Peptide cysteine thiol functionalities

The simplest approach to disulfide bond formation of chemically synthesized peptides and proteins involves (i) complete deprotection including that of the cysteine thiol functions, and (ii) mild oxidation of the thiol groups to form the folded product with the native cystine connectivity. With careful attention to experimental conditions, the native-type folding of the peptide can be accomplished however, misfolded disulfide isomers are often produced as the main products in spite of efforts to optimize the reaction conditions. 

Similarly, the mixed homodetic/heterodetic bicyclic structures containing a disulfide bridge are preferably produced as monocyclic compounds that are then typically oxidized in solution adopting standard procedures as described in Section 6.1.1 (Scheme 23, path Bl). If even disulfide formation is performed on resin by the procedures reported in Section 6.1.2, an additional level of selective protection is required for the cysteine thiol functions. The synthetic paths Bl and B2 are also applicable for the synthesis of type II and III bicyclic peptides, independent of whether only lactam bridges are produced or mixtures of lactam/ disulfide bridges. 

Scheme 13. Conjugate addition of cysteine thiol group to the maleiimido functionality for the synthesis of protein-peptide constructs...

Besides being presently the most efficient method for site-directed interchain disulfide bridging of two different cysteine peptides, this procedure is also recommended for a controlled peptide-protein conjugation by reacting Npys-protected cysteine peptides with properly thiol-functionalized protein carriers. 1761 In this conjugation procedure the amount of peptide grafted to the protein is quantitatively determined by measuring spectro-photometrically at 430 nm the amount of 3-nitropyridine-2(l//)-thione liberated in the reaction. An example of this approach is outlined in Scheme 18. 

Figure 3 Different approaches for the introduction of lipid functionalities, here exemplified via the farnesyl group, into peptides. (A) Lipidated amino acid building blocks. (B) Substitution of bromoalanine with a nucleophile. (C) Alkylation or acylation of a free thiol functionality of a cysteine. (D) Conjugate addition of a nucleophile (e.g., farnesylthiolate) to a dehydroalanine. (E) Conjugate addition of a nucleophile to aziridine-2-carboxylic acid containing...

Organic carbon-centered radicals from alcohols seem to react mostly with thiol functions in amino-acids (cysteine) or small peptides such as glutathione (121). The reaction is the so-called "repciir process"... 

Lipopeptides, peptides modified with lipid residues which are preferentially bound at the thiol function of cysteine or at the a-amino group of N-terminal amino acids. The a-subunits of heteromeric G proteins and non-tyrosine receptor kinases contain N-myristoylated N-terminal glycine residues together with S-palmitoylation of a neighboring cysteine residue. Lipid moieties are necessary to recruit and anchor peptides and proteins to the membrane. Furthermore, it has been postulated that lipidation of proteins represents an event in signal transduction. The synthesis of lipid-modified peptides is not easy to perform as all coupling and deprotection reactions must be carried out under very mild conditions [S. Moffet et al., EMBO J. 1993, 12, 349 D. Kadereit et al., Chem. Fur./. 2001, 7,1184]. 

Fluoro-3-nitropyridine also reacts with the thiol function. Cysteinyl peptides react quantitatively with this reagent in a few minutes, whereas the reaction with amino groups is much slower. S-3-Nitropyridyl-L-cysteine derivatives show an absorption maximum at about 365 nm. In the CD spectra, a positive Cotton effect is centered at about the same wavelength. When thiol and amino moieties are in suitable relative positions (free L-cysteine), they react as a common functional group, but the Cotton effects are separated sufficiently (at 365 and 425 nm, respectively) and are both diagnostic of the configuration of cysteine residues. 

Special chemical derivatives free thiol functions of cysteine can be problematic because of post-synthetic uncontrolled oxidation. To avoid this, you can replace Cys by serine (Ser), alanine (Ala), or u-aminobutyric acid (Abu). Alternatively, choose the hydrophilic Cys(Acm) and leave protected. For the simultaneous preparation of peptides of different size with free amino terminus, couple the terminal amino acid as /V-Boc derivatives so that they will not become acetylated during the normal elongation cycle. Boc is removed during the final side chain deprotection procedure (Protocol 4). Special labels can be attached to the N-termini by spotting respective derivatives in an additional coupling cycle. We have succes lly added biotin via the in situ formed HOBt-ester (normal activation procedure) or fluorescein via the isothiocyanate (FITC) dissolved in NMP. 

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