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Structure and deformation ofa crosslinked poly(α-aminoacid)Tsutomu Sugie a & Anne Hiltner aa Department of Macromolecular Science , CaseWestern Reserve University , Cleveland, Ohio,44106Published online: 19 Aug 2006.

To cite this article: Tsutomu Sugie & Anne Hiltner (1980) Structure anddeformation of a crosslinked poly(α-amino acid), Journal of MacromolecularScience, Part B: Physics, 17:4, 769-785, DOI: 10.1080/00222348008212836

To link to this article: http://dx.doi.org/10.1080/00222348008212836

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J. MACROMOL. SC1.-PHYS. B17(4), 769-785 (1980)

Structure and Deformation of a Crosslinked Poly(a-amino Acid)

TSUTOMU SUGIE and ANNE HILTNER Department of Macromolecular Science Case Western Reserve University Cleveland, Ohio 44106

A B S T R A C T

The relationship between the chain conformation and the mechan- ical response is a n important consideration in understanding the solid state properties of block and random copolymers of a- amino acids. In order to eliminate intermolecular effects, crosslinked poly -2-hydroxy ethy l- L -glutam ine was prepared, and the mechanical properties of swollen f i lms were determined. In water, the f i lms take the random coil conformation and the mechanical behavior is elastomeric. In methanol, the chains are a-helical and plastic deformation is observed. Two molec- ular processes accompany the deformation of the a-helical net- work. At low strains, orientation of the a-helical chain seg- ments in the draw direction is observed. At higher strains, some of the a-helical chains are converted to the @-sheet con- formation. This latter process probably accounts for the ob- served nonrecoverable deformation. The behavior in mixed sol-

769

Copyright 0 1980 by Marcel Dekker, Inc.

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770 SUGIE AND HILTNER

vents showed that the nonhelical chain segments provided suf- ficient flexibility to the chain that the properties of f i lms with up to 75% helicity were elastomeric.

I N T R O D U C T I O N

It is well known that copolymer properties can be profoundly influ- enced by the presence of blocks, particularly if these are long and numerous enough to segregate into separate phases. Phase separa- tion of block copolymers in which both components are amorphous has been the subject of extensive experimental and theoretical re- search. This work has been reviewed in several monographs [I, 21. The presence of a crystallizable block, as is the case with the poly- urethane elastomers, has a profound effect on the tendency toward domain formation. Because of the strong intermolecular interactions, even very short sequences demonstrate their individual identity and function as blocks. Polymers of a-amino ac ids constitute another c l a s s of polymers which exhibit ordering phenomena. These poly- m e r s exhibit conformational stability, and the chain conformation of many poly(a-amino acids) has been well-characterized for both the solution and solid state. In the latter case, the possibility exists for further organization of the molecules in a crystalline a r r a y .

Structural studies of random copolymers of a-amino ac ids have shown that copolymers of two amino ac ids which in homopolymers assume the same conformation (e.g. a-helix) cocrystallize in iso- morphic structures, although the side chains may vary substantially in size and composition [3]. On the other hand, copolymers where one monomer is a-helix and the other P-sheet directing are found to be amorphous although local regions of conformational order exist [4].

these polymers cannot be stained by conventional methods for elec- tron microscopic examination, the solid-state s t ructure has been characterized by dynamic mechanical and wide-angle x-ray diffraction methods [4-6]. When both blocks are a-helical, phase-separation was observed in f i lms cast from a preferential solvent, however mix- ing of the two components was observed when a nonpreferential solvent was used. Conversely, when the blocks were a-helical and p-sheet, respectively, phase separation was always observed. Conformation should thus be considered a primary factor in determining the solid- state s t ructure of a-amino acid polymers.

Block copolymers of a-amino ac ids have been prepared. Although

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CROSSLINKED POLY (WAMMO ACID) 771

I t is apparent that conformation will a l s o affect the solid-state properties of a-amino acid polymers but this relationship has not been examined systematically. Recently we have undertaken a n investigation of this relationship. To minimize intermolecular effects, a cross- linked polymer was prepared and conformationai changes induced by altering the swelling medium.

