendicular to the magnetic field produced rather complicated spectra (Figure 1C and D). The large side bands were dependent upon the spinning rate and easily recognizable. The computed results (Figure 1D) showed that the central parts of the spectra simply corresponded to systems having all dipole-dipole interactions reduced to of those for the nonspun samples. Deuteron Resonance for Dichloromethane-d2 and i ,2Dickloroethane-de. The deuterium nmr spectra for CD2Clz and CD2ClCD2Clin the polypeptide solutions The Journal of Physical Chemistry
I
I
I
I
.I5
.20
.25
Mole fraction of peptide Figure 2. The proton dipole-dipole interaction ((D) = 2Av/3) as functions of PBLG concentration for nonspun samples. Geminal coupling, 0;vicinal coupling, a.
showed two peaks due to nonzero quadrupole intera ~ t i o n . The ~ variation of the deuteron quadrupole splitting with the concentration of PBLG is plotted in Figure 3. Chlorine-36 Resonance for Dichloromethane and 1,2Dichloroethane. The 35Clmagnetic resonance spectra obtained from PBLG solutions in CH2Clz and CHr ClCHzCl showed three equally separated peaks.6 Two of the spectra are shown in Figure 4. The line width of the central peak was markedly narrower in comparison with the two wings. The intensity ratio of the three peaks estimated by treating the spectra as Lorentzian curves was approximately 3 :4:3 for all cases. The separation was dependent upon the concentration of the peptide. It is related to the quadrupole coupling constant e2q&/hby lo
3 e2g& 1 (3 cosv 4 h I ( 2 I - 1)
A v = -__
- 1)
(1)
for a nucleus with spin I and zero asymmetry parameter. The angle e between the symmetry axis and the magnetic field is not constant in liquid crystal; therefore the angular part in (1)represents the motional averaged value. The data for the *C1 quadrupole splitting are plotted in Figure 5. (9) P.J. Black, K. D. Lawaon, and T. J. Flautt, J . Chem. Phys., 50, 542 (1909). (10) M.H. Cohen and F. Reif, Solid State Phys., 5, 321 (1967).
85
NMRSTUDY OF POLY- BENZYL-L-GLUTAMATE
I
.05 I
.O5
I
1
I
.I5 .20 Mole fraction of peptide
.IO
Figure 3. The deuteron quadrupole splitting (AY) of CDaC12 ( 0 )and CD&lCD2Cl (0)as functions of PBLG concentration.
100
kHz
Figure 4. Chlorine-35 magnetic resonance of PBLG solutions of CHzCla (spectrum A, peptide mole fraction = 0.140) and CHaClCHzCl (spectrum B, peptide mole fraction = 0.086). The resonance frequency was 6.67 MHz. The tilted base lines were due to accumulation of small dc components during the repeated scans.
Other Resonance Study. I4N and deuterium (in the NH p.osition by exchange with CF&OOD-chloroform solution) resonance study of PBLG has been attempted. No detectable signals were obtained even after 100 scans of computer averaging.
Discussion The proton nmr spectrum of PBLG in CDzClCD2Cl consisted of a broad, structureless peak. The lack of structure is likely due to many different inter-
I
.IO .I5 Mole fraction of peptide
I
I
.20
Figure 5. The chlorine-35 quadrupole splitting ( A Y ) of CHzClz ( 0 )and CH2ClCHzCl (0) as functions of PBLG concentration.
actions in the complicated polypeptide molecule which give rise to completely overlapping peaks. The “superLorentzian” shape of the spectra, as already mentioned, is typical for many mesomorphic systems.’ The second moment of the polypeptide in the liquid crystalline state was about 1.6% of that in the solid state, which is also characteristic of those systems.’ These features of the spectra indicate that the motion of the polypeptide in the liquid crystalline solution is not much different from that in a single-component liquid crystal. The spectra of the solvents, namely CH2C12, CH2ClCH2C1, and their deuterated analogs, show dipoledipole and nuclear quadrupole interactions.3-6 For 1,2-dichloroethane, there are six dipole-dipole and six indirect spin-spin interaction constants for the four protons. By comparing the experimental and computed nmr spectra, it was shown that the two geminal dipole-dipole interactions had to be the same, and the four vicinal dipole-dipole interactions were likewise equal. These results imply that the rotation of the C-C bond is fast on the nmr time scale. For the indirect spin-spin interaction, the sign and magnitude for the geminal protons are unimportant because they do not affect the transition frequencies. The four vicinal spin-spin couplings were set to be equal because of fast rotation, although small variations gave similar results for the computed spectra. The reduction of the proton dipole-dipole splitting to 1/4 upon spinning was first observed by SobajimaS for CH2C12 and CH2Br2. From that result he concluded that the molecular axes of all the solvent molecules were parallel to the magnetic field. On the other hand, Panar and Phillips4 argued that the reduction factor of ‘/4 “is precisely that expected if, prior to spinning, the time-averaged solvent dipoledipole vector and Ho are parallel.” However, those results are not necessarily a consequence of the alignment of the proton-proton vector either along or perVolume 74,Number 1
January 8, 1970
B. M. FUNG, M. J. GERACE, AND L. S. GERACE
86 pendicular to the field. It is sufficient that the solvent molecules have a definite averaged orientation with respect to the polypeptide, and the polypeptide molecules slowly align in the magnetic field such that after a long time their molecular axes become parallel to the field. Weber” showed that the factor (3 cos26 - 1) which appears in the formulas for dipolar and quadrupolar splittings (eq 1) in liquid crystals can be decomposed into the product of several terms. For the present case where the macroscopic ordering is perfeci in a sufficiently high magnetic field
(3 COS^ e
- 1) = 1/~(30052 5
- 1) x
(3 cos2S
- 1)(3
COS’ y
- 1)
+ 2B) - 3/2T]pz + 1
(1/2T)p43 dp32/dt = (iA dpda/dt
[i(A - 2B)
+i d c
- 1/T)p3z + i2C
- 3/2T]p43 +
(1/2T)ai
+i d c
(3)
(4)
- wo = sweeping frequency Larmor frequency (6)
- l)/86
