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Staining and fixation of unsaturated membrane lipids by osmium tetroxide: c rystal structure of a model osmium (VI) intermediate
Biochimica et Biophysica Acta, 320 (1973) 745-747 © Elsevier Scientific Publishing Company, Amsterdam - Printed in The Netherlands
BBA Report BBA 21380 Staining a n d f i x a t i o n o f u n s a t u r a t e d m e m b r a n e lipids b y o s m i u m t e t r o x i d e : crystal structure of a model osmium(VI) intermediate
R. COLLIN, W.P. GRIFFITH, F.L. PHILLIPS and A.C. SKAPSKI Department of Chemistry, Imperial College, London SW7 2A Y (Great Britain)
(Received July 27th, 1973)
SUMMARY The crystal structure of a cyclic mono-ester of osmium(VI) has been determined, and is shown to be dimeric with a double oxo bridge. The relevance of this structure as a possible model for the staining and fLxation of lipids by osmium tetroxide is discussed.
OsO4 is extensively used as a staining and fixation agent for electron microscopy of tissues 1,2 and as a specific reagent in organic chemistry for the cis-hydroxylation of olefinic double bonds 3 . It is thought that the first step in both processes is the formation of an osmium(VI) mono- or di-ester. For cis-hydroxylation the ester is then hydrolysed to give the cis-diol, but in staining neither the nature of subsequent processes nor the extent of reaction with other functional groups has been elucidated. It has been shown, however, that OsO4 reacts in a 1 : 1 ratio to each olefinic double bond, with a wide variety of unsaturated tissue lipids and phospholipids, suggesting the formation of mono-esters4 ,s. Various structures have been proposed for such mono-esters; that most generally accepted involves four-coordinate osmium(VI) (Fig. 1) 6 ,7, but other polymeric structures involving octahedral osmium(VI) have also been proposed (Becker, R., quoted in ref. 5, ref. 8). As part of a study of the reaction of osmium tetroxide with model systems of biological relevance we report here the structure of bis-[/a-oxo-oxo(tetramethylethane-1,2diolato)osmium(VI)], [OsO2 (02 C2 Me4)]2, prepared by allowing stoichiometric quantities of OsO4 and tetramethylethylene to react in carbon tetrachloride (a modification of the procedure described by Criegee et al. 7). The compound was selected for study because of its crystallinity; it has so far not been possible to obtain suitable crystalline esters of more complex olefms or of lipids. The complex, which is diamagnetic, was shown to contain osmium(VI) by a titration method developed from that of Crowell and Kirschmann 9 ; elemental analyses were in accord with the formula OsO4 C4 H12.
746
BBA REPORT
Os
Me
oj Fig. 1. Structure of four-coordinate osmium(Vl) mono-esters. Fig. 2. The centrosymmetric structure of [OsO2(02 C2Me4)]2. The distance between mid-points of the two ester C-C bonds is approx. 7.7 A.
The black elongated prisms obtained from carbon tetrachloride solution are monoclinic with unit-cell dimensions a = 8.145, b = 14.008, c = 7.811 A,/3 = 93.24 °; space group P21/n and Z = 2. The structure has been determined on the basis of 1321 independent reflections measured on a Siemens four-circle diffractometer. Least-squares refinement has now reached R --- 0.040. The centrosymmetric structure of the dimeric molecule is shown in Fig. 2. The osmium has a square-based pyramidal coordination, two of the four basal oxygen atoms being from the planar Os2 02 bridge (mean OS--Obridg e distance 1.92 A) and the other two from the ester linkage (mean Os-Oester distance 1.87 A). The apical atom is the terminal oxo ligand and the very short Os-Oterminal distance of 1.675(8) A indicates that there is extensive multiple bond character in this linkage. A feature of particular interest is the large Oterminal--Os--Obasal mean angle of 111°; the average Oterminal--Obasa 1 distance of 2.95 A is appreciably larger than the mean Obridge--Oeste r distance of 2.59 A. This distortion probably arises from repulsions by the strongly 1r-donating terminal oxo ligand (we have observed an analogous effect in [OsNC14 ]- ref. 10). Despite the far greater complexity of osmium compounds with membrane lipids in biological systems there is spectroscopic evidence which suggests that this complex may be a viable model for such species. The infrared spectra of the compound and its 180substituted analogues show that the Os-Oterminal stretching frequency is at 982 cm-1 and the two Os--Obridg e stretches are at 752 arid 631 c m - 1 , repectivetyal. Osmium(VI) monoesters of larger olefins, e.g. camphene, indene and cholesterol are dimeric and also have bands in these three regions 11, indicating that they have similar structures (monomeric structures as in Fig. 1 would show no such bridging bands but would have two strong bands near 850 cm -1 associated with the cis terminal oxo ligands 12). It is known that osmium(VI) esters derived from unsaturated lipids and phospholipids have strong infrared bands near 980 c m - 1 (refs 4, 13). We believe that the unexpected dimeric structure of this complex is significant in two respects. Firstly, it may be of help in clarifying the role of OsO4 in the fixation of
