Toward Semisynthetic Lipoproteins by Convergent Strategies Based on Click and Ligation Chemistry

Cell-membrane proteins are anchored to the lipid bilayer by single or multiple insertion of transmembrane helices or by regioselective single or dual lipidation in coand post-translational enzymatic processes, including acylation with fatty acids, prenylation, and rather commonly C-terminal amidation with glycosylphosphatidylinositols (GPI). Procedures for native and neolipidation of peptides have been comprehensively reviewed, and, more recently, even the synthesis of complex GPIs has been reported. However, lipidation of proteins at defined sites and particularly grafting of GPIs or related mimetic structures to the C termini of proteins still represent formidable long-term goals of chemistry and molecular biology. So far, lipoproteins have been obtained by chemical ligation of synthetic lipopeptides with recombinant protein fragments by the highly selective and efficient maleinimide/thiol addition reaction, as shown for the RAS protein, for example, or by total synthesis through orthogonal protection schemes for regioselective lipidation of side-chain amino groups, for example, with 1,2-dipalmitoyl-glycero-3-succinate. In view of the recently developed efficient procedures for the semisynthesis of proteins by native-chemical [6] and expressed-protein ligation, we have performed model studies toward C-terminal lipidation of proteins by exploiting the copper(i)-catalyzed Huisgen’s 1,3-dipolar cycloaddition of terminal alkynes to azides to form a stable triazole product and the transthioesterification between peptide thioesters and N-cysteinyl-lipopeptides followed by intramolecular S! N acyl shift as the synthetic strategy set forth in Scheme 1. Aside from validating the methodology of a combined click and ligation chemistry, incubation of HeLa cells with the micellar solution of the lipopeptide confirmed its fast uptake, as visualized by confocal fluorescence microscopy. It is well established that dual vicinal lipid chains, as present in the di-fattyacyl glycerol moiety of natural GPI anchors are required for an almost irreversible capture of peptides and proteins by lipid bilayers. Correspondingly, to properly mimic the GPI anchor, phosphatidylethanolamine was converted into the corresponding azide 1 by CuSO4-catalyzed diazotransfer with triflyl azide to produce the key intermediate for subsequent application of the click chemistry. The crystalline azide 1 was then used for the 1,3-cycloaddition reaction with the Sprotected model peptide 2, which contained a C-terminal propargylglycine (Pra) residue as suitable reaction partner (Scheme 1). The azide–alkyne cycloaddition was performed with CuI as catalyst in organic solvent, and the lipopeptide derivative 3 was isolated by silica gel chromatography in yields of 70–75 %. Upon removal of the acid-labile S-trityl and NBoc groups from 3 with TFA, the subsequent native chemical ligation of the cysteinyl-lipopeptide with N-dansylor N-rhodamine B-labeled Gly-Pro-Gly-Gly-SPh ester 4 was performed in micellar solutions of 2 % octyl-b-d-glucopyranoside. Ligation was found to proceed smoothly in the presence of tris(2-carboxyethyl)phosphine (TCEP) if excess of the thioesters was carefully avoided to prevent bisacylation as a side reaction. HPLC served to isolate the fluorescence-labeled lipopeptides 5 a,b in yields of 60–70 % as analytically well-characterized compounds, as shown in Figure 1 for compound 5 b. The lipopeptide 6 was obtained in practically quantitative yield by treatment of the C-terminal propargylglycine residue with azide 1 in aqueous–organic media and in the presence of [a] H. -J. Musiol, Dr. S. Dong, Dr. M. Kaiser, Prof. Dr. L. Moroder Max-Planck-Institut f r Biochemie Am Klopferspitz 18, 82152 Martinsried (Germany) Fax. : (+ 49) 89-8578-2847 E-mail : moroder@biochem.mpg. de [b] Dipl. -Phys. R. Bausinger, Dr. A. Zumbusch Institut f r physikalische Chemie Butenandtstraße 5–13, 81377 M nchen (Germany) [c] Dr. U. Bertsch Zentrum f r Neuropathologie und Prionforschung, LMU M nchen Feodor Lynen Straße 23, 81377 M nchen (Germany)