7000 tor was determined with A',"-hexamethylenedimaleimide at 360, 334, and 313 nm. Quenching of the Cyclomerization. Quenching of the cyclomerization with biacetyl was carried out with monochromatic light of 334 f 7 nm. Solutions of 5 X M dioxycoumarins in dichloromethane, containing concentrations of biacetyl varying from 0.5 X to 1 X 10-1 M. were degassed, thermostated at 20", and irradiated to obtain 3 ~ 1 0 %conversion. Biacetyl does not absorb light under these conditions. The solutions were stirred vigorously by means of a magnetic stirrer. The conversion was determined after dilution by measuring the optical density at 324 nm. The difference in optical density before and after irradiation was compared with the difference in a reference sample without biacetyl, giving directly a value of W/@. Preparative Quenching Experiments. A Msolution of IC in dichloromethane is degassed in the presence of variable biacetyl concentrations and irradiated with a Bausch and Lomb monochromator at 334 i 7 nm with up to 12 =t 3 % conversion. The solvent and biacetyl were evaporated under reduced pressure, and the residue was dissolved in DMSO-&. Nmr spectra of the reaction mixture were obtained by accumulation (25 scans) on a XL 100 nmr, and the ratios of the isomer were determined by the ratio of the area of part of the nmr absorptions of the protons Hs, He, and H8in the head-to-head cyclomer over the area of the absorption g in the head-to-tail cyclomer. The validity of this of proton H
determination was confirmed by comparison with mixtures prepared from pure cyclomers and starting materials. Production Distribution. The relative ratios of cyclomer as a function of the excitation wavelength were determined by an analogous method as described in the preparative reaction quenching.
Acknowledgments. The authors t h a n k the National Science Foundation of Belgium for financial support of the laboratory and for two fellowships (J. P. and L. L.) and the IWONL for a fellowship to H. L. Dr. S. Toppet is t h a n k e d for assistance by t h e nmr interpretation. Supplementary Material Available. A detailed kinetic analysis and data for obtaining Figure 6 will appear following these pages in the microfilm edition of this volume of the journal. Photocopies of the supplementary material from this paper only or microfiche (105 X 148 mm, 24X reduction, negatives) containing all of the supplementary material for the papers in this issue may be obtained from the Journals Department, American Chemical Society, 1155 16th St., N.W., Washington, D. C. 20036. Remit check or money order for $3.00for photocopy or $2.00 for microfiche, referring to code number JACS-74-6994.
A Carbon-13 Nuclear Magnetic Resonance Study of the Visual Chromophores and Model Compounds Robert Rowan, 111, and Brian D. Sykes* Contribution from the D e p a r t m e n t of Chemistry, Harvard University, Cambridge, Massachusetts 02138. Received M a r c h I , 1974
nmr spectra of all-trans, g-cis-, 1I-cis-, and 13-cis-retinals, p-ionone, and cis- and trans-crotonaldehydes have been obtained in acetone-do solution. The 3C nmr spectra are also reported for 6-ionone and alltrans-, 1I-cis-, and 13-cis-retinals in cyclohexane-d12 solution. Striking differences in chemical shifts were found among the protonated carbons of the polyene chain portion of the different retinal isomers. Many of these differences are attributed t o the steric polarization effect. In comparing the chemical shifts of the olefinic carbons of each cis isomer relative t o the corresponding carbons of all-trans-retinal, the 11-cis isomer was found not to follow the pattern set by 9-cis- and 13-cis-retinals. Spin-lattice relaxation times TI are reported for p-ionone, alltrans- and 13-cis-retinal, and cis- and trans-crotonaldehydes. The TI values imply that (a) the methyl groups of the retinal polyene chain rotate rapidly compared with the overall tumbling of the molecule, and (b) the rotational diffusion of 13-cis-retinal and presumably al~-truns-retinalis considerably anisotropic, with 011= 4 . 6 0 1 for 13cis-retinal,
Abstract: The
The
conjugated polyene aldehydes 1I-cis- a n d alltrans-retinal play a crucial role in vision. In the only p h o t o c h e m i c a l reaction in the vision process 11cis-retinal is isomerized to the trans isomer, while attached through an i m i n e linkage to the protein opsin. This initiates a sequence of reactions resulting ultim a t e l y in the sensation of vision.' The polyene aldehyde 9-cis-retinal is also a b l e to c o m b i n e with o p s i n , 2 and 13-cis-retinal has been implicated as t h e chromophore of the membrane protein bacteriorhodopsin. As p a r t of a program to establish retinal as a nmr p r o b e of the active site of rhodopsin, we h a v e obtained t h e 13C nmr spectra o f all-trans-, 9-cis-, 1 1-cis-, a n d 13cis-retinals free i n solution. In addition, P-ionone and cis- and trans-crotonaldehyde h a v e been studied as (1) G. S. Wald, Science, 162,230(1968). (2) R . Hubbard and G. Wald, J . Gen. Physiol., 36,269 (1953). (3) D. Oesterhelt, M. Meentzen, and L. Schuhmann, Eur. J . Biochem.,
40,453 (1973).