BBA REPORT
747
membrane lipids, since cross-linking of erstwhile double bonds via an Os20~ bridge would be expected to enhance the coherence of the lipid structure. Secondly, the requirement of an approx. 7.7 A distance between the two C - C ester bonds may severely distort the arrangement of unsaturated lipids; thus electron micrographs of osmium-stained materials may not accurately reflect the original disposition of membrane lipids. Although the steric requirements of the substrate could induce formation of a similarly oxo-bridged dimer in which the terminal oxo ligands are cis rather than trans to each other as in this structure, the requirement of a 7 - 8 3, distance between the C=C bonds would remain. (Such a cis configuration would not materially alter the spectroscopic data). In the event o f osmium(VI) di-esters being formed 13 in preference to mono-esters, such cross-linkage distortion may be even more severe 14. In addition to studying model osmium(VI) di-esters, we are now investigating the nature of reactions between OsO4 and other reactive groups found in membrane and tissue systems. We thank the Science Research Council for a Postdoctoral Fellowship (to R. Collin), the University of Ghana for a Postgraduate Scholarship (to F.L. Phillips), and Johnson, Matthey Ltd, for the loan of osmium t e t r o x i d e . REFERENCES 1 Riemersma, J.V. (1970) Some Biological Techniques in Electron Microscopy, p. 69, Academic Press, New York 2 Hayat, M.A. (1970) Principles and Techniques of Electron Microscopy, Vol. 1, p. 36, Van Nostrand, London 3 Cairns, J.F. and Roberts, H.L. (1968) J. Chem. Soc. (C] 640-642 4 Stoeckenius, W. and Mahr, S.C. (1965) Lab. Invest. 14, 1196-1207 5 Riemersma, J.C. (1968)Biochim. Biophys. Acta 152, 718-727 6 Criegee, R. (1936)Ann. 522, 75-96 7 Criegee, R., Marchand, B. and Wannowius, H. (1942)Ann. 550, 99-133 8 Griffith, W.P. and Rossetti, R. (1972)J. Che~ Soc. (Dalton) 1449-1453 9 CroweU, W.R. and Kirschmann, H.D. (1929)J. Am. Che~ Soc. 51,175-179 10 Fletcher, S.R., Griffith, W.P., Pawson, D., Phillips, F.L. and Skapski, A.C. Inorg. Nucl. Chem. Lett. in the press 11 CoUin, R. and Griffith, W.P.J. Chent Soc. (Dalton), submitted 12 Griffith, W.P. (1969)J. Chem. Soc. (,4) 211-218 13 Korn, E.D. (1967) J. CellBiol. 34,627-638 14 Adams, C.W.M. (1965) Neurohistochemistry, p. 39, Elsevier, New York
BBA Report BBA 21380 Staining a n d f i x a t i o n o f u n s a t u r a t e d m e m b r a n e lipids b y o s m i u m t e t r o x i d e : crystal structure of a model osmium(VI) intermediate
R. COLLIN, W.P. GRIFFITH, F.L. PHILLIPS and A.C. SKAPSKI Department of Chemistry, Imperial College, London SW7 2A Y (Great Britain)
(Received July 27th, 1973)
SUMMARY The crystal structure of a cyclic mono-ester of osmium(VI) has been determined, and is shown to be dimeric with a double oxo bridge. The relevance of this structure as a possible model for the staining and fLxation of lipids by osmium tetroxide is discussed.
OsO4 is extensively used as a staining and fixation agent for electron microscopy of tissues 1,2 and as a specific reagent in organic chemistry for the cis-hydroxylation of olefinic double bonds 3 . It is thought that the first step in both processes is the formation of an osmium(VI) mono- or di-ester. For cis-hydroxylation the ester is then hydrolysed to give the cis-diol, but in staining neither the nature of subsequent processes nor the extent of reaction with other functional groups has been elucidated. It has been shown, however, that OsO4 reacts in a 1 : 1 ratio to each olefinic double bond, with a wide variety of unsaturated tissue lipids and phospholipids, suggesting the formation of mono-esters4 ,s. Various structures have been proposed for such mono-esters; that most generally accepted involves four-coordinate osmium(VI) (Fig. 1) 6 ,7, but other polymeric structures involving octahedral osmium(VI) have also been proposed (Becker, R., quoted in ref. 5, ref. 8). As part of a study of the reaction of osmium tetroxide with model systems of biological relevance we report here the structure of bis-[/a-oxo-oxo(tetramethylethane-1,2diolato)osmium(VI)], [OsO2 (02 C2 Me4)]2, prepared by allowing stoichiometric quantities of OsO4 and tetramethylethylene to react in carbon tetrachloride (a modification of the procedure described by Criegee et al. 7). The compound was selected for study because of its crystallinity; it has so far not been possible to obtain suitable crystalline esters of more complex olefms or of lipids. The complex, which is diamagnetic, was shown to contain osmium(VI) by a titration method developed from that of Crowell and Kirschmann 9 ; elemental analyses were in accord with the formula OsO4 C4 H12.
