Sept. 20, 1963
COMMUNICATIONS TO THE EDITOR
2869
Imahori and Tanaka.' The idea t h a t the decrease in the extinction coefficient a t this wave length could be simply correlated with the extent of helix formation was called into question by Wada,2 A p p l e q ~ i s t ,and ~ Miyazawa4on the grounds t h a t the extinction coefficient for poly-L-glutamic acid (PGA) was not simply correlated with the optical rotation and in fact seemed to follow more nearly the degree of ionization of the carboxyl groups. It was recognized, however, that the various experiments being compared were carried out under different conditions, particularly regarding concentration of polypeptide, and that the results of the ,CH2CH2N A0 comparison were therefore inconclusive. I n order to obtain more decisive information, we have carried out u c1 C=N optical rotation, ultraviolet absorption, and titration I measurements on identical solutions of PGA. PGA (Pilot Chemicals Lot G-13, intrinsic viscosity = 1.56 dl./g. in 0.2 M NaC1, pH 7) was dissolved with the aid of dilute NaOH to a concentration of 0.50 g.,/dl. (based on carboxyl form) in the presence of 0.2 M XaC1. Titration of the solution with 0.8 AbfHCl k2 I1 was followed with a Beckman Llodel G pH meter. The The infrared and n.m.r. spectra of I and I1 were convolume change due to addition of acid from pH 7 to 4 sistent with the imine formulations. was less than 4y0and was not corrected for in the conCompounds I and II had different ultraviolet absorpcentration. Optical rotations a t 589 mM were meastion spectra: I, A",tH 248 mp (E 25,3(JO); 11, ":A:: ured with a Rudolph spectropolarimeter in a 20-cm. 233 ( 6 27,l(JO),362 mp ( e 4,840). The a- and p-oximes polarimeter tube, giving measured rotations from of 2-amino-5-chlorobenzophenone (111 and IV) whose -0.1' to -1' with a precision of =t(J.Ol'. Absorpconfigurations have been firmly established3 as syn tion spectra were measured on a Cary Model 14 and anti, respectively, to the substituted phenyl had spectrophotometer in a cell of 0.01 cm. path length the following ultraviolet absorption spectra : 111, (Quaracell Products, Inc.). At 190 mp absorb":,A: 246 mp ( e 21,700); IV, "::A: 231 (26,000); ances were in the range of 2 to 3. Large errors a t this 346 mp ( 6 8,500). Compound I, therefore, has a syn wave length due to noise and chloride absorption configuration to the substituted phenyl with I1 having (arising from slight mismatching of sample and solvent an anti arrangement. The absorption bands above cells) precluded accurate work, and we chose instead to 320 mp in I1 and IV are believed to be due to the chromake measurements on the side of the absorption band, mophores arising from conjugation of the o-aminowhere the hypochromic effect is still easily observable. phenyl group with the imino bond which is possible The wave length 200 mM seemed optimum for this puronly when the nitrogen substituent is anti. Steric pose, as the absorbances were in the range of l t o 2 crowding does not permit the rings to be ~ o p l a n a r . ~ and the precision +0.005. Extinction coefficients The assigned configurations are supported by the showed less precision, however, due to variations in absorption spectrum of N-(2-amino-5-chloro-a-phenyl- path length which occur when the cell is dismantled benzylidene) glycine, sodium salt, ::A: 240 mp (E 17,for refilling. Rotation and extinction coefficient measX U ) , which was obtained5 by opening 7-chloro-5urements were therefore of comparable precision. phenyl- 1,3-dihydro-ZH-1,4-benzodiazepin-2-one with The results are shown in Fig. 1. I t is seen that the alkali and which must have its nitrogen substituent syn change in extinction coefficient follows rather closely to the amino substituted phenyl group. the change in rotation, and that neither of these is a Further, the absorption spectrum of benzophenone linear function of the degree of ionization. I t , theremorpholinoethylimine, b.p. 180-183' ( 0 . 3 mm.) (Anal. fore, appears t h a t the discrepancies noted earlier can be Calcd. for C19H,,r\jZO: C, 77.51; H , 7.53; N, 9.52. attributed to differences in conditions of measurement. Found: C, 77.74; H , 7.54; E, 9.23.), in which the Furthermore, it is now evident t h a t Imahori and imino bond can only be in conjugation with an unTanaka were correct in attributing the hypochromic substituted phenyl group, exhibited no maximum effect to interactions in the helical conformation, since above 300 mp. This is true also for benzophenone the change in rotation has been shown5 to result from oxime. helix formation a t low pH. This conclusion is of ( 9 ) I. H Sternbach. S. Kaiser. and E. R e e d e r , J . A m . Chem Soc., 82, 475 particular interest in view of the success of Tinoco, ( 1 9 6 0 ) , T . S . Sulkowski and S J Childress, J Oyg Chem., 26, 4424 (1962) et uL.,~in predicting the magnitude of the hypochromic 1 1 ) P A . S Smith and E P. Antoniades, I'elrohrdvon, 9 , 210 (1960). effect from dipole-dipole interactions in the helix. I t l a ) S C Bell, T S Sulkowski, C Gochman and S J Childress. J. Org. Chrm , 2 6 , 562 (1962). does not follow from these observations t h a t either rotation or absorption is linearly related to the helix conRESEARCH A N D DEVELOPMENT DIVISION STAXLEY C. BELL WYETHLABORATORIES, INC. GEORGE L. CONKLIK tent, but merely t h a t both are similar functions of helix R A D N O RPEXNSVLVANIA , SCOTT J . CHILDRESS content. The linear relationship generally assumed is RECEIVED J U L Y 31, 1963 neither confirmed nor contradicted. benzophenone and aminoethylmorpholine in xylene under reflux in the presence of zinc chloride. Following solvent removal, fractional crystallization of the residue from alcohol and hexane gave a less soluble a-form I, m.p. 140-142' (Anal. Calcd. for C19H22CINsO: C, 66.36; H , 6.45; K , 12.22. Found: C, G(i.13; H , 6.29; N , 12.09.) and a more soluble p-form 11, m.p. 112-114' (Found: C, 66.17; H , 6.50; N, 12.43). Both I and I1 upon treatment with aqueous acid returned the starting materials. Heating I1 without a solvent a t 140-150' for 10 min. afforded I.
a"'
Comparison of Hypochromic Effect and Optical Rotation in Poly-L-glutamic Acid
Sir: L e wish to report some data which resolve a controversy over the relationship between the helical conformation of polypeptides and the hypochromic effect in the amide absorption band a t 190 mp discovered by
(1) K Imahori and J T a n a k a , J M o l B i o i , 1, 3.59 (l!X9) (2) A Wada, in " P d y a m i n o Acids, Polypeptides, and Proteins," 11. A . Stahmann, Ed , University oi lt'iscmsin Press, h l a d i s i m Wisc
