1642
CATHERINE Y. S. CHENAND CHARLES A. SWENSON
The Ultraviolet Absorption Spectra of
Lactamsla
by Catherine Y. S. Chenlb and Charles A. Swensonlo Department of Biochemistry, University of Iowa, Iowa City, Iowa 66840 (Received August 83,1068)
The ultraviolet absorption spectra of a series of lactams with ring sizes of 5, 6, 7, 8, 9, 10, 11, and 13 have been studied in both water and cyclohexane. Solvent shifts allowed a partial separation of the n-n* band from the T-T* band in cyclohexane and enabled an estimate of its position and intensity. The effects of the configuration of the peptide group as well as the conformation of the molecule on the absorption maxima and the intensities of the T-T* and n-n* bands are discussed. Introduction Recently, Litman and Schellman2 studied the ultraviolet absorption and the optical properties of L-3aminopyrrolid-Zone (3-amino derivative of butyrolactam) in various solvents. Their most interesting observation is that this substituted lactam shows a Cotton effect in nonpolar solvents at 231 mp, which is similar to helical peptides. The study by Balasubramanian and Wetlaufera on the optical rotatory properties of various diketopiperazines has also revealed a Cotton effect around 230 mp in several solvents. These two studies strongly suggest that this n-x* Cotton effect of the peptide chromophore is not unique to the or-helical structure. Theoretical studies on the optical rotatory dispersion of peptides and proteins and the analysis of rotatory dispersion curves are greatly aided by knowledge of the optical properties, such as the absorption maxima, the band widths, the dipole strengths, and the oscillator strengths of model compounds. These properties are especially needed for the weak but important n-n* transitions, as few have been studied. The n-r* transition is usually hidden beneath the very intense T-T* transition; however, it is possible to locate it in nonpolar solvents. Solvent shifts for these transitions in simple amide^,^'^-^ peptidesJ2and polypeptides6 can be interpreted in terms of the general studies by I The Journal of Physical Chemietry
9. The n = 9 lactam exists in both the cis and trans configurations. Spectral studies on lactams in a homologous series will permit a comparison of the properties of the n-T* and a-r* transitions in both the cis- and trans-peptides with differing degrees of structural rigidity. Although the lactams we used are not optically active, they can be made so by substitution on the a carbon. Previously, the ultraviolet absorption of lactams has been studied down to 210 mp by Huisgen and coworkers.l' Experimental Section Materials. Cyclohexane, Eastman spectrograde and Baker Analyzed GC spectrophotometric quality, was used as solvent without further purification. Butyrolactam (n = 5) and valerolactam (n = 6) from K & K Laboratories were purified by vacuum distillation a t 90
0.063 0.087 0.071 0.120 0.099 0.092 >0.085
0.0013 0.0018 0.0014 0.0024 0.0020 0.0019 >0.0018
D,and f are 10-15%.
260
(mp)
Figure 4. Ultraviolet difference spectra of lactams in cyclohexane us. water. Ring size (n)and absorption maximum (mp), respectively, are A, 11 (226.3); B, 13 (226.3); C, 7 (233.7); D, 9 (232.5).
