2268
R.H. KARLSON, K . S. NORLAND, G , D. FASMAN A N D E.It. Br,onr
[COKTRIRUTION P R O X
THE IIARORATORY O F THE
CHILDREN'S CANCER RESEARCH I'Ol*NDATION SCIIOOL, B O S T O S , MASS.]
A K D THE
17nL S:! IIARVARD hIEDIC.11.
The Helical Sense of Poly-P-benzyl-L-aspartate. Synthesis and Rotatory Dispersion of Copolymers of p-Benzyl-L and D-Aspartate with r-Benzyl-L-glutamatel HI' I 0
20
40
60
80
MOLE % B E N Z Y L ASPARTATE.
I
4
100
,
,
I
I
,
,
0
20
40
60
80
IO0
MOLE ‘tb BENZYL ASPARTATE.
Fig. 5.-The bo values for the wave length range 365 t o 578 mp (from eq. 1 assuming A0 = 212 mp) of copolymers of 0benzyl-L-aspartate:?-benzyl-L-glutamate, 0-0-0 , and copolymers of &benzyl- D-aspartate :r-benzy l-l-glutain dichloroacetic acid solutions. mate, A-A-A,
conformational stability of the random chain for
Fig. 4.-The bo values for the wave length range 365 to the me~opolypeptide.~4For PBG in CHC13:DCA 575 mp (from eq. 1 assuming A0 = 212 mfi) of copolymers of the stability of the helix relative to the random and P-benzyl-L-aspartate:?-benzyl-L-glutamate, 0-0-0, chain was shown to be inversely proportional to of 8-benzyl-D-aspartate:?-benzyl-L-gluta- the fraction of D-isomer down to L / ( D copolymers L) = mate, A-A-A, in chloroform solutions. While all these studies were performed with
+
0.5.22 form (Fig. 2 ) the optical rotation, in the series D,L-isomers of the same amino acid, the qualitative L-B.4 :L-BG, is non-linear and markedly depend- features would be expected to remain in a copolymer ent, particularly above ?’()’% L-BA, upon the mole of two different amino acids, when the respective yo benzyl aspartate in the copolymer. On the pure polypeptides are helical. Thus i t might be argued that if L-PBA and Lother hand, in the series D-BA:L-BG, the optical rotation varies in a regular manner with the mole PBG are helices of the opposite sense of twist, the yo benzyl aspartate. In dichloroacetic acid solu- optical rotation and bo of their copolymers should tion both the D and L series of benzyl aspartate: change markedly over a relatively narrow range benzyl-L-glutamate copolymers show a linear of copolymer composition, representing a region of variation of [a1646 with mole yo benzyl aspartate transition from one helix sense to the other. Furthermore, since i t has been demonstrated that the (Fig. 3). When the optical rotatory dispersion of the co- L-benzyl aspartate helix is less stable than that polymers in chloroform solution is fitted to eq. 1, of L-benzyl glutamate the transition should occur the coefficient of the second term, bo, is found to a t less than 50% L-BG. On the other hand the vary in the manner shown by the optical rotation, optical rotation and bo of D-BA:L-BG copolymer ;.e., marked dependence upon mole % benzyl as- in the helical conformation should show a regular, partate in the L-BA:L-BG series above 70 mole almost linear, dependence on composition which yo and a relatively linear dependence in the D- would be evidence that no change of helix sense BA:L-BG series (Fig. 4). Similarly, bos deter- occurs in this series. This is indeed the case as mined in dichloroacetic acid solution parallel the seen in Fig. 2 and 4, and therefore we conclude that behavior of the optical rotation in this solvent; there is a change of helical sense in the series the dependence of bo upon mole yobenzyl aspartate L-BA:L-BG while no change in sense of twist occurs in the D-BA:L-BG series. was approximately linear for both series (Fig. 5). I t is noted that there is a departure from linearity The substitution of D-isomers into a helix composed of L-residues has been used to estimate the in the optical properties in chloroform solution of contribution of a right-handed helix to the optical the D-BA:L-BG series which may be correlated rotation of polypeptide^.^^^^^^^^ As a result of these with the varying viscosities of the copolymers investigations i t was concluded that a single sense of shown in Table 11. In addition, the average enhelix was maintained from L / ( D L ) = 1.O (where vironment of a D-BA or L-BG residue in a copolyand L and D refer to the fraction of L and D isomer mer would differ from the environment in the pure respectively) to L / ( D L ) E 0.9-0.6 depending on polypeptide, influencing the mean residue rotation the particular poly-a-amino acid being studied. in a manner analogous to the solvent effects often The fact that the optical rotation, a criterion of found with optically active compounds. In DCA solution, Fig. 3 and 5, both copolymer helix of a single sense was not linear over the complete range of L / ( D L ) (from 1.0 + 0.5) was as- series exhibit the relatively linear dependence of cribed to (a) the increasing thermodynamic stabil- optical rotation and bo anticipated for random chain ity of a mixture of right and left-handed helices as conformations. One further point should be the fraction of D-isomer increased or (b) the greater noted about the bo values found in strong hydrogen
+
+
+
sumably right-handed helix.26?’ Since the anorn;Jy oljscrvetl with T,-PRA is opposite in s i p , it lends support to the argument that the sense of helix in L-PBA is the opposite of that in L-PBG. The displacement of the anomaly to the ultraviolet might be accountcd for by the expected levorotation of a left-liaiided helix above ca. 285 in,u.25 Qualitatively the change to positive rotation would then be a result of the dextrorotation of the left-handed a-helix below C U . 253 m u .