Journal of the American Chemical Society
96.22
model compounds. In particular t h e protonated carbons of the polyene chain portion of e a c h of t h e retinal isomers have been independently and unequivocally assigned, without reference to model c o m p o u n d s , additive parameters, or substituent effects. We discuss the observed results in terms of substituent and conformational effects previously observed in systems other t h a n conjugated polyenes. We a l s o present a n d discuss longitudinal relaxation t i m e measurements f o r alltrans-retinal, 13-cis-retinal, a n d the m o d e l c o m p o u n d s .
Experimental Section fl-Ionone, all-trans-retinal, 9-cis-retinal, 13-cis-retinal, and transcrotonaldehyde were purchased from Eastman. 11-cis-Retinal was generously supplied by P. K. Brown, Acetone-de and cyclohexanedlz were purchased from Stohler Isotope Chemical Co. Most samples were prepared in the concentration range 0.35-0.75 M . Samples were filtered through sintered glass, degassed with at least five freeze-pump-thaw cycles, and sealed under vacuum. Samples SO prepared, protected from light, and stored at - 15' were stable for
1 October 30, 1974
7001 Table I.
a
13C Chemical
Shifts in Acetone-d8
Carbon
p-Ionone
1c 2c 3c 4c 5c 6C 7c 8C 9c 1OC 11c 12c 13C 14C 15C 1,l ‘CHs 5CH3 9CHa 13CH3
32.61 38.36 17.54 31.84 133.43 134.69 140.66 130.74 195.52
cis-Crotonaldehyde
trans-crotonaldehyde
146.33 129.49 189.01
152.54 133.26 191.93
12.09
16.56
27.03 19.74 25.18
All-trans
9-Cis
11-Cis
13-Cis
32.89 38.38 17.88 31.64 128.56 136.49 127.77 136.25 139.37 128.71 131.14 133.81 153.02 127.77 189.10 27.32 19.96 11.03 11.03
32.81 38.26 17.85 31.61 128.67 136.63 129.28 128.49 138.12 127.17 129.88 133.16 153.22 127.77 189.24 27.30 19.98 18.98 11.08
32.88 38.38 17.90 31.61 128.49 136.52 127.87 136.52 139.74 124.96 129.63 129.63 153.87 128,67 189.19 27.28 19.89 10.43 15.93
32.86 38.35 17.85 31.61 128.54 136.44 127.71 136.20 139.49 128.78 132.01 125.84 152.60 126.55 187.92 27.28 19.92 10.95 18.97
All shifts =t0.05ppm, relative to internal HMDS.