band^.^,^ Each method has its own disadvantages; however, different methods do give Amax'swhich agree to within 1-2 mp, If it is assumed that the -R--R* band in cyclohexane has the same general shape as the total absorption band in water, the slope of the long-wavelength side of the T-T* absorption in cyclohexane can be synthesized according to the band shape observed in water. This is shown as the dotted curve in Figure 3. The differences between the absorption in cyclohexane and the curve drawn in this manner showed the n-a* absorption to be centered at about 230 mp for most lactams. One set of such difference spectra obtained for the lactams is shown in Figure 4. The asymmetric band shapes are caused by the larger uncertainties in the absorbances for the short-wavelength side. This method generally locates A,, to within 1-2 mp for different sets of spectra (Table 111). In addition to the error due to the change in molar absorptivities in different solvents, which is shown in previous section, any error in the concentration of solutions used will also affect the final results. The molar absorptivities of this absorption are seen to be about 0.02-0.01 that of the T-T* transition. The Journal of Physical Chemistry
Xmax
-226 233 23 1 233 23 1 231 230 228
83 f 10 69& 8
i 1.2
The possible errors in the estimated values of
220
_---_____--
om-'
Another independent method of separation by curve fitting was done with a DuPont 310 curve resolver. Although it is still difficult since only part of the absorption curve is available, here we have the advantage of changing various parameters in numerous ways to get the best resolution of the band. The instrument is very sensitive with regard to the change in position or height of each peak. The method used is described below. The function generators were adjusted to fit the ultraviolet spectrum of a 1 X lov3 M solution measured in cells of 1-mm path length. The curve was generally a slightly modified Gaussian with increased wing intensity. This was repeated for five channels. With two channels on, the spectrum for a solution of twice the concentration was obtained. The base line was then lowered by adjusting the vertical position knob so that the peak coincided with the peak of the recorded Cary spectrum. Keeping this vertical position, the height of each channel was adjusted such that the curve was identical with the two times spectrum obtained previously. The combined spectrum from five channels so adjusted was used as the a--R* spectrum at 1-cm path length. A Gaussian curve was then generated on a sixth channel. The height, width, and position of this curve were adjusted to give the best fit of the 1-em spectrum. The curve for the n--R* absorption and one of the five curves for the -R--x* absorption were used as reference for integration (100%). The per cent of area for each channel and for a standard Gaussian curve was then recorded. From the known area of the standard Gaussian curve, the area of the n--R* absorption in terms of extinction coefficient and wavelength units was calculated. Table I11 summarizes the absorption maxima and the molar absorptivities at the absorption maxima, together with the dipole strengths and the oscillator strengths estimated from the above analysis. The absorption maxima agree with those by graphical method to within 1-2 mp, The estimated error in absorption maximum is around 2 mp. The integrated areas of the n--R* bands are about 0.02-0.01 that of the -R-P* band. This agrees with the ratio of extinction coefficients. Values for the dipole strengths are slightly
1647
THEULTRAVIOLET ABSORPTION SPECTRA OF LACTAMS
2401
3
n-r* absorption maxima, A preliminary study by Huisgen, et aZ.,li obtained similar curves. Because they did not observe the absorption maxima, wavelengths for molar absorptivity of 200 and 400 were plotted as a function of ring size.
Summary
4
The far-ultraviolet spectra of lactams with ring sizes of 5 , 6, 7,8, 9, 10, 11, and 13 were measured carefully in I both water and cyclohexane. This is the first detailed study for such a series of lactams. z 2 It is found that butyrolactam (n = 5) which contains ! i ring strain is not a good spectroscopic model for a cispj 200. peptide. The absorption maxima for the n-r* and the UJ m B a-r* absorptions seems to agree with the solvent effects a I I as given by Bayliss and McRae, with the n = 6 lactam r, A ' as the only exception. The absorption maxima of the r-r* transition in water are 195-197 mp for the cis-lactams and 191-193 I I I I I my for the trans-lactams. I n cyclohexane, it is shifted ' 1 T I I L to 194-196 mp for the cis and 188-190 mp for the trans. RING SIZE OF LACTAMS n The maximum molar absorptivity is higher in water (7000-8000 M - l cm-l) than in cyclohexane (5400-7000 Figure 5 . Ultraviolet absorption maxima of lactams as a function of ring size: A, T-T* absorption in cyclohexane; M-' cm-1). The dipole strengths and oscillator B, T-T* absorption in water; C, n-T* absorption strengths are about 10 D2 and 0.24, respectively, in in cyclohexane. water, and 8 D2and 0.21, respectively, in cyclohexane for all lactams studied. Absorption maxima and oslower than those reported for amides (0.12 D) by Nielcillator strengths of the cis- and trans-lactams bear the sen and S ~ h e l l m a n . ~However, the orders of magnisame relation as those of poly-L-proline I and I1 (cis and tude for the dipole strength and oscillator strength are trans polymers). similar to those for amides4 and are about 100 times For the n-r* transition in cyclohexane, the absorpsmaller than those for t'he r-r* transition. tion maxima are about 233 mp for the cis and 227-230 Another interesting feature in the ultraviolet study mp for the trans. The molar absorptivity and intensity of this band are 0.02-0.01 that of the a-r* of lactams is the dependence of the absorption maxima on ring size and cis-trans isomerization. In Figure 5 , band. The dipole strengths and oscillator strengths the absorption maxima are plotted as a function of ring range from 0.06 to 0.12 D2 and 0.001 to 0.002, respecsize to show the parallelism between the r-r* and the tively.
r 2
220-
Volume 73,Number 6
June 1969
