Synthesis of Lipidated Ras Peptides

Isoelectric point or pi is the pH at which in one molecule the negative and positive 

Fluorescence lifetime imaging microscopy (FLIM)-based guantitative fluorescence resonance energy transfer (FRET). Direct detection of biomolecular interactions is possible with FRET measurements, where a donor fluorophore transfers the energy to an acceptor fluorophore in case they are close in space. The combination of this technique together with FLIM, based on the decrease in donor fluorophore lifetime that is induced by FRET, has recently enabled the quantitative assessment of the protein-interacting fractions [12]. 

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Triola G, Gerauer M, Gormer K et al (2010) Solid-phase synthesis of lipidated ras peptides employing the Elhnan sulfonamide linker. Chem Eur J 16 9585-9591... 

The solid-phase synthesis of lipidated Rab peptides has relied on the hydrazide linker, similar to the lipidated Ras peptides (15). By employing geranylgeranylated or fluorescent labeled lipidated cysteines as building blocks, a highly flexible... 

As an illustrative example for the successful realization of this concept, here the interplay between three disciplines, organic synthesis, biophysics and cell biology in the study of protein lipidation and its relevance to targeting of proteins to the plasma membrane of cells in precise molecular detail is described. The interplay is highlighted using the Ras protein as a representative example. Included herein is the development of methods for the synthesis of Ras-derived peptides and fully functional Ras proteins, the determination of the biophysical properties, in particular the ability to bind to model membranes, and finally the use of synthetic Ras peptides and Ras proteins in cell biological experiments. 

Scheme 2 General considerations for the synthesis of lipidated peptides of Ras family proteins. PG protecting group...

The suitability of the Aloe group for the construction of lipidated peptides is emphasized by the synthesis of the maleimidocaproyl-modified, S-palmi-toylated and farnesylated heptapeptide 16 which corresponds to the N-Ras C-terminus (Scheme 10).1211 In contrast to classical urethane-type protecting groups, the Aloe group can be removed in the presence of additional functional groups and under neutral conditions. It is therefore a very convenient protecting group for the synthesis of very hydro-phobic lipid-modified peptides, which are not soluble in the aqueous media required for enzyme catalyzed transformations. 

For the synthesis of a small library of palmitoylated and isoprenylated N-Ras peptides in solution, a modular strategy was adopted, with the tetrapeptide MGLP 38 as a key intermediate. This tetrapeptide allowed further elongation at its C-terminus with lipidated or nonlipidated cysteine methyl esters, as well as the addition of various N-terminally MIC-labeled dipeptides, consisting of different GC lipidated units 39—41... 

In summary, the hydrazine linker is currently the most suitable linker system for the synthesis of C-terminally methylated lipidated peptides. Allowing the incorporation of different types of lipid chain in combination with the MIC group at the N-terminus the hydrazine linker gives access to a variety of lipidated peptides of the Ras superfamily. 

A large body of work has been devoted to the synthesis of the C -terminal lipidated peptides of the small GTPases H-/N-Ras. As such, these peptides have been synthesized in solution, via combined solution and solid support approaches and completely on solid phase. 

Figure 5 Solid-phase synthesis of an N-Ras lipidated peptide on hydrazide resin. (A) Fmoc-Cys(Far)-OH, FIBTU, FIOBt, IMP, CFI2CI2/DMFS (1 1). (B) piperidine/DMF (1 4). (C) Fmoc-AA-OH, HBTU, HOBt, DIPEA, DMF. (D) piperidine/DMF (1 4). (E) Fmoc-Cys(Pal)-OH, HBTU, HOBt, TMP, CH2CI2/DMF (1 1). (F) DBU (1 %) in DMF. (G) Fmoc-Gly-OH, HATU (5 eg.), DIPEA (20 eq.) CH2CI2/DMF (7 1). (H) Cu(OAc)2 (0.5 eq.), pyridine (30 eq.), acetic acid (SO eq.), methanol (215 eq.) CFI2CI2, O2.

In the next pages the synthesis of C-terminal lipidated peptides corresponding to Ras and Rab proteins will be described in detail. First of all, the synthesis of the required building blocks will be shown followed by different solid-phase strategies for the synthesis of a large variety of monolipidated, double lipidated, labeled, or differently functionalized peptides. 

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