+12000 -
t1oooo-
B\
w
+eo03 -
iI
t4000-
I
re1
i \
+POOO-
I
160,--
‘7
\
0
eo -
0
0 -6000
‘ 0
40-
I
240
260
280
300
320
\
340
/
/
x
A (mp)
Fig. 6.-The ultraviolet rotatory dispersions of poly-Pand poly-y-benzyl-L-glutabenzyl-L-aspartate, A-A-A, mate in chloroform solutions. The specific rotations, [ a ] ~ are plotted as a function of wave length in mp.
bonding solvents. I n DCA solution bo -250 for L-PBA, l 9 in trifluoroacetic acid solution i t differs only slightly (bo = - 270, [a]646 -5”). Since it is known that solvents of this type break down more stable polypeptide helices, e.g., L-PBG, there is no doubt that the L-PBX helix is also transformed to a random chain in these solvents. Therefore the bo values observed with PBR in these strong hydrogen bonding solvents must be characteristic of the residue and thus the bo observed for L-PBA in weakly hydrogen bonding solvents should reflect both helical and residue contributions. Ultraviolet Rotatory Dispersions.-The optical rotatory dispersions of L-PRG and L-PBA from ca. 240 to ca. 340 mp are shown in Fig. 6. The dispersion of L-PBA is clearly anomalous.25 In contrast to L-PBA, the dispersion of L-PBG is not anomalous in the region 240 to 3GO mp but is comple~.~~ Since i t is known that the optical rotatory dispersion of L-PBG is anomalous in the visible and fits eq. 1, i t was of interest to determine whether this relation held in the ultraviolet and in particular whether the anomalous dispersion found for LPBA could be described by the Moffitt equation. It was found that the data for L-PBG could be fit to eq. 1 from 240 to GOO mp, bo = -660, Fig. 7. However the optical rotatory dispersion of L-PBA only fit eq. 1 down to 265 mp (bo = +740) and a t shorter wavelengths deviated markedly, particularly below 250 mp. (Fig. 7). Returning now to a consideration of the data in Fig. 6,it should be emphasized that both L-PBA and L-PBG show anomalous rotatory dispersion; for L-PBd in the ultraviolet, for L-PBG in the visible region. The observed anomaly for L-PBG has been correlated with its existence as a pre-
/
\“
“/.v’v
-4Ob //’
-8C.\
- I00 -
365
0
260 rr&
T+l
.
Lli-1
i
:2
~
L--
30
~
I
1.
4C
,Az->,:)-
.
5 :
x
1
6C
~
1
73
~~~
~
2
2L
\. 30
c6.
Fig. 7.-Moffitt plot (from eq. 1 using XO = 212 m p ) of the optical rotatory dispersion of poly-P-benzyl-L-aspartate, A-A-A, and poly-r-benzyl-L-glut3mate, 0-0-0, in chloroform solutions over the wave length range 237 to 578 m p .
