Cysteinyl peptides

Raftery, M.A., and Cole, R.D. (1963) Tryptic cleavage at cysteinyl peptide bonds. Biochem. Biophys. Res. Comm. 10, 467-472. 

Aminopeptidases that catalyze the hydrolysis of cysteinyl peptides are known. The membrane-bound aminopeptidases are glycoproteins, usually with molecular weights of about 100,000 daltons. They appear to be metalloproteins, one of the better known being a zinc-containing enzyme. Other enzymes, such as the leucine aminopeptidase, are cytosolic but, at least in this case, are also zinc-containing. The substrate specificity of these enzymes varies but most are relatively nonspecific. 

Watanabe and Snell40 isolated the labeled cysteinyl peptide from l4C-bromopyruvate-inactivated apotryptophanase. Comparison of this peptide with the total amino acid sequence indicated that Cys-298 was the alkylated residue. Honda and Tokushige 63> found that Cys-298, Cys-148 and Cys-352 reacted with A -(4-anilino-1 -naphthyl)-maleimide and that Cys-298 was the most extensively modified. 

To better simulate the behavior of Fe-S clusters in the protein environment, a logical step is to ligate the clusters to actual cysteinyl peptides. Peptide-ligated clusters can be prepared via ligand exchange reaction (17) or cluster reconstitution using FeCb, Na2S, a reductant, and the peptide in aqueous buffer. 

Wang, H. et al. (Goh et al. 2007) 2007. Characterization of mouse proteome using global proteomic analysis complemented with cysteinyl peptide enrichment. J. Proteome Res. 10, 1-13. 

Figure 4. Depiction of ligand substitution reactions of [Fe4S4(S-t-Bu)4] with cysteinyl peptides to yield peptide clusters 11-14 and their conversion to [Fe4S4(SPh)4] , in MejSO solution (78).

Clusters (6b) with L = halide have been obtained from (6a) by reaction (4) , and are useful starting materials for the preparation of clusters (6b) with L = ArO and C4H4N , as in ligand substitution reaction (5) . This reaction has also been used with NaSR to prepare thiolate clusters (6, including those containing short cysteinyl peptides as closer models of protein site (1). Peptide clusters are also accessible by reaction (3). Bis(pentasulfide) cluster (6c) is obtained by the reaction of [Fe(SPh)4] with five equivalents of (PhCFl2S)2S. ... 

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. 

The acyl oxazolidinone stracture 25 was reported as another active structure, in which an NS acyl shift reaction can be performed under neutral conditions, and ligation with cysteinyl peptide 7 can then proceed [59]. Although the synthesis of hBNP32-NH2 was successfully performed, the acyl oxazolidinone was partially decomposed by piperidine treatment and was potentially racemized during peptide chain elongation by Fmoc SPPS (Scheme 7). 

Anilides are slightly more active than alkyl amides. (V-Sulfanylethylanilide (SEAhde) 27 can be transformed into the thioester via intramolecular N-S acyl shift under both acidic and neutral conditions [60, 61]. Phosphate salts are needed for thioester formation under neutral conditions, in which ligatimi with the cysteinyl peptide is performed (Scheme 8). 

Recently, ligation via an N-S acyl shift at the a-methylcysteine site was reported [78]. By using an a-methylcysteine residue, the ligation with a cysteinyl peptide 7 was performed successfully, not only at the glycine or histidine residue but also at the leucine residue under neutral, aqueous conditions (Scheme 15). 

An initial search for the thioester-forming sequence indicated that a peptide 67 containing a Cys-Pro-Cys/Ser sequence appeared to be converted into the DKP thioester. An N-SIO acyl shift at the second Cys or Ser residue (path b) would produce a Cys-Pro (thio)ester structure 68b. Once the CPE structure is formed, the DKP thioester 52 would be obtained via an N-S acyl shift at the first Cys residue (path a) followed by DKP formation (path c). The order of reactions a and fe would not be critical for the overall reaction. As of this writing, DKP thioester formation from the CPC peptide was demonstrated only in model systems (Scheme 22). When the CPC peptide, H-Ala-Lys-Leu-Arg-Phe-Gly-Cys-Pro-Cys-NH2 (70), was treated at 110°C under acidic conditions, dilute hydrochloric acid or heptafluorobutyric acid (HFBA), the corresponding DKP thioester 71 was obtained. Although epimerization in the DKP moiety was observed, the reaction mixture was reacted with cysteinyl peptide, H-Cys-Tyr-NH2 (72), to produce 73 as a single isomer. 

Fig. 5 RP-HPLC of (a) reaction mixture of the thioesterification of CPE peptide 74, (b) reaction mixture of the ligation of CPE peptide 75 and cysteinyl peptide 76, (c) final reaction mixture after removal of the protecting groups. Column YMC-Pack ProClS (4.6 x 150 mm) (a) or YMC-Pack C8 (4.6 X 150 mm) (b, c), eluent aq CH3CN containing 0.1% TEA, flow rate 1.0 mL/min. 80 H3 (44-135).

Spahr, C.S. Susin, S.A. Bures, E.J. Robinson, J.H. Davis, M.T. McGinley, M.D. Kroemer, G. Patterson, S.D. Simplification of complex peptide mixtures for proteomic analysis reversible biotinylation of cysteinyl peptides. Electrophoresis 2000, 21, 1635-1650. 

The synthesis of Mo-Fe-S clusters as models for the active site of the enzyme has been possible.The clusters consist of two M0FC3S4 cubes bridged through the molybdenum corners by three S atoms but cannot be true analogues because they differ in the Fe S Mo ratio. Mononuclear Mo complexes of cysteinyl peptide ligands have also been reported. 

Figure 3. Thiazolidine formation in ligation site by two steps. (A) Oxidation of a C-terminal 1,2-propanediol peptide or an N-terminal serinyl peptide by NalOt to form a C-terminal or an N-terminal peptide aldehyde, respectively. (B) An aldehyde ligated with an /V-cysteinyl peptide to form a thiazolidine peptide with different orientations of the thiazolidine ring.

In thiazolidine ligation, the reaction is performed between a glycolaldehyde peptide ester and an A/"-cysteinyl peptide. The capture step is based on... 

Figures. Thiol-thioester exchange to form an amide bond. Nucleophilic attack takes place by the side chain thiol functionality in a cysteinyl peptide at a thioester segment to form an intramoiecular thioester, which undergoes intramolecular S N acyl transfer to form an amide bond.

Peptidyl thioester TQFCFH-SCHjCHjCONH, (synthesized by BocKihemistry on thio-ester resin) and cysteinyl peptide CKVLTTGLPALISW (synthesize by Fmoc-chemistry)... 

Add the cysteinyl peptide solution to the peptidyl thioester (94 ig, 0.1 (Linol). 

Introduction. Methoxycarbonylsulfenyl chloride (Scm-Cl), easily prepared from equimolar amounts of methanol and chlorocarbonylsulfenyl chloride (eq 1), finds many applications, based on the reactivity of alkoxycarbonylsulfenyl chlorides against thiols. Brois and co-workers realized the value of methoxycarbonylsulfenyl chloride in the synthesis of asymmetric disulfides, which led to important applications in the chemistry of cysteine and its derivatives, particularly cysteinyl peptides. Other applications include preparation of aromatic thiols, symmetric trisulfides, etc. The applicability of Scm-Cl surpasses its drawbacks which include relatively short storage life (1-2 months at —20°C in sealed ampules) and relatively rapid hydrolysis in the protic media that some of its most important uses require. 

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