The w -hy d r ox ya lky 1 - L -g luta mine polymer s are non ion iza ble , water-soluble poly(a-amino acids) whose conformation depends on various factors including length of the side chain, molecular weight, temperature and solvent. Lotan and colleagues [7] have shown f rom ORD and CD studies that poly-2-hydroxyethyl-L-glutamine (PHEG) takes the random coil conformation in water and the a-helical con- formation in methanol. In order to examine the relationships be- tween conformation and condensed state properties, crosslinked PHEG was prepared. The solvent-induced helix-coil transition of the crosslinked PHEG in aqueous mixtures of methanol was examined by swelling measurements and CD studies. The shape of the stress- strain curve w a s found to depend on the conformation. The possibility that stress may produce conformational changes during deformation was examined by polarized infrared spectroscopy.

E X P E R I M E N T A L

P o l y m e r s

Poly(y-benzyl-L-glutamate) (PBLG) was prepared by polymeriza- tion of the N-carboxyanhydride of y-benzyl L-glutamate in a methylene chloride-benzene solution with triethylamine as the initiator. The molecular weight of the PBLG was about 250,000 by viscosity measurements.

F i lms of PBLG were converted to the crosslinked poly-2-hydroxy- ethyl-L-glutamine (PHEG) in a single step. F i lms of PBLG were cast from benzene because this solvent produces f i lms with low crystallinity [6]. However PHEG fi lms prepared from highly crystalline PBLG fi lms cast from chloroform showed no measureable differences in physical properties. To prepare the crosslinked PHEG film, the PBLG film was immersed in a solution of ethanolamine and 1,12-dodecadiamine (cross- linker) at 55°C for 4 days. The progress of the exchange reaction was followed by W spectroscopy and by the change in dry mass and was

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772 SUGIE AND HILTNER

complete in 4 days. The f i lms were washed with ethanol and methanol to remove excess ethanolamine and diamine, and then equilibrated in water, methanol, o r water-methanol mixtures.

The crosslink density was varied by changing the ratio of c ros s - linker to ethanolamine. Typically, the crosslinker concentration was in the range of 1-4 mole ?(I. The uniformity of crosslinking was tested by treating f i lms which varied in thickness up t o 250 pm. No effect of fi lm thickness on the swelling in water was observed.

with the crosslinker omitted. Linear polymer (DP = 150) was prepared by the same procedure

C i r c u l a r D i c h r o i s m S p e c t r o s c o p y ( C D )

Optical measurements were recorded at 20°C in the 200-260 nm wavelength range by using a JASCO 5-20 recording spectrometer. Fo r measurements on swollen f i lms in aqueous mixtures of methanol, the f i lms were placed between two quartz cel ls in order to keep the environment constant during the measurement.

S w e l l i n e R a t i o

The swelling was determined by equilibrating specimens in water, methanol o r a water-methanol mixture at room temperature. When a constant weight was achieved, the specimen was removed, blotted t o remove surface water and weighed. The film w a s then dried in a vacuum oven. The swelling rat io q is defined as the ratio of the wet to dried weight of the crosslinked film.

T e n s i l e S t r e s s - S t r a i n M e a s u r e m e n t s

The s t ress-s t ra in curve was determined with the f i lms immersed in methanol, water, o r methanol-water mixtures at room temperature. An Instron Tester was used with a strain rate of 25%/min. The speci- men geometry was ASTM Micro-tensile with a width of 0.5 c m and a n effective length of 2.0 cm.

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CROSSLINKED POLY( (Y-AMINO ACID) 7 73

I n f r a r e d S p e c t r o s c o p y

The chain conformation and orientation were determined by polar- ized infrared measurements. The spectra were obtained with a Digi- lab Fourier transform spectrometer with a Perkin-Elmer wire grid polarizer.

R E S U L T S A N D D I S C U S S I O N

C i r c u l a r D i c h r o i s m

The chain conformation in the swollen state w a s characterized by circular dichroism spectroscopy. Figures 1 and 2 show the CD spec- tra of linear PHEG and crosslinked PHEG f i lms in methanol-water mixtures. Both the linear and crosslinked PHEG are essentially a- helical in methanol and random coil in water. In methanol, the crosslinked fi lm has a strong negative peak at 223 nm, and in water has a weak negative peak a t 235 nm and positive peak at about 216 nm. The a-helical conformation is usually characterized by two negative peaks: the n - II* transition is a broad peak centered around 223 nm and the 71 - T * transition appea r s at about 209 nm. The 'II - T* tran- sition appears as a shoulder on the broad peak at 223 nm for l inear PHEG and could not be resolved for the crosslinked PHEG films.

