trans isomers of retinal analogs by high-performance proton NMR method

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GENERAL METHODS FOR RETINAL PROTEINS

[70]

ticulate silica gel (10/zm). The eluate is passed through a UV flow detector operating at 254 or 340 nm. Ether (0.3-0.7%, v/v) in cyclohexane is used as the eluting solvent at a flow rate of 1-3 ml/min. The conditions of flow rate and ether content of the solvent will vary with the column but are adjusted to give efficient separation with a standard sample of retinals. Typical elution volumes for retinal isomers using 0.5% ether are as follows: ll-cis, 61.5ml; 13-cis, 72.6ml; 9-cis, 96.6ml; all-trans, 112.5 ml. The desired isomers are collected separately and the solutions are concentrated with a rotary evaporator and then transferred to small conical tubes. Further concentration to a smaller volume ( - 5 0 / z l ) is achieved with a stream of nitrogen. At this point an aliquot is transferred to the device used to introduce solid samples into the direct inlet of a mass spectrometer. If the sample holder has been previously cleaned in acid, it is imperative that it be washed with considerable distilled water, ammonia, and then distilled water before drying. Traces of residual acid in contact with retinal at elevated temperature lead to substantial decomposition and a considerably altered 285/284 ratio. After transfer, the solvent is evaporated off in a stream of nitrogen. The process is repeated until the whole solution is transferred to the sample holder and all the solvent is removed. The sample is introduced into the direct inlet system and the sample is analyzed. Typical conditions for the Hitachi-Perkin Elmer RMU-7 mass spectrometer are as follows: sample source chamber temperature, 60°; ionization chamber, 250°; photomultiplier voltage, 1.25 kV; ionizing voltage, 80 eV. The source pressure is generally 1 x 10-6 mm during measurement. Prior to mass spectral analysis of the unknown, pure control samples of all-trans-retinal and/or 13-cis-retinal are analyzed to verify that the system is operational and to measure the 285/284 ratio in the absence of isotopic enrichment.

[70] Identification of cis/trans Isomers of Retinal Analogs by High-Performance Proton NMR Method

By PAUL TOWNER and WOLFGANG GARTNER The first absolute identification of the isomers of retinal relied on their stereospecific synthesis; this afforded the assignment of an isomeric configuration to the chromatographic bands of retinal isomers when separated by thin-layer chromatography (TLC). 1 Further developments led to i C. von Planta, U. Schwieter, L. Chopard-dit-Jean, R. R0egg, and M. Kotter, Helv. Chim. Acta 45, 548 (1962). METHODS IN ENZYMOLOGY, VOL. 88

Copyright © 19~2 by Academic Press, Inc. All rights of reproduction in any form reserved. ISBN 0-12-181988-4

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547

the identification of structure by using N M R methods, particularly in the lucid works of Patel 2 and Rowan et al., 3 who accomplished the complete characterization of retinal using 220-MHz machines. In this article we describe an unambiguous method of identification of the isomers of retinal and retinal analogs using a high-performance N M R technique. The essential role retinal plays in bacteriorhodopsin and rhodopsin is undisputed; however the mechanisms by which these proteins fulfill their function is still not clearly understood. Consequently many scientists interested in retinal protein interaction use retinal analogs as probes of the retinal binding sites, which in the case of bacteriorhodopsin should give valuable clues as to the mechanism of light-induced proton transport. In such experiments it is imperative to know the isomeric configuration of any analog used, and in some cases this has been done by inference to the properties of retinal. The chromatographic order of retinal isomers is known to be all-trans, 7-cis, 9-cis, 11-cis, and 13-cis with increasing Rf value; furthermore the apo-protein of rhodopsin (opsin) forms chromoproteins with 7-, 9- and l l-cis-retinal, 4,5 whereas bacterio-opsin reacts readily with all-trans- and 13-cis-retinal, 6 and to a lesser extent with 7-cis. 7 Thus a pure but unknown isomer of an analog can be isolated from a TLC plate, mixed with opsin and bacterio-opsin and the hma x value of any new chromophores observed, giving an inference of the isomeric type. However, as a means of identification this is clearly unsatisfactory since some retinal analogs are found to behave differently than retinal. A case in point is 13-demethylretinal where the chromatographic order of the ll-cis and 13-cis isomers is reversed, and where the ll-cis isomer readily reacts with bacterio-opsin. To obtain unequivocal identification of retinal analog isomers the use of high-resolution N M R machines with magnetic fields of 400 and 500 MHz was found to yield excellent results with as little as 90/zg material, which required 40 min for accumulation of a Fourier transform spectrum. As an example the 400-MHz proton NMR spectrum of 11-cis13-demethylretinal is shown in Fig. 1. The signals in this spectrum fall into three categories: (1) the aldehyde proton at 9.5 ppm, (2) the olefinic protons of the chain (6.0-7.6 ppm), and 2 D. J. Patel, Nature (London) 221,825 (1969). 3 R. Rowan, III, A. Warshel, B. D. Sykes, and M. Karplus, Biochemistry 13, 970 (1974). 4 W. J. DeGrip, R. S. H. Liu, V. Ramamurthy, and A. Asato, Nature (London) 262, 416 (1976). 5 G. Walt:l, Nature (London) 219, 800 (1968). e D. Oesterhelt and L. Schuhmann, FEBS Lett. 44, 262 (1974). T K.-D. Kohl and W. Sperling, Annu. Meet. Dtsch. Ges. Biophys., 1979 (1979). s W. G~rtner, H. Hopf, W. E. Hull, D. Oesterhelt, D.Scheutzow, and P. Towner, Tetrahedron Lett. 21, 347 (1980).

