N to S acyl shift

Peptide Thioester Formation via an Intramolecular N to S Acyl Shift for Peptide Ligation... 

A fourth method involves an intramolecular O-to-S or N-to-S acyl shift process following peptide chain assembly (Scheme 5). Typically, thiol-functionalized ester... 

It is noteworthy that there is another limiting factor in the choice of amino acid types at the junction sites which affect the enzymatic process of the intein. For example, in the case of SceVMA (also called PI-Seel) from the IMPACT system, proline, cysteine, asparagine, aspartic acid, and arginine cannot be at the C-terminus of the N-terminal target protein just before the intein sequence. The presence of these residues at this position would either slow down the N-S acyl shift dramatically or lead to immediate hydrolysis of the product from the N-S acyl shift [66]. The compatibility of amino acid types at the proximal sites depends on the specific inteins and needs to be carefully considered during the design of the required expression vectors. The specific amino acid requirements at a particular splicing site depends on the specific intein used and is thus a crucial point in this approach. 

Figure 4 Mechanism of trans-protein splicing, (a) Initial association of the intein halves to form a functional intein. (b) Activation of the N-terminal splice-junction via an N-S acyl shift, (c) Formation of a branched intermediate upon transthioesterification. (d) Branch resolution and intein release by succinimide formation. Spontaneous S-N acyl rearrangement yields the processed product with a native peptide backbone.

As shown, under the reducing ligation conditions, 0—>S acyl transfer occurred at the o-disulfide phenolic ester as previously described (see above), to provide the thioester, which underwent transthioesteriflcation with the thiol-containing glyco-peptide (upon in situ reduction of the auxiliary disulfide bond) the transient intermediate underwent an S N acyl transfer to generate the thermodynamically favored amide bond of the doubly glycosylated peptide adduct. The auxiliary was subsequently removed through sequential methylation of the fi-ee thiol to prevent the reverse acid-mediated N S acyl shift, followed by TFA treatment. 

In order to stabilize the thioester intermediate at the cysteine residue, the amino group, produced as a result of an N-S acyl shift, needs to be protected. An interesting reaction was reported in 1985, in which the acyl cysteinylproline active... 

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. 

The palmitoyl group is highly reactive towards a nucleophilic attack, and once deprotected, the palmitoyl group can easily undergo an S- to IN -acyl shift. Specific methods are required for coupling as well as for the N-terminal Fmoc deprotection in order to minimize the formation of side products. 

In line with the above-mentioned reactivity of cysteine thioesters is the occurrence of a S, N-acyl shift of the palmitoyl group from the thiol side chain to the a-amino group, when this amino group is present as a free amine. 

To prevent the S—>N acyl shift, S-palmitoylation with palmitoyl chloride is performed in anhydrous solvents on N -aminoacylated cysteines, e.g. dipeptide building blocks, whereby the residual functionalities of the peptide have to be protected taking into account the base-lability of the thioester bond. 

Due to the S—N acyl shift, the stepwise synthesis using a S-palmitoylated cysteine derivative is not advisable. A more convenient approach is to assemble the peptide sequence and to acylate the cysteine thiol group upon its selective deprotection, retaining, however, the protection of other reactive groups. For this purpose the Cys(StBu) and Cys(Acm) deriv-... 

The splicing mechanism, which is illustrated for this intein, is shown in the accompanying equations.1 1 Step a is an N —> S or N —> O acyl shift. This is followed by transesterification (step b) which involves either thioesters (as illustrated) or oxygen esters. Formation of a succinmide intermediate (step c) releases the intein and the spliced protein. The latter must undergo an S—> N or O—> N acyl shift (step d), and the succinimide in the extein must be hydrolyzed to complete the process. 

Figure 1 Native chemical ligation (NCL) between two unprotected peptide segments. The initial transthioesterification reaction leads to an intermediate that undergoes an S to N-acyl shift via a five-membered cyclic transition state and generates a native amide bond at the ligation site.

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