A. Succinyl and Malonyl Spacer 4432 B. Phthaloyl and Isophthaloyl Spacer 4434 C. Rigid Spacer Concept 4435 D. Peptide Spacer 4437 V. Synthesis of Cyclic Glycosides 4437 VI. A Unique Case Termed "Intramolecular Glycosidation" 4440 VII. Conclusion 4440 VIII. References
Mixed protein-surfactant micelles are used for in vitro studies and 3D crystallization when solutions of pure, monodisperse integral membrane proteins are required. However, many membrane proteins undergo inactivation when transferred from the biomembrane into micelles of conventional surfactants with alkyl chains as hydrophobic moieties.Here we describe the development of surfactants with rigid, saturated or aromatic hydrocarbon groups as hydrophobic parts. Their stabilizing properties are demonstrated with three different integral membrane proteins. The temperature at which 50% of the binding sites for specific ligands are lost is used as a measure of stability and dodecyl-β-D-maltoside ("C12-b-M") as a reference for conventional surfactants. One surfactant increased the stability of two different G protein-coupled receptors by approximately 10°C compared to C12-b-M. Another surfactant yielded a stabilization of the human Patched protein receptor by 13°C.In addition, one of the surfactants was successfully used to stabilize and crystallize the cytochrome b 6 f complex from Chlamydomonas reinhardtii. The structure was solved to the same resolution as previously reported in C12-b-M. Declaration of Interest statementThe authors report no declarations of interest. Mixed protein-surfactant micelles are often used for in vitro studies when solutions of pure, monodisperse integral membrane proteins are needed (Garavito and Ferguson-Miller, 2001). Because integral membrane proteins evolved in a lipid bilayer environment, the exchange of lipid for surfactant frequently causes a destabilization of the protein. This can result in increased activity followed by increased rate of denaturation, increased susceptibility to protease attack, accelerated heat denaturation, oxidative damage, loss of activity regulation and lack of crystallization (Tanford and Reynolds, 1976;Tate, 2010). Europe PMC Funders GroupAt the beginning of membrane protein research only surfactants developed as detergents for consumer or industrial use were available many of which were inhomogenous. In-vitro studies of solubilized membrane proteins and structural work (Rosenbusch et al., 1981;Michel and Oesterhelt, 1980) led to an increasing demand in tailored surfactants. In the micellar solution the protein should be functional and stable for extended periods of time and the micellar belt attached to the protein should be small in order to leave much of the polar protein surfaces uncovered for crystal lattice contacts (Michel, 1983).Studies of the micellar mass revealed the relation between the chemical structure of a surfactant and the size of its micelle. It was found to depend upon the ratio of the polar cross-section and the length of the extended hydrophobic chain (Israelachvili et al., 1976). In case of spherical micelles the latter matches the radius of the hydrophobic core. With alkyl chains as hydrophobic moieties of surfactants proteins require a length between an extended octyl and a dodecyl chain (Iwata, 2003).Small micelles are for...
Reaction of the benzyl-and acetyl-protected a-trichloroacetimidates 1 and 6 a with dialkyl and diary1 phosphates in the presence of traces of acid affords stereoelectively the thermodynamically more stable a-L-fucopyranosyl phosphates 2, 7 and 8, respectively, in high yields. The use of very pure, recrystallized dibenzyl phosphate results in the stereoeletive formation of the fi-L-fucopyranosyl phosphates 3 and 9. In each case separation of the anomers is not required because of the very high stereoselectivity of the reactions. After deprotection the fucose 1-phosphate 12 is coupled with GMP morpholidate 10 to yield GDP-fucose 13. After the development of a new purification procedure for GDP-fucose 13 we have obtained a very pure compound suitable for biochemical investigations. Analytical and preparative HPLC has been performed on reversed-phase columns using a volatile buffer system (triethylammonium hydrogen carbonate) as the eluant.L-Fucose is included in bacterial lipopolysaccharides and in blood-group substances2') or other mammalian glycosphingoIipids4*'). Fucosylated glycolipids have antigenic properties and play an important role in cell-growth regulation and cell differentiation6). Therefore, the metabolism, development during cell growth, and oncogenic transformation of glycolipds especially in tumor cells have been intensively studied2,6). GDP-fucose is the substrate for fucosyl transferases involved in the biosynthesis of fucose-containing oligosaccharides. Although much GDP-fucose is required for these biochemical investigations, but only one chemical synthesis has been reported 'I. Enzymatic synthesesspi0) proceeding via fucose 1-phosphate or via GDP-mannose are suitable only for the preparation of very small quantities.The key intermediate in the chemical synthesis of GDPfucose is fucose 1-phosphate, whose availability is the limitating factor. a-Fucopyranosyl phosphate can be readily synthesized according to the chlorophosphoamidite method"). The reported syntheses, however, of P-fucopyranosyl p h o~p h a t e '~,~) proceed with low yields and low stereoselectivity, probably due to its increased lability against acids 13). So far, enzymatic syntheses8,") of P-fucopyranosyl phosphate are not suitable for large-scale preparations. Owing to the good results obtained by use of trichloroacetimidates in the synthesis of glycosyl phosphates 15,16) we have developed new syntheses for a-and 0-fucopyranosyl phosphates operating with high stereoselectivity. The trichloroacetimidates of fucose are also useful as glycosyl donors in the chemical synthesis of fucose-containing oligosaccharides ").Using the easily available benzyl-protected trichloroacetimidate 1 17), we have synthesized the benzyl-protected fucosyl phosphates 2 and 3 in high yields. Reaction of 1 with dibenzyl phosphate in the presence of traces of acid yields stereoselectively the thermodynamically more stable a anomer 2. The use of very pure recrystallized dibenzyl phosphate resulted in the stereoselective formation of the P an- Starting from ...