548

GENERAL METHODS FOR RETINAL PROTEINS

TM~

~5~ 0

•

10.0

,II

.I I,

9.5

[70]

ZC) 6.0 2.0 chemical shift (ppm)

~

Jr

1iO

0:0

Fits. 1. 400-MHz [1H]NMR spectrum of l l-cis-13-demethylretinal in deuteriochloroform. The chemical shifts of some of the protons of this spectrum differ from those presented in the table because of changes in concentration and temperature.

(3) the aliphatic protons of the ring and the methyl groups (1.0-2.0 ppm). It is the olefinic group of signals that reveals the cis/trans nature of the four double bonds of 13-demethylretinal; these signals are shown in expanded form in Fig. 2. Their interpretation first requires a short description of two basic NMR principles. First, it must be understood how the signal of a proton is influenced by neighboring protons on adjacent carbon atoms by spin-spin coupling, which effects splitting of the signal of the proton. Second, we explain how beginning with an unambiguously identified signal, an assignment of the residual signals to the other protons, is H8

H13

7.7

6.8

Z5

6.6

6.4 6.2 6.0 chemical shift (ppm) Fro. 2. The olefinic proton resonances of 1 l-cis-13-demethylretinal in expanded form.

[70]

HIGH-PERFORMANCE PROTON NMR

549

determined by the double-resonance technique. These features can be best explained by reference to the partial olefinic structure I of 13-demethylretinal shown below. H I

H I

HI STRUCTURE

The effect of spin-spin coupling observed for any proton depends on (1) the number of protons bound to the adjacent carbon atoms, and (2) the type of carbon-carbon bond. The coupling between protons over three bonds is the most meaningful in interpretation of 1H-NMR spectra; however coupling over four or more bonds can occur and yield useful information, but since this coupling is in the range of 1 Hz it is not totally resolved by machines of less than 250 MHz and leads only to a broadening of the signals. If the coupling constants between the protons are similar, the splitting follows the rule m -- n + 1, where m is the number of peaks in the signal group of a proton and n the number of proton neighbors on adjacent carbons. The coupling constant gives information on the type of bond between the carbon atoms. Thus the proton at position 2 undergoes splitting first to a doublet via coupling to proton at C-1 and is further split, with a different coupling constant, to a double doublet by simultaneous interaction with the proton at C-3. This is illustrated schematically in Fig. 3. The symbol iJk,l designates the apparent coupling constant where i is the number of bonds over which coupling takes place and k,1 signify the coupled protons. In structure I, 3J2.~, which is over a single carbon-carbon bond, has a value of l l - 12.5 Hz; 3J1,2 is over a trans double bond and

(a)

FIG. 3. Schematic representation of proton coupling. (a) Single resonance peak of the uncoupled proton at C-2 in structure I. (b) A doublet is formed by coupling with proton at C-l. (c) A multiplet is formed by simultaneously coupling to proton at C-3.

550

GENERAL METHODS FOR RETINAL PROTEINS

[70]

equals 14-16 Hz, and if the double bond was cis substituted, then 3J1, 2 is 11-13 Hz. Thus a knowledge of the coupling constants of protons across double bonds gives valuable information on the cis/trans nature of a polyene chain. The coupling constants in hertz can be directly measured from the spectrum as the distance between two peaks in a signal group, since 1 ppm (part per million) is by definition 400 Hz in the case of a 400-MHz machine. The assignment of signals to the distinct protons is found by decoupiing experiments using the double-resonance technique. This is dependent on the fact that a split proton signal, due to coupling with another proton, will collapse to a single peak when the resonance frequency of the effector proton is disturbed by irradiation. The signal pattern of the protons at C-l, C-2, and C-3 in structure I are drawn in Fig. 4a. Upon irradiation at the frequency of the resonance of proton at C-3, only the residual coupling between protons 1 and 2 is found; the signal pattern of proton 1 remains largely uninfluenced (Fig. 4b). The signals of the protons in a polyene chain could be anticipated to overlap, causing a spectrum of higher order to arise, which requires computer-assisted interpretation. NMR machines with large magnetic fields have the advantage that they produce well-defined first-order spectra from which valuable information of the proton interactions can be obtained at a glance. As a general rule, a spectrum can be classified as first order when the ratio of the difference in the chemical shift, in hertz, to the coupling constant between two protons is greater than or about 10. By reference to Fig. 2, protons 10 and 11, which have a coupling constant of

(a)

H2

FIG. 4. Double-resonanceexperiment. (a) In structure I proton at C-2 couples with both H-1 and H-3, with differentcouplingconstants. (b) Decouplingby irradiationat H-3 leaves the residual couplingbetween H-2 and H-1.