746
BBA REPORT
Os
Me
oj Fig. 1. Structure of four-coordinate osmium(Vl) mono-esters. Fig. 2. The centrosymmetric structure of [OsO2(02 C2Me4)]2. The distance between mid-points of the two ester C-C bonds is approx. 7.7 A.
The black elongated prisms obtained from carbon tetrachloride solution are monoclinic with unit-cell dimensions a = 8.145, b = 14.008, c = 7.811 A,/3 = 93.24 °; space group P21/n and Z = 2. The structure has been determined on the basis of 1321 independent reflections measured on a Siemens four-circle diffractometer. Least-squares refinement has now reached R --- 0.040. The centrosymmetric structure of the dimeric molecule is shown in Fig. 2. The osmium has a square-based pyramidal coordination, two of the four basal oxygen atoms being from the planar Os2 02 bridge (mean OS--Obridg e distance 1.92 A) and the other two from the ester linkage (mean Os-Oester distance 1.87 A). The apical atom is the terminal oxo ligand and the very short Os-Oterminal distance of 1.675(8) A indicates that there is extensive multiple bond character in this linkage. A feature of particular interest is the large Oterminal--Os--Obasal mean angle of 111°; the average Oterminal--Obasa 1 distance of 2.95 A is appreciably larger than the mean Obridge--Oeste r distance of 2.59 A. This distortion probably arises from repulsions by the strongly 1r-donating terminal oxo ligand (we have observed an analogous effect in [OsNC14 ]- ref. 10). Despite the far greater complexity of osmium compounds with membrane lipids in biological systems there is spectroscopic evidence which suggests that this complex may be a viable model for such species. The infrared spectra of the compound and its 180substituted analogues show that the Os-Oterminal stretching frequency is at 982 cm-1 and the two Os--Obridg e stretches are at 752 arid 631 c m - 1 , repectivetyal. Osmium(VI) monoesters of larger olefins, e.g. camphene, indene and cholesterol are dimeric and also have bands in these three regions 11, indicating that they have similar structures (monomeric structures as in Fig. 1 would show no such bridging bands but would have two strong bands near 850 cm -1 associated with the cis terminal oxo ligands 12). It is known that osmium(VI) esters derived from unsaturated lipids and phospholipids have strong infrared bands near 980 c m - 1 (refs 4, 13). We believe that the unexpected dimeric structure of this complex is significant in two respects. Firstly, it may be of help in clarifying the role of OsO4 in the fixation of
BBA REPORT
747
membrane lipids, since cross-linking of erstwhile double bonds via an Os20~ bridge would be expected to enhance the coherence of the lipid structure. Secondly, the requirement of an approx. 7.7 A distance between the two C - C ester bonds may severely distort the arrangement of unsaturated lipids; thus electron micrographs of osmium-stained materials may not accurately reflect the original disposition of membrane lipids. Although the steric requirements of the substrate could induce formation of a similarly oxo-bridged dimer in which the terminal oxo ligands are cis rather than trans to each other as in this structure, the requirement of a 7 - 8 3, distance between the C=C bonds would remain. (Such a cis configuration would not materially alter the spectroscopic data). In the event o f osmium(VI) di-esters being formed 13 in preference to mono-esters, such cross-linkage distortion may be even more severe 14. In addition to studying model osmium(VI) di-esters, we are now investigating the nature of reactions between OsO4 and other reactive groups found in membrane and tissue systems. We thank the Science Research Council for a Postdoctoral Fellowship (to R. Collin), the University of Ghana for a Postgraduate Scholarship (to F.L. Phillips), and Johnson, Matthey Ltd, for the loan of osmium t e t r o x i d e . REFERENCES 1 Riemersma, J.V. (1970) Some Biological Techniques in Electron Microscopy, p. 69, Academic Press, New York 2 Hayat, M.A. (1970) Principles and Techniques of Electron Microscopy, Vol. 1, p. 36, Van Nostrand, London 3 Cairns, J.F. and Roberts, H.L. (1968) J. Chem. Soc. (C] 640-642 4 Stoeckenius, W. and Mahr, S.C. (1965) Lab. Invest. 14, 1196-1207 5 Riemersma, J.C. (1968)Biochim. Biophys. Acta 152, 718-727 6 Criegee, R. (1936)Ann. 522, 75-96 7 Criegee, R., Marchand, B. and Wannowius, H. (1942)Ann. 550, 99-133 8 Griffith, W.P. and Rossetti, R. (1972)J. Che~ Soc. (Dalton) 1449-1453 9 CroweU, W.R. and Kirschmann, H.D. (1929)J. Am. Che~ Soc. 51,175-179 10 Fletcher, S.R., Griffith, W.P., Pawson, D., Phillips, F.L. and Skapski, A.C. Inorg. Nucl. Chem. Lett. in the press 11 CoUin, R. and Griffith, W.P.J. Chent Soc. (Dalton), submitted 12 Griffith, W.P. (1969)J. Chem. Soc. (,4) 211-218 13 Korn, E.D. (1967) J. CellBiol. 34,627-638 14 Adams, C.W.M. (1965) Neurohistochemistry, p. 39, Elsevier, New York