However the dextrorotations measured for LPBA a t wave lengths below 245 mp are in excess of those anticipated for a left-handed helix.29 This excess dextrorotation may be a consequence of the onset of a positive Cotton effect arising from an interaction between the asymmetric a-C and the
No
-C-0- chroniophore which absorbs around 220-230 nip. The existence of a Cotton effect would imply
Yo
that the free rotation of the -C-0-R
group is re-
( 2 R ) D . D .Fitts and J. G. Kirkwood, Proc. N a f l . Acad. Sci. U . s.,43, 1046 (1957). (27) P . Doty and J. T. Yang, THISJOURNAL, 78, 498 (1956). (28) Using the values no = +680, bo = 500, XO = 212 mfi found by Doty and Lundbergli for a presumably right-handed a-helical optical rotatory dispersion, it is found that the contribution of a right-handed a-helix is positive above ca. 285 m p and becomes highly negative below. A left-handed a-helix would be expected to mirror this behavior. (29) From the constants given above (ref. 28) the optical rotation of a right-handed a-helix a t 240 m p is estimated t o be [m’]be1ix34a -3700O. Then the optical rotation of a left-handed a-helix would be ca. +3700°, much less than the observed rotation for L-PBA at this wave length ([m’]L-PBAno u +17.000°).
-
May 5 , 19130
SYNTHESIS OF 0-BENZYL-L AND D-ASPARTATE
~ t r i c t e d . ~On ~ the other hand a positive Cotton effect around 225 mp is inconsistent with the excess levorotation observed in the visible for helical L-PBA unless i t is assumed that there is a net large levorotatory contribution from absorption bands lying a t shorter wave lengths. However, it should be noted a t this point that all the optical effects observed for L-PBA cannot be explained simply by the presence of oriented chromophores in the side chain but almost necessarily involve a change of helical sense compared to L-PBG. Finally, measurements have been made in the region 240 to 360 mp on 9 : 1 copolymers of L-BA : LRG and D-BA:L-BG. The results are shown in Fig. 8 plotted on a log scale. The anomaly observed and noted above for L-PBA is completely lost upon the incorporation of 10 mole % L-BG (Fig. 8). In contrast to this observation the incorporation of 10 mole % L-BG in D-PBA does not result in a loss of the anomaly observed with D-PBA. Thus the rotatory dispersions shown in Fig. 8 indicate that a structural change is involved when 10 mole % L-BG is incorporated in L-PBA, whereas no such change is found upon incorporation of L-BG in D-PBA. These data offer additional support for the conclusions reached above concerning the opposite sense of helix in L-PBA and L-PBG on the basis of optical rotatory dispersion measurements a t wavelengths longer than 360 mp. Conclusions From the interpretation of the rotatory dispersions of the fifteen polymers and copolymers reported here i t is concluded that the sense of helix in poly-P-benzyl-L-aspartate is opposite to that observed in poly-y-benzyl-L-glutamate. Consideration has been given to possible effects of rotatory
I !
240
2275
/
260
280
X
300
IN
320
340
360
rn4.
Fig. 8.-Ultraviolet rotatory dispersions in chloroform poly-0-benzyl-L-aspartate ; B, solutions: A , A-A-A, O-O--O, 9 : 1 copolymer of P-benzyl-L-aspartate:?-benzylL-glutamate; C, A-A-A, poly-P-benzyl-D-aspartate; D, 0-0-0, 9: 1 copolymer of 8-benzyl-D-aspartate:?-benzylL-glutamate.
//O
aspartate are due in part to the -C-0- group in the p position. Optical rotatory dispersion measurements in the ultraviolet (240 to 360 mp) reveal effects not seen in previous rotatory work on poly//O contributions due to the -C-0- group and to the peptides carried out in the more easily accessible benzyl chromophore. It is suggested that the region between 360 and 600 mp. optical rotations observed with poly-P-benzyl-LAcknowledgment.-We are pleased to acknowledge the support of this work by the Office of the (30) We have consiclrred t h e possibility t h a t the benzyl groups in Surgeon General, Department of the Army and by the side chains may be oriented. Howrver since t h e absorption of this U. S. Public Health Service Grant #A2558. We chromophore did not allow measurement around its absorption maxi= 258 mw) and since it would be expected t h a t this groupmum (A, also acknowledge the technical assistance of Carole ing would make only a weak contribution, if any, to the optical rotaLindblow and the stimulation provided by dislory dispersion ( W . Kauzmann, “Quantum Chemistry,” Academic cussions with Drs. Carolyn Cohen, Tatsuo MiyaPress, Inc., New York, 1957, p. 718) we can only conclude t h a t t h e effect of this chromophore is small. zawa and Lubert Stryer.