many months. For all samples except 9-cis-retinal and Il-cisretinal, which were prepared in 5-mm sample tubes, 12 mm tubes were used. In most cases amber sample tubes were used (Wilmad Glass). Gd(fod), was purchased from Bio-Rad Labs. Solvents were used as purchased except for the G d ( f ~ d expe$nents; )~ in this case the C6D12 was dried over previously heated 3 A molecular sieve (Linde). cis-Crotonaldehyde was prepared by irradiation of trans-crotonaldehyde, which had been purified by distillation, for 24 hr in a water-cooled Ace Glass Co. photoreactor cell, using a 500-W mercury arc source held in a quartz sleeve. The resulting mixture of cis- and trans-crotonaldehyde was purified by distillation at reduced pressure. The distillate was analyzed by lH nmr and found to be 24 % cis-crotonaldehyde and 76 trans-crotonaldehyde, with no evidence of impurities. Natural abundance nmr spectra were obtained using a VarianXL-100-15 spectrometer equipped with a Varian 620i computer and operating in the Fourier transform mode. Depending on the sample from 128 to 9000 transients were accumulated, with acquisition time and spectral digitization typically 0.8 sec and 1.25 Hz/pt, respectively. Tl measurements were made under conditions of full 1H noise decoupling using both progressive saturation ( ~ O - T ) ,and ~ inversion-recovery (T-l80-~-90),6 sequences. The x pulse length averaged 76 psec, corresponding to -yHl = 6500 Hz. The maximum spectral width for relaxation time experiments was 5000 Hz for progressive saturation and 3000 Hz for inversion-recovery. Freeman and Hill5 have shown that for offsets up to 0.8yH1, and properly calibrated tip angle, the TI measured by progressive saturation deviates not more than 5 % from the actual Tl. This was verified in the present experiments by measuring the TI of a given peak at both small and maximum offset from the carrier frequency. The relaxation time measurements were analyzed using either a two or a three parameter non-linear least-squares program with the equilibrium peak height, spin-lattice relaxation time Tl, and sometimes the effective tip angle as parameters. The ambient temperature in the probe was maintained at 31 during all experiments including decoupling. O
Results Using a variety of techniques including (1) singlefrequency and noise-modulated resonant and offresonant proton decoupling, (2) TI relaxation time measurements, and (3) the use of the relaxation reagent Gd(fod)o, all of the carbons in all-trans-retinal, 9-cisretinal, 11-cis-retinal, 13-cis-retinal, and in the model compounds P-ionone and cis- and trans-crotonaldehyde (4)D. E. McGreer and B. D. Page, Can. J. Chem., 47,866 (1969). (5) R. Freemanand H. D. W. Hil1,J. ChemPhys., 54,3367(1971). (6) R. L. Vold, J. S. Waugh, M. P. Klein, and D. E. Phelps, J . Chem. Phys., 48,3831 (1968).
i3 CH3
14
13
15
5CH3
Figure 1. Visual chromophores and model compounds showing the numbering scheme used in the text and in the tables: (a) p-ionone, (b) all-trans-retinal, (c) 9-cis-retinal, (d) 11-cis-retinal, ( e ) 13-cis-retinal, (f) cis-crotonaldehyde, (8) trans-crotonaldehyde.
have been assigned. The nmr chemical shifts of all of these compounds in acetone-& solution are listed in Table I (see Figure 1 for numbering of carbons). The assigned 13C nmr spectra of the polyene chain carbons of the four retinal isomers, excluding the aldehyde carbon, are shown in Figure 2. Since there is some disagreement between these assignments and those reported earlier for P-ionone and trans-retinyl acetate,’ a compound which might be expected to have some (7) M. Jautelat, J. B. Grutzner, and J. D. Roberts, Proc. Nar. Acad.
Sci. U.S., 65,288 (1970).