Although quantitative interpretation of solid-state CD spectra is not possible, because of uncertainties in precise film thickness, frac- tional changes in conformation were obtained by comparing measure- ments on a single film. The helical content was estimated from the depth of the negative peak [8], The helical content of samples relative to that in methanol is shown in Fig. 3. The S-shaped curve shows the helix-coil transition at a water-methanol ra t io of about 40-60, es- sentially the same composition at which the conformational transition is observed fo r solutions of the linear polymer.

S w e l l i n g

The swelling ratios of crosslinked samples in methanol-water mix- t u r e s are shown in Fig. 4. The swelling rat io dec reases as the helicity

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774 SUGIE AND HILTNER

I PHEG SOLUTION

200 220 240 260 W A V E L E N G T H ( n m )

FIG, 1. Circular dichroism spectra for linear PHEG in methanol- water mixtures.

increases. However, the swelling curve does not have the typical S- shape of the CD curve, although the midpoint in the swelling change occurs at a water-methanol composition close to that of the solvent induced helix-coil transition. In the random coil conformation, the crosslinked f i lms in water can be expected to exhibit behavior s im- ilar to that of a swollen elastomer. Figure 5 shows the decrease in swelling ratio in water with increasing percent crosslinker in the feed. The slope of the log-log plot has the value of -3/5. When the swelling rat io is large, q is given {9] by Eq. (1):

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CROSSLINKED POLY(a-AMINO ACID) 7 75

200 220 240 260

W A V E L E N G T H (nm)

FIG. 2. Circular dichroism spectra for the crosslinked PHEG film in methanol-water mixtures.

where M is molecular weight of the repeat unit, n is the number of 0 C

repeat units between crosslinks, V is molar volume of solvent, and x is the interaction parameter. Since the observed slope in Fig. 5 is that predicted by Eq. (I), it appears that the effective crosslink den-

sity (n -') is proportional to the percent crosslinker in the feed. Ab-

solute values of n can be obtained by combining swelling measure-

ments with a n independent determination of the modulus.

C

C

S t r e s s - S t r a i n

The deformational behavior of the a-helix and random coil confor- mation was examined by immersing specimens in methanol and water

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776 SUGIE AND HILTNER

0 50 100 M e O H ( v o l % ) i n H z O

FIG. 3. Relative percent helicity for l inear and crosslinked speci- mens in methanol-water mixtures crosslinked PHEG film: (0) linear

3 2 PHEG solution; (0) PHEG solution, [el =27 X 10 deg-cm /dmole in methanol.

respectively. Tests were also made in methanol-water mixtures. Figure 6 shows the stress-strain curve of the water-swollen samples. Here the crosslinked fi lms show typical elastomeric behavior: The stress-strain curve is independent of strain rate (5%/min to 250%/ min), elongation up to 75% i s almost completely recoverable, and no stress-relaxation is observed at constant elongation.

The average number of repeat units between crosslinks, n was

calculated from the combined swelling and stress-strain date [9]: C'

where 7 is the retractive force referred to the swollen cross-sec-

tional a rea and a i s the elongation. a

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CROSSLINKED POLY (a-AMINO ACID) 777

2 0 0 I- 4 t

* 10 f -I -I W

f 5

I

'

I 0 50 100

M e O H ( v o l % ) i n H z 0

FIG. 4. Dependence of swelling ratio of the crosslinked PHEG on percent crosslinker in the feed: (0) 1.22%; ((3) 2.44%; (0) 3.66%.

slope - q / s ,\ u

I 2 5

X C R O S S L I N K E R

FIG. 5. Log-log plot of swelling ratio of the crosslinked PHEG film versus percent crosslinker in the feed. For original PBLG film cast from various solvents: (0) chloroform; (0) dioxane; (0) benzene.

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778 SUGIE AND HILTNER

P H E G I N WATER 3.6 % C R O S S L I N K E R

N I

% 2

150

P E R C E N T S T R A I N

FIG. 6. Stress-strain curves of crosslinked PHEG fi lms in water. F i lms were treated with various percent crosslinker as indicated in the figure.

