Peptides C-terminal thioester

Ingenito, R., Bianchi, E., Fattori, D. and Pessi, A. (1999) Solid phase synthesis of peptide C-terminal thioesters by Fmoc/t-Bu chemistry. Journal of the American Chemical Society, 121(49), 11369-11374. 

Scheme 7 Synthesis of partially protected peptide C-terminal thioesters.

Fmoc-protected amino acid fluorides 307 were found to be excellent reagents for the acylation of sulfonamide safety-catch linkers (SCLs) 306 suitable for the subsequent preparation of peptide C-terminal thioesters (Equation 57). This generally applicable approach enables high loadings on different types of resins using short reaction times with low levels of racemization <20020L1187>. 

Preparation of Peptide C-Terminal Thioesters by Fmoc-SPPS... 

Scheme 10 Direct ligation of a peptide C-terminal thioester with another N-terminus...

Other very important applications of C-terminal thioester-functionalized peptides include their usage in the condensation of large unprotected peptide fragments (ligation see Vol. E22a, Section 4.1.5). 4,5,74 In this process the thioester-modified unprotected peptide reacts with the N-terminal cysteine of a second unprotected peptide giving a thioester intermediate. This step is followed by a rapid intramolecular S—>N shift with formation of the thermodynamically favored amide bond at the ligation site. 

In this method, the cysteine-thioester cyclization generates a cyclic peptide 86a (see Scheme 23) with a Xaa-Cys bond whose thiol moiety is then used for tethering to the core through an S-alkylation reaction.191 The requirement for the cyclization reaction is a linear precursor 84 containing both an N-terminal Cys and a C-terminal thioester. Such a peptide precursor can be conveniently synthesized by a stepwise solid-phase synthesis on a thioester resin 81 using Boc chemistry (Scheme 22). Cleavage by HF after assembly of the peptide sequence will produce the desired precursor with an N-terminal Cys and a C-terminal thioester 84. The crude peptide is then purified by RP-HPLC and the purified unprotected peptide is then circularized in aqueous conditions buffered at pH > 7.0. 

Fig. 3. Intein-mediated protein ligation. The IMPACT system allows affinity purification of proteins fused to an intein-CBD tag and their further isolation with a C-terminal thioester moiety (A), or an N-terminal cysteine (B). (A), N-terminal intein splicing for thioester isolation. Target protein (protein 1) is expressed in E. coli with C-terminally located intein-CBD tag. After specific binding to the chitin resin, the thiol reagent provokes the cleavage of the peptide bond between the target protein and the intein. Whereas the intein-CBD tag remains bound to the chitin resin, the protein thioester is eluted from the column. (B), C-terminal cleavage to obtain N-terminally...

Beligere, G. S. and Dawson, P. E. (1999) Conformationally assisted protein ligation using C-terminal thioester peptides. J. Am. Chem. Soc. 121, 6332-6333. 

The native chemical ligation (NCL) has enjoyed tremendous success as one of the few methods for C-terminal protein modification. In its most general form, this approach is a ligation between a peptide that contains a C-terminal thioester and a... 

Figure 1 Relative Native Chemical Ligation Rates of C-Terminal Thioester Peptides Corresponding to L-Y-R-A-X (X is the designated amino acid) Percent Ligation Product (Filled Symbols) and Phenylmeth-anethiol-Thioester Exchange Intermediates (Unfilled Symbols) as a Function of Timel ...

The sensitivity of thioester groups to aminolysis by piperidine has prevented the use of thioester-based linkers for Fmoc SPPS. Two groups d have adapted a modified form of Kenner s acylsulfonamide safety-catch linkerf for the synthesis of C-terminal thioester peptides using Fmoc protocols (Scheme 8). This approach is promising and future studies will more fully analyze the limitations of the method. 

Scheme 8 Synthesis of C-Terminal Thioester Peptides Using an Fmoc-Compatible Linkerl Fmoc-Xaa, PyBOP, DIPEA Fmoc ...

Fig. 13 a The principle of native chemical ligation. Peptide 1, containing a C-terminal thioester, undergoes a nucleophilic attack by the cysteine residue at the N-terminus of peptide 2. The intermediate rearranges spontaneously to form the native peptide bond, b Native chemical ligation without the requirement of a cysteine residue using the l-phenyl-2-sulfanylethyl auxiliary group... 

In order to perform a native chemical ligation, two prerequisites have to be fulfilled. First, the C-terminal fragment (2) has to contain a Cys residue (which in principle could also be Ser, although it is less reactive) at the N-terminus. The N-terminal fragment (1) possesses a C-terminal thioester. The reactivity of this activated carboxyl group can be tuned by proper selection of the thiol substituent. For example, aromatic groups are more reactive than alkyl groups. The mechanism of peptide bond formation encompasses two steps. In a first reversible reaction the... 

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