Rowan, Sykes
1
Nmr Study of the Visual Chromophores
7002 Table 11. 13C Spin-Lattice Relaxation Timesa Carbon
p-Iononed
1c
p-Iononebtc
1 7 . le 1.32 0.99e 1.29 15.7 23.5h 3.85 3.64 14.81
2c 3c 4c 5c 6C 7c 8C 9c
1oc
23.7
1.85 1.66 1.94 29.8 43.0 5,26 4.94 44.0
all-transRetinald
all-transRetinab
9.46 0.75 0.65 0.82 8 .70e 11.3 1.07 1.05 9.366 0.97
17.2 1.59 0.696 1.56 17.1 23.10 2.39i 2.36 21 . 6 6 2.18 2.18 2.36 25.2 2.39j 3.43 1.35 4.99 6 . W 6.004
11c
1 .oo
12c 13C 14C 15C 1,l'CHa 5CH3 9CH3 13CH3
1.02 9 . 80e
1.10 1.44 4.94 7.70
1.42 0.87 3.57 4.05k 4.05k
2.07 6.17 10.2
134s-
RetinalC 14.7 1.22 0.93e 1.36 18.4 30.7 2.37 2.56 27.4 2.26 2.45 2.53 16.8 1.52 2.14 1.65 3.85 6.44 3.97
trans-Crotonaldehydec
cis-Crotonaldehydec
22.5 28.7 25.7e
29.2 27. 2e 33 .O
36.2
46. 3i
a Relaxation times TI in seconds. Samples were degassed unless otherwise noted. Error limits given are the standard deviations of the All values are 1 < l O % unless otherwise noted. b Not degassed. c In acetone-& nonlinear least-squares fits. Most values are + < 5 7 & In C6DI2. e 1 < 1 5 % . &5.1 sec. 1 4 . 6 sec; corrected for partial overlap of 8c resonance. * 1 3 . 8 sec. 1 2 0 . 9 sec. 2 7C and 14C are coincident. 9CH3 and 13CH3are coincident. Q
4
i
I1
C)
156
6
152
''c
148
I44
140
CHEMICAL SHIFT
I36
132
I28
124
(ppm from HMDS)
Figure 2. nmr (25.16 MHz) spectra of the four retinal isomers in acetone-&, showing the olefinic, nonaldehyde carbon region: (a) all-rrans-retinal, (b) 9-cis-retinal, (c) 11-cis-retinal, (d) 13-cis-
retinal.
Peak marked x in spectrum b is an impurity.
spectral similarity to all-trans-retinal, our assignments are discussed in detail below. In the following discussions of IH decoupling experiments, reference will be made to characteristics of the corresponding lH nmr spectra. These have been reported by several authors and include all of the retinal
isomers in CDC18,* all-trans- and 1 1-cis-retinal in acetone-d6,' 9 4 s - and 13-cis-retinal in acetone-&, lo all four retinal isomers in C6D,, and ethano1-ds,l0 P-ionone (neat), and cis- and trans-crotonaldehyde neat4 and in aCetOne-ds.'* We first consider the assignment of the 13C nmr spectrum of P-ionone. The single quaternary carbon, lC, is identified immediately in the saturated carbon region of the nmr spectrum by its long T 1 value (Table 11). Single frequency off-resonance 'H decoupling distinguishes the resonances of the three methylene carbons 2C, 3C, and 4C from three methyl carbon resonances. 4C is identified by single frequency on-resonance decoupling of 4H,H'. Since the 'H nmr spectrum of 2H,H' and 3H,H' is tightly coupled at 100 MHz, 2C and 3C were only tentatively distinguished from each other by 'H decoupling. Confidence in this choice of assignments for 2C and 3C is strengthened by comparing the observed nmr chemical shifts with those predicted using the parameters of Savitsky and Namikawa. I 3 The methyl carbon resonances corresponding to l,1'CH3, 5CH3, and 9CH3 were easily assigned by single-frequency irradiation of the 'H methyl resonances. Of the five unsaturated carbons, three are unprotonated and thus distinguished by their long TI'S (c$ Table 11). The carbonyl carbon 9C was immediately identified by its chemical shift. 5C and 6C were assigned on the basis of their Tl values. Given that the 13Crelaxation times are dominated by intramolecular dipole-dipole interactions with neighboring protons, l 4 and assuming that the same rotational correlation time characterizes (9) R. Rowan, 111, A. Warshel, B. D. Sykes, and M. Karplus, Biochemislry, 13,970 (1974). (10) R. Rowan, 111, Ph.D. Thesis, Harvard University, 1974. ( 1 I ) M. Mousseron-Canet and J. C. Mani, Bull. SOC.Chim. Fr., 3285 (1966). (12) R. Rowan, 111, J. A. McCammon, and B. D . Sykes, J. Amer. Chem. SOC.,96,4173 (1974). (13) G. B. Savitsky and I