Calculated values of n and x from Eqs. (1) and (2) are shown in C

Table 1. Calculated n values are about a factor of four larger than

the value predicted by the feed composition. Several explanations are possible: the reactivity of the diamine and hydroxyamine may differ or partitioning in the heterogeneous system may change the effective concentration of reactants in the film. Crosslinker with only one amine reacted and intramolecular crosslinker a l s o will contribute to the low apparent crosslink density. A crit ical analysis of the var- ious factors must await development of chemical methods for c ros s - linker determination.

The methanol-swollen f i lms exhibit a typical sigmoidal stress- strain curve (Fig. 7). The f i lms deform uniformly to elongations greater than 100% and permanent set is observed at the high elonga- tions. The s t ress-s t ra in curve is strain-rate-dependent, with higher

C

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CROSSLINKED POLY (@-AMINO ACID) 779

TABLE 1

Evaluation of 77 f rom Linear Deformation and Swelling c'

1'22

1.83

2.44

3.66

~

0.80 13.5 207

1.08 10.5 167

1.30 8.5 149

1.85 6.5 114

0.46

0.47

0.48

0.48

2oos y 0 1 5 0 1

c

5.6 %

C R O S S LI N KER

2.4 96 PHEG I N METHANOL

P E R C E N T S T R A I N

FIG. 7. Stress-s t ra in curves of crosslinked PHEG fi lms in meth- anol. Fi lms were treated with various percent crosslinker as indi- cated in the figure.

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780 SUGIE AND HILTNER

s t resses observed at higher strain rates, and s t r e s s relaxation is ob- served at constant stain. This behavior is characteristic of plastic deformation, and when compared with the elastic deformation of the water-swollen films, suggests that different deformation mechanisms a r e operative for the a-helix and random coil conformations.

7 I

P H E D , Corrected fo r t h e

D r y C r o s s - s e c t i o n a l Area

% CROSSLINKER r /9 1

0 50 100 M c O H ( v o l % 1 In H e 0

FIG. 8. Dependence of modulus, referred to the dry cross-sec- tional area, on percent methanol in water: (0) 3.6% crosslinker; (a) 2.4% crosslinker; (0) 1.2% crosslinker.

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CROSSLINKED POLY(C~-AMINO ACID) 781

The stress-strain curve of specimens swollen in methanol-water mixtures is intermediate between methanol and water. Figure 8 shows the modulus relative to the dry cross-sectional area in meth-

6 anol-water mixtures. The modulus in water is about 8 X 10 dynes/

cm and increases gradually with increasing methanol concentration. At about 70% methanol there is a sharp increase in E

7 2 X 10 dynes/cm in pure methanol. The sharp increase in modulus does not coincide with the coil-to-helix transition in Fig. 3. The 70% methanol composition corresponds to a helical content of about 75%.

Figure 9 shows the amount of permanent set in crosslinked speci- mens drawn to various strains in water-methanol mixtures. The un- recoverable strain is negligible in mixtures up to 70% methanol, then increases sharply with increasing methanol content. The Same patter1 is shown in the rate of stress relaxation at 50% and 75% strain

2

to about 5 D

(Fig. 10).

0 5 0 100

MeOH ( v o l % ) i n Y 2 0

FIG. 9. Dependence of the unrecoverable strain, after (0) 50% or (0) 75% strain, on percent methanol in water.

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782 SUGIE AND HILTNER

0 50 100 M e O H ( ~ 0 1 % ) i n H 2 0

FIG. 10. Dependence of stress relaxation after (0) 90 or (0) 600 sec on percent methanol in water.

The data presented in Figs. 8-10 show that the mechanical response resembles that of a random coil even though the helical content may be as high as 75%. This suggests that the nonhelical regions provide suf- ficient flexibility to the chain that the mechanical response is elasto- meric. The curves in Figs. 8-10, with the abrupt change in proper- ties, parallel the change in intrinsic viscosity with helicity reported in isopropanol-water mixtures [lo]. The relationship between in- tr insic viscosity and molecular dimensions [lo-121 further supports the hypothesis that the interrupted helix has effectively the flexibility of the random coil.

