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1 p1 of 10 mM oxalic acid. To some samples, valinomycin was added in such a concentration (2 pI of the 0.1 mg/ml solution) that the steady state value of the proton transport was i n c r e a~e d '~. To each sample, nigericin was added in such an amount ( I pI of the 0.1 mg/ml solution) that the AjiH of the membrane was destroyed 14. l 4 K . HellinywerS, Ph. D. Thesis, Amsterdam (1979).Preparation of 11,14-epoxy-bridged and isomeric chain-demethylated retinals. 13-Demethyl-ll,14-epoxy-, 9-demethyl-, 13-demethyl-and 9,13-bisdemethyl-retinals Abstract. 9-Cis-and all-trans-l3-demethyl-l I , 14-epoxyretinyl acetate were prepared via a Wittig coupling between P-ionylidenetriphenylphosphonium bromide (1) and 5-(acetoxymethyl)furfural. Saponification of these acetates, subsequent oxidation and HPLC separation afforded pure 9-cisand all-trans-] 3-demethyl-1 1,lCepoxyretinal. 1 1-Cis-and all-trans-9-demethyl-, 13-demethyl-and 9,13-bisdemethyl-retinaI were prepared via the same scheme, based on the Pommer vitamin A acetate synthesis. Their photochemistry shows the same type of solvent dependence as retinal. HPLC separation of the photostationary mixtures of these retinals yielded the all-trans, 13-, I I-, 9-and 7-cb isomers in 98% purity. IntroductionIn the preceding paper' we described the preparation of modified bacteriorhodopsins by the reaction of 13-demethyl-] 1,14-epoxyretinal and geometric isomers of 9--demethyl-, 13-demethyl-and 9,13-bisdemethylretinals with bacterioopsin. Investigations into the possible light--driven proton pump action and light/dark adaptation of these pigment analogues led to new information concerning the conformation of the chromophore and the process of proton pumping. In this paper, the syntheses of the required retinals are described. A simple method, based on the Wittig reaction, is given for the preparation of all-trans-and 9-cis-13--demethyl-1 1,14-epoxyretinal. For the synthesis of all--trans-and 1 l-cis-9-demethyl, 13-demethyl-and 9,13-bisdemethylretinal, we applied the Wittig reaction. The isomers were separated by preparative HPLC. Irradiation of the all-trans isomer of the demethylated retinals in a suitable solvent gives rise to photostationary mixtures of 7-, 9-, 11-and 13-cis isomers from which preparative amounts with over 98 % chemical purity could be isolated by HPLC. SynthesisFor the preparation of 9-cis-13-demethyl-l l ,14-epoxyretinal (5) and all-trans-13-demethyl-l l, 14-epoxyretinal (6) we used the approach depicted in Scheme 1. The synthesis of the Wittig salt 1, starting from j3-ionone, has been previously described*. 5-(Acetoxymethyl)furfural (2) is commercially available. The Wittig coupling between 1 and 2, using butyllithium as base, gives a I : I mixture of the bridged retinyl acetates 3 and 4. Saponification of 3 and 4 and subsequent oxidation with MnO, affords a I : 1 mixture of the 11,14-epoxy-bridged retinal 5 and 6 (overall yield 78 %). Both retinals could be separated by HPLC into pure 5 and 6. They were characterized by 'H N M R spectroscopy, NOE difference ...