2-Hydroxy-4,6-dimethoxyacetophenone (4) was glycosylated with 0-(2,3,4,6-tetra-O-benzy~-a-~-glucopyranosyl) trichloroacetimidate (5) and trimethylsilyl triflate as promoter to yield directly the C-glycoside 6. Construction of the flavone system by application of a Baker-Venkataraman-type rearrangement followed by deprotection yielded isoembigenin (2). Glycosylation of 4,6-bis(tert-butyldimethylsilyloxy)-~-hydroxyacetophenone (17) with the trichloroacetimidate 5 afforded the 0-glycoside intermediate 18 which was converted via Fries rearrangement into the C-glycoside 21. Applying again the Baker-Venkataraman rearrangement and cyclization gave isovitexin and vitexin derivatives 25 and 26, which were completely deprotected to yield isovitexin (lb) and vitexin (la), respectively.
6-Su~fo-a-~-quinovopyranosyl phosphate was reacted with different nucleoside monophosphate morpholidates to form ADP-, CDP-, GDP-and UDP-sulfoquinovose. Analytical and preparative HPLC of these nucleotides was performed on reversed-phase columns using volatile buffer systems as eluant. The isolated compounds were characterized by NMR spectroscopy (except the CDP derivative) and used for an investigation of sulfolipid biosynthesis by chloroplasts. For this purpose intact spinach chloroplasts were biosynthetically preloaded with radioactive diacylglycerol to provide a sulfoquinovosyl acceptor. When sulfosugar nucleotides were added to such prelabelled intact organelles, the background levels of sulfolipid biosynthesis did not rise. On the other hand, after osmotic shock of prelabelled chloroplasts sulfolipid labelling was significantly increased by the addition of UDP-or GDP-sulfoquinovose. The same stimulation was observed with isolated envelope membranes, and UDP-sulfoquinovose proved to be twice as active as the GDP derivative. From these results it was concluded that the final step in sulfolipid biosynthesis is catalyzed by a UDP-sulfoquinovose: 1,2-diacylglycero13-0-a-D-sulfoquinovosyltransferase. This chloroplast enzyme cannot use exogenously supplied sulfosugar nucleotides, which as membrane-impermeable compounds are expected to be formed in viwo within chloroplasts.The plant sulfolipid 1 ',2'-di-0-acyl-3'-0-(6-deoxy-6-sulfoa-D-glucopyranosy1)-sn-glycerol [I] is a membrane lipid found in higher plants, cyanobacteria and some other photosynthetic procaryotes [2]. With regard to the sulfur budget of plants, the sulfolipid represents a major component which accounts for about half of the organic sulfur in leaves [3]. In higher plants this unique glycolipid is confined to plastids [4]. In these organelles it is located in the two surrounding envelopes as well as in the inner thylakoid membranes [5], where part of it may be firmly bound to specific protein complexes [6 -81.After application of radioactive sulfate to plants, the sulfolipid is rapidly labelled [I, 2, 91. Despite this specific and easy labelling, several steps in sulfolipid biosynthesis have not been resolved and at present two alternative routes are discussed. It has been suggested that a sulfoglycolytic sequencc [lo -121 produces 3-sulfo-~-lactaldehyde which instead of 3-phospho-~-glyceraldehyde can be used by aldolase [lo] to form 6-deoxy-6-sulfo-~-fructofuranosyl 1-phosphate. Subsequent hydrolysis and isomerisation to 6-sulfoquinovose (quinovose = 6-deoxy-~-glucose), rephosphorylation at C-I and activation by a corresponding pyrophosphorylase could result in a nucleoside diphospho-sulfoquinovose. Such a com-
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