[70]

HIGH-PERFORMANCE PROTON NMR

551

12 Hz, have a difference in chemical shift of 0.2 ppm; the ratio then equals about 7. Using a knowledge of the structure of 13-demethylretinal, the signals of the olefinic protons can be constructed. Protons 7 and 8 are each observed as doublets, as is proton 10, whereas protons 11, 12, 13, and 14 show double doublets. The aldehyde proton, via coupling with proton 14, would also be present as a doublet at very low field at 9.5 ppm. Double resonance at this recognized site would change the signal form of proton 14, and if this proton in turn were subjected to double resonance it would lead to the identification of proton 13. This process can be repeated until protons 12, 11, and 10 are located. At position 9 the methyl group disrupts a link to protons 7 and 8; thus these two isolated protons can be recognized only by inference. Once the signal pattern has been correlated to specific protons and their coupling constants measured, the isomeric configuration of the 7/8, 11/12, and 13/14 double bonds can be determined. The assignment of cis or trans stereochemistry to the 9/10 double bond is impossible using the preceding method since no vicinal coupling is present; however there is a remarkable downfield shift of proton 8 for the cis configuration, as is found with retinal, z Using these methods the chemical shifts of the olefinic protons of allt r a n s and all four m o n o - c i s isomers of 13-demetfiylretinal can be measured. The coupling constants enabled the cis/trans geometry of the double bonds to be evaluated. These results are presented in the accompanying table. Using these methods we have recorded the 1H-NMR spectra of a-retinal, 5-demethylretinal, and 9-demethylretinal. The interaction of these analogs with opsin and bacterio-opsin was then studied and their biological activity determined. 9 As a practical guide we have usually used about 10 mg material, which substantially reduces the time required for accumulation of a Fourier transform spectrum. When loading samples into the machine, we were always careful to use dim light conditions. With small quantities of material ( - 100/xg) the relatively high temperature (30°) of the sample holder did not cause destruction or isomerization of the sample during the measuring time; thus we have not found it necessary to connect a cooling unit during experiments. The main advantage of the sensitive 400- and 500-MHz machines is their ability to provide highly resolved spectra from small quantities of sensitive material. It is thus possible to extract retinal from a chromoprotein sample and use the extract directly for NMR analysis. 9 p. Towner, W. G~irtner, B. Walckhoff, D. Oesterhelt, and H. Hopf, FEBS Lett. 117, 363 (1980).

552

[71]

GENERAL METHODS FOR RETINAL PROTEINS

[~H]NMR DATA OF 13-DEMETHYLRETINAL MONO-CISISOMERS IN DEUTERIOCHLOROFORM Chemical shift (ppm)

allqrans

7-cis

9-cis

1l-cis

13-cis

7-H 8-H 10-H ll-H 12-H 13-H 14-H 15-H 1-CH3 5-CH 3 9-CHa

6.38 6.17 6.19 7.07 6.46 7.21 6.14 9.56 1.04 1.72 2.02

6.03 6.15 6.26 7.01 6.43 7.20 6.16 9.57 1.04 1.51 1.92

6.37 6.67 5.99 7.16 6.40 7.20 6.04 9.54 1.05 1.75 2.03

6.40 6.24 6.62 6.84 6.24 7.68 6.17 9.63 1.05 1.74 2.02

6.37 6.18 6.22 6.96 7.20 7.04 5.83 10.19 1.05 1.74 2.04

16 11.5 14.5 11.5 15 8

12.5 11.5 14.5 11.5 15 8

16 12 14.5 11.5 15 8

16 12 11 11.5 15 8

16 12 14 12 11 8

Coupling constants (Hz) J(7,8) J(10,11) J(11,12) J(12,13) J(13,14) J(14,15)

Acknowledgments The authors are grateful for the NMR facilities provided by Bruker Messtechnik, Karlsruhe, and the encouragement offered by Dr. W. E. Hull. We thank Professor H. Hopf for helpful discussions and Professor D. Oesterhelt for his interest and provision of facilities. This work was supported by the Deutsche Forschungsgemeinschaft.

[ 7 1 ] Methods for Extraction of P i g m e n t Chromophore By

MOTOYUKI TSUDA

I. Introduction Visual pigments, retinochrome, and the purple membrane protein of the halobacteria (bacteriorhodopsin, BR) are chromoproteins consisting of a retinal molecule covalently bound to the protein. The isomeric conformation of retinal varies from one pigment to another; rhodopsin

METHODS IN ENZYMOLOGY,VOL. 88

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