I n f r a r e d S p e c t r o s c o p y

The molecular deformation processes which accompany the plastic deformation of the a-helical conformation were characterized by

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CROSSLINKED POLY( a-AMINO ACID) 7 83

w c) z Q m a

m a

0 v)

1658(d und e f or m e d P H E G

1550 (d) w V z a m a

m a

0 ul

100% stretch

I 7 5 0 1650 1550 1450 1750 1650 1550 1450

w A V E N U M B ER ( c m-9 W A v E N u M B E R (c m-')

FIG. 11. Polarized infrared spectra of the amide I and amide I1 region of the methanol swollen f i lms for (left) undeformed film and (right) stretched film. (-) E vector parallel to direction of stretch- ing: (...) E vector perpendicular to direction of stretching.

polarized infrared spectroscopy (Fig. 11). The undrawn film shows

an amide I band at 1650 c m - l and amide I1 band at 1550 cm-l . These frequencies a r e characterist ic of the @-helical conformation. Upon

deformation, new bands a r e observed at 1630 and 1520 cm" with polarized light perpendicular and parallel to the draw direction, re- spectively. The amide I band is associated with N-H stretching, the transition moment is largest in the direction parallel to the hydrogen- bond direction. Therefore, for the a-helical conformation, the amide I band is polarized parallel to the chain direction, while for the p-sheet conformation polarization is perpendicular to the chain direction. The amide I1 band is associated with N-H deformation and as in nylon 66 [13], its transition moment is normal to that of the N-H stretching mode.

The presence of a weak band at 1695 cm indicative that the P-sheet structure is antiparallel.

-1 with parallel polarization is

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784 SUGIE AND HILTNER

-i 1.3 . U

U 2

0 1.2 -

2 " 1.1

c

. -

1 0 5 0 100 150

P E R C E N T S T R A l N

FIG. 12. Dichroic ratio of the amide I1 band at 1550 cm-l -1 (a-helix, I), at 1520 c m

strain. (@-sheet, 11) as a function of percent

Figure 12 shows the dichroic ratio (A /Al) of the amide I1 band at II 1550 and 1520 cm-l as a function of strain. The dichroic ratio at 1550

-1 cm

then remains almost constant. The band at 1520 cm . (&sheet, 11) appears for the samples deformed 70% o r more and its dichroic ratio increases with stretching. I t appears that two molecular processes accompany the deformation of the a-helicaI network. At low strains, orientation of the a-helical chain segment in the draw direction is ob- served. At higher strains, some of the a-helical chains a r e converted to the @-sheet conformation. The @-sheet chains a r e well-oriented in the draw direction. This latter process probably accounts for the non- recoverable deformation.

(a-helix, I) decreases with stretching up to about 70% strain, -1

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CROSSLINKED POLY(CY-AMINO ACID) 785

A C K N O W L E D G M E N T

This research was generously supported by the National Science Foundation, Grant DMR-7820341, Polymers Program. The authors are indebted to J. M. Anderson for h i s invaluable assistance in the synthesis aspects of this work.

R E F E R E N C E S

JJ A. Manson and L. H. Sperling, Polymer Blends and Compos- - ites, Plenum Press, New York, 1976. ~

D. C. Allport and W. H. Janes, Eds., Block Copolymers, John Wiley, New York, 1973. A. Hiltner, J. M. Anderson, and E. Borkowski, Macromolecules, 5. 446 (19721. - F. Uralil, T. Hayashi, J. M. Anderson, and A. Hiltner, Polym. Eng. Sci., 17, 515 (1977). T. Hayashi, A. G . Walton, and J. M. Anderson, Macromolecules, - 10, 346 (1977). - T. Hayashi, J. M. Anderson, and P. A. Hiltner, Macromolecules, - 10, 352 (1977). N Lotan, A. Yaron, and A. Berger , Biopolymers, 4, 365 (1965). N. Greenfield and G. D. Fasman, Biochemistry, 8, 4108 (1969). P. J. Flory, Principles of Polymer Chemistry, Cornell Univ. Press, Ithaca, N. Y., 1953. K. Okita, A. Teramoto, and H. Fujita, Biopolymers, - 9, 717 (1970). K. Nagoi, J. Chem. Phys., 34, 887 (1961). W. G. Miller and P. J. F l o r E J. Mol. Biol., 15, 298 (1966). C. W. Bunn and E. V. Garner, Proc. Roy. Socr(London), A189 39 (1947).

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