The binding to bacterioopsin of the all-trans isomers of retinal analogues lacking the six-membered ring and differing in length of the conjugated chain, as well as the light-driven action of the proton pump of the resulting bacteriorhodopsin analogues, were studied. The 'opsin shifts' in these modified bacteriorhodopsins are all around 2700 cm-' and do not depend on the number of double bonds in the chromophore. These experimental results suggest that the 4800 cm-' 'opsin shift' in unmodified bacteriorhodopsin consists of a contribution of about 2700 cm-' due to the interaction of the protonated Schiff-base with the counterion. The extra 2100cm-shift in bacteriorhodopsin is due to the specific interaction of the cyclohexene ring and the protein. Only the bacteriorhodopsin analogue with the same number of conjugated double bonds in the chromophore as bacteriorhodopsin itself shows light-driven proton pump action.Bacteriorhodopsin (hereafter bR), the only protein in the purple membrane of the halophilic microorganism Halobacterium halobium, occurs in a dark-adapted ( A, , , = 558 nm) and a light-adapted form ( A, , , = 568 nm), which are interconvertible [l]. The chromophoric part of native bR is derived from all-trans retinal and 13-cis retical in dark-adapted bR, and from all-trans retinal in light-adapted bR [2]. The chromophore is protonated Schiff-base bound to the lysine-216 residue [3,4]. Light-adapted bR functions as a light-driven proton pump, pumping protons from the inside to the outside of the bacterial cell thereby creating a proton gradient across the bacterial membrane to generate the energy for ATP synthesis [5 -81. The release of the chromophore from the bR binding site can be achieved without denaturation by irradiation of bR with visible light in the presence of hydroxylamine, leaving a chromophore-free apoprotein (called bacterioopsin) and all-trans retinal oxime [9]. Addition of all-trans retinal to bacterioopsin results in immediate formation of the so called 430 -460-nm intermediate complex which rapidly converts into all-trans bR [lo] (A, , , = 568 nm). This regenerated bR, after incorporation into phospholipid vesicles pumps protons upon illumination as efficiently as native bR [6]. The red shift in the absorption maximum of dark-adapted bR (in cm-') relative to the protonated all-trans retinal Schiff-base model compound (in cm-', A,,,=440 nm), which is called 'opsin shift' [ll], is believed to be caused by special proteinchromophore interactions. Contributing factors for this 'opsin shift' are an interaction of an anion located near the cyclohexene part of the chromophore in addition to the interaction of the counterion with the protonated Schiff-base part [I I].In order to study the importance of the six-membered ring and of the 5,6 and 7,8 double bond in the chromophore for the purple colour and proton pump activity of bR, our straAbbreviations. bR, bacteriorhodopsin; bR(1) -bR(5), bacteriorhodopsin containing the retinal analogues 1 -5. tegy was to investigate the binding to bacterioo...

42M. Muradin-Szweykowska et at. / Bacteriorhodopsins with a chemicalb modified chrornophore Bacteriorhodopsins with a chemically modified chromophore. The light-driven proton pump action of [ 13-demethyl-1 l,lCepoxy]-, [9-demethyl]-, [13-demethyl]-and [9,13-bisdemethyl]-bacteriorhodopsinAbstract. The binding of the isomers of 13-demethyl-l I , 14-epoxy-, 9-demethyl-, 13-demethyland 9,13-bisdemethylretinaI to bacterioopsin has been studied. The bacteriorhodopsin analogues formed were incorporated into phospholipid vesicles and their light-driven proton pump action examined.[9-Demethyl]-bacteriorhodopsin, with a similar light-dark adaptation as the unmodified bacteriorhodopsin, shows 37% of the proton pumping ability of the native pigment. Interestingly, all-trans-13-demethylretinal does not form [13-demethyl]-bacteriorhodopsin, whereas both the 11-and 13-cis isomer form a dark-adapted [13-demethyl] pigment analogue. This [ 13-demethyl]-bacteriorhodopsin analogue neither shows light adaptation nor pumps protons, which is in agreement with the finding that only light-adapted bacteriorhodopsin can exert such a function. The 13-demethyl-l l, ICepoxyretinal forms a light-adapted bacteriorhodopsin analogue with a proton pump action. It does not show a dark-adapted form. In the 9,13-bisdemethylretinal, the perturbation at position 9 is considerably larger than in the previous cases. The specificity of the protein-chromophore interaction no longer being present, all-trans, 9-cis, 1 I-cis and 13-cis isomers form the corresponding bacteriorhodopsin analogues with proton pump ability. These results indicate that the chromophore in light-adapted bacteriorhodopsin can only occur in the 12-s-cis conformation. Furthermore, it would seem that isomerisation around the 13-double bond is not as important in proton pumping as has